The Cerebellum Revisited

Rodolfo Llinas Constantino Sotelo

Editors

The Cerebellum
Revisited
With 209 Illustrations, 5 in Full Color

Springer-Verlag
New York Berlin Heidelberg London Paris
Tokyo Hong Kong Barcelona Budapest
Rodolfo Llinas Constantino Sotelo
Department of Physiology/Biophysics INSERM U-106
New York University Medical Center H6pital de la Salpetriere
550 First Avenue 47 Boulevard de I'H6pital
New York, NY 10016 75013 Paris
USA France

Library of Congress Cataloguing-in-Publication Data

The Cerebellum revisited/Rodolfo Llim'ts, Constantino Sotelo,

editors.
p. cm.
Based on the Cajal Centenary Meeting on The Neuron Doctrine held
in 1988 in Barcelona, Spain.
Includes bibliographical references and index.
ISBN-13:978-1-4612-7691-3
1. Cerebellum-Physiology-Congresses. I. Llinas, Rodolfo R.
(Rodolfo Riascos). II. Sotelo, Constantino. III. Cajal
Centenary Meeting on The Neuron Doctrine (1988:Barcelona, Spain)
[DNLM: 1. Cerebellum-physiology-congresses. WL 320 C414 1988]
QP379.C46 1991
599'.0188-dc20
DNLM/DLC 91-5226

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Preface

This book is organized into three parts that correspond with the main
groups of chapters delivered during the Cajal Centenary Meeting on
The Neutron Doctrine. These chapters represent important aspects of the
morphology, development, and function of the cerebellum and related
structures. Clearly an exhaustive analysis of all aspects of the cerebellar
system, as they relate to the legacy of Ramon y Cajal, would be impossible
to contain in just one volume, given its far-reaching impact. Instead, we
deliberately steered away from the traditional handbook approach that
some of us have taken in the past and selected those aspects of cerebellar
research currently under vigorous study that would also represent the
widest scope of interest for neuroscientists in general and for cerebellar
specialists in particular.
In particular, we felt that as the discrete anatomy of the cerebellum is
quite well known, only certain aspects of the structure should be discussed
here. For example, the organization of the pontocerebellar pathways, we
felt, would be particularly interesting given the enormity of the system in
higher vertebrates. Also of interest is the distribution and development
of the synaptology and neurotransmitter properties in this cortex. Indeed,
from the point of view of cerebellar development, this may represent one
of the clearest paradigms in the understanding of rules for neurogenesis
for the central nervous system. More specifically, aspects relating to the
precise organization of the shape of Purkinje cells and the epigenetic
restrictions that govern the crystal-like structure of the cerebellar cortex
are being concretized and are expected to be generalizable to other regions
of the nervous system. This is particularly significant given the precise
crystal-like organization of the morphology of the cerebellar cortex.
Purkinje cells are isoplanar and strictJy regimented with regard to each
other, thereby producing maximum divergence and maximum convergence
for their parallel fiber input. Equally precise is the distribution of the
granular cell-parallel fiber system and the rostrocaudal distribution in
the olivocerebellar system.
On the other hand, the simplicity of connectivity that characterizes this
cortex throughout evolution and the fact that the basic cerebellar circuit
described by Cajal recurs at different levels of phylogeny from the lower
vertebrates to man indicates the presence of powerful laws of morphogenesis

v
VI Preface

where those variables that determine the final structure of neural nets
may be studied more dearly. With regard to the functional aspects of the
cerebellum, some specific issues may be mentioned concerning the electro-
physiology of the Purkinje and the olivary cells that are of general interest
to electrophysiologists and of special interest to those who wish to under-
stand integrative cell physiology 'or the organization of rhythmic para-
computation in the olivocerebellar system.
In terms of electrophysiology, Purkinje cells are the largest neural
elements in the brain and the epitome of neuronal interaction. They have
been studied intracellularly with single and double penetrations, where
the electrical activity of both the soma and dendrites of a given neuron
are simultaneously recorded. Biophysically, the use of the patch-damp
techniques have yielded descriptions of the voltage- and ligated-dependent
currents in these neurons with a degree of detail seldom obtained in other
neuronal elements of the brain. By contrast, the inferior olivary cells are
the prototypical oscillating neurons of the vertebrate brain with their ionic
conductances and are organized in a very special fashion. They represent
the other extreme of neuronal functioning from the Purkinje cell. Their
ability to integrate and transmit information is limited by their powerful
intrinsic oscillatory properties. Thus, it is only through studies of neuron
ensembles, as may be studied in slices, and more particularly with the

Figure 1. Vertical section of the cerebellar folium in chicken stained with the
Golgi method. The three parenthesis (to the right) show, in A the molecular layer,
in B the granular layer, and in C the white substance. (A) is the body of the
Purkinje cell; (B) the Deiters prolongation of the neuron (the axon); (D) a small
stellate cell; (L) the axonal prolongation of those elements; (C) the terminal
branches of this prolongation; (S) the hole left by the descending branch where
the body of the Purkinje cell is lodged; (H) small elements of the granular layer
with the axonal cylinder directly upwards; (F) large stellate cell of the granular
layer; (G) axonal cylinder of that cell. (Reprinted from S. Ramon y Cajal, May
1988. Estructura de los centros nerviosos de las aves. I. Cerebelo. Revist. trimestr.
de Histol. norm. y patol., 1. Mayo de 1888.)
 Preface Vll

Figure 2. Section of the cerebellar folium in pigeon stained with the Golgi method.
For the sake of clarity, the Purkinje cells are not depicted. (A) neuroglial cells
from the molecular layer; (B) neuroglial cells from the granular layer; (C) tree-like
neuroglial cells; (D) large stellate cell with its axon terminating in a large number
of varicose ramifications; (E) nodose fiber (mossy fiber) with small ramification;
(F and G) varicose vertical fibers, (P) stellate elements of the molecular layer,
(0) axon from the cell in P. (Reprinted from S. Ramon y Cajal, May 1888. Estructura
de los centros nerviosos de las aves. 1. Cerebelo. Revist. trimestr. de Ristol. norm.
y patol.. 1. Mayo de 1888.)

chimeric hardware-neuronal hybrids developed by Y. Yaron-that we

begin to understand the properties of such neuronal groupings.
The third aspect of the book relates to the physiology of cerebellar
systems, most particularly, the way in which neuronal afferents interact
at the level of the cerebellar cortex. Because the cerebellar cortex is close
to homogeneous along its surface, many of the details of functional
diversity encountered relate to the nature of information that reaches the
cerebellar cortex as well as the nature of afferents that reach it.
The cerebellar cortex may be viewed as a radial with fractured somato-
topy when considering the mossy fiber system and rostrocaudal bonded
projection considering the climbing fiber systems (see Figs. 1 and 2).
Although published in 1888, Figures 1 and 2 depict the cellular elements
in the cerebellar cortex as we know them today. They are also the first
description of the neuron as a distinct cellular element having a body, a
dendritic tree, and axon. These nerve cells were described as "contacting,"
without direct continuity, their target cells, i.e., the author indicated that
the cells maintain their individuality.
Finally, beyond the understanding of the cerebellar mapping and the
different distribution of cerebellar afferents, the issues relating to the role
of the cerebellum in motor coordination continue to challenge us. Indeed,
Vlll Preface

the precise manner in which the neuronal circuits, so well understood,

ultimately control the execution of movement is far from clear.
Nevertheless, progress has occurred; many ideas currently related to
overall cerebellar functioning are under investigation. In short, then, this
book required a personal choice of topics and an incomplete one at that.
And yet we believe we have assembled a comprehensive collection of
writings covering fundamental topics of cerebellar neuroscience from the
molecular to the systems level that we hope would please both the specialist
and the generalist in accordance with what we consider to be the intel-
lectual revolution that commenced a century ago with Ramon y Cajal at
the helm.
Contents

Preface v

Contributors xi

Part 1. Organization, Development, and Repair

of the Cerebellar Circuits 1
1. Purkinje Cell Heterogeneity: Its Role in Organizing
the Topography of the Cerebellar Cortex Connections 5
Marion Wassef, Pierre Angaut, Leonor Arsenio-Nunes,
Franck Bourrat, and Constantino Sotelo
2. Zebrins: Molecular Markers of Compartmentation
in the Cerebellum 22
Richard Hawkes, Gino Brochu, Louise Dore, Claude Gravel,
and Nicole Leclerc
3. Cerebellar Granule Cells and the Neurobiology
of Excitatory Amino Acids 56
Robert Balazs, Nicola Hack, and Ole S. Jprgensen
4. Microtubule-Associated Proteins
in Cerebellar Morphogenesis 72
Andrew Matus, Richard P. Tucker, and Christopher Viereck
5. Cerebellar Grafting as a Tool to Analyze New Aspects
of Cerebellar Development and Plasticity 84
Constantino Sotelo and Rosa-Magda Alvarado-Mallart
6. Light and Electron Microscopic Immunocytochemistry
of Putative Neurotransmitter Amino Acids
in the Cerebellum with Some Observations
on the Distribution of Glutamine 116
Ole P. Ottersen and Jon H. Laake
7. The Expanding Role of the Basilar Pontine Nuclei as a Source
of Cerebellar Afferents 135
G.A. Mihailoff, R.J. Kosinski, S.A. Azizi, H S. Lee,
and E.G. Border

IX
x Contents

Part 2. Electrophysiology of Purkinje and Inferior

Olivary Neurons 165
8. The Electrophysiology of the Cerebellar Purkinje Cell Revisited 167
Rodolfo R. LUnas and Mutsuyuki Sugimori
9. Voltage- and Transmitter-Gated Channels in Purkinje Cells
from Organotypic Cerebellar Cultures 182
Isabel Llano, Beat H. Giihwiler, and Alain Marty
10. Electroneuronal Hybridization: A Novel Approach to
Investigate Rhythmogenesis in the Inferior Olivary Nucleus 201
Yosef Yarom

Part 3. Electrophysiology of Movement 213

11. Cerebellar Control of Saccadic Eye Movements
in the Pigmented Rat 215
Piergiorgio Strata, Leonardo Chelazzi. Filippo Tempia.
Ferdinando Rossi, and Mirella Ghirardi
12. A Possible Connection Between the Mossy
and Climbing Fiber Systems at Precerebellar Level 226
Francisco J. Rubia
13. Eye Movements and the Zonal Structure of the Rabbit Flocculus 255
John I. Simpson, Johannes Van der Steen, and Joep Tan
14. The Dynamic Selection Hypothesis: A Proposed Function
for Cerebellar Sagittal Zones 267
James R. Bloedel and Thomas M. Kelly
15. Cerebellar Output: Multiple Maps and Modes of Control
in Movement Coordination 283
W. Thomas Thach, S.A. Kane, J.w. Mink, and H.P. Goodkin
16. The Role of the Cerebellum in Voluntary and Reflexive
Movements: History and Current Status 301
John P. Welsh and John A. Harvey

Index 335
Contributors

Rosa-Magda Alvarado-Mallart. Ph.D. INSERM U-106, Hopital de la

Salpetriere, 75013 Paris, France.
Pierre Angaut. Ph.D. INSERM U-106, Hopital de la Salpetriere, 75013
Paris, France.
Leonor Arsenio-Nunes, M.D. INSERM U-106 Hopital de la Salpetriere,
75013 Paris, France.
S.A. Azizi, Ph.D. Department of Cell Biology, University of Texas
Southwestern Medical School, Dallas, TX 75235, USA.
Robert Balazs. M.D., Ph.D. Netherlands Institute for Brain Research,
1105 AZ Amsterdam, The Netherlands.
James R. Bloedel. M.D .• Ph.D. Division of Neurobiology, Barrow
Neurological Institute, Phoenix, AZ 85013, USA.
B.G. Border, Ph.D. Department of Neurology, University of Texas
Southwestern Medical School, Dallas, TX 75235, USA.
Franck Bourrat. Ph.D. INSERM U-106, Hopital de la Salpetriere, 75013
Paris, France.
Gino Brochu, M.Sc. Laboratory of Neurobiology, Faculty of Medicine,
Laval University, Ste-Foy, Quebec GAl 1Z4, Canada.
Leonardo Chelazzi. Ph.D. Istituto de Fisiologia Umana, University of
Verona, Strada Le Grazie, 37134 Verona, Italy.
Louise Dore. M.D.. Ph.D. Laboratory of Neurobiology, Faculty of
Medicine, Laval University, Ste-Foy, Quebec GAl 1Z4, Canada.
Beat H. Giihwiler. Ph.D. Brain Research Institute, CH-8029, Zurich,
Switzerland.
Mirella Ghirardi. Ph.D. Dipartimento di Anatomia e Fisiologia Umana,
Universita degli Studi di Torino, C.so Raffaello 30, 10125 Torino, Italy.
H.P. Goodkin. M.D .• Ph.D. Department of Anatomy and Neurobiology,
Washington University Medical School, St. Louis, MO 63110, USA.

xi
Xll Contributors

Claude Gravel, Ph.D. Institut de Recherches Cliniques de Montreal,

Montreal, Quebec, P.Q., Canada.
Nicola Hack, Ph.D. Netherlands Institute for Brain Research, 1105 AZ
Amsterdam, The Netherlands.
John A. Harvey, Ph.D. Division of Behavioral Neurobiology, Depart-
ment of Pharmacology, Medical College of Pennsylvania, Philadelphia,
PA 19129, USA.
Richard Hawkes, Ph.D. Department of Anatomy and Neuroscience
Research Group, Faculty of Medicine, University of Calgary, Calgary,
Alberta T2N 4N1, Canada.
Ole S. j¢rgensen, Ph.D. Psychochemistry Institute, Rigshospitalet, DK-
2100 Copenhagen (j), Denmark.
S.A. Kane, Ph.D. Department of Anatomy and Neurobiology, Washington
University Medical School, St. Louis, MO 63110, USA.
Thomas M. Kelly, Ph.D. Department of Physiology, University of
Arizona, Tucson, AZ 85721, USA.
R.J. Kosinski, Ph.D. Department of Cell Biology, University of Texas
Southwestern Medical School, Dallas TX 75235, USA.
Jon H. Laake, M.D. Department of Anatomy, Institute of Basic Medical
Sciences, University of Oslo, N-0317 Oslo 3, Norway.
Nicole Leclerc, Ph.D. Center for Neurologic Disease, Brigham and
Women's Hospital, Boston, MA 02115, USA.
H.S. Lee, Ph.D. Department of Cell Biology, University of Texas South-
western Medical School, Dallas, TX 75235, USA.
Isabel Llano, Ph.D. Laboratoire de Neurobiologie, Ecole Normale
Superieure, 46 Rue d'Ulm, Paris 75005, France.
Rodolfo R. Llinas, Ph.D. Department of Physiology/Biophysics, New
York University Medical Center, New York, NY 10016, USA.
Alain Marty, Ph.D. Laboratoire de Neurobiologie, Ecole Normale
Superieure, 46, Rue d'Ulm, Paris 75005, France.
Andrew Matus, Ph.D. Friedrich Miescher-Institut, 4002 Basel, Switzerland.
G.A. Mihailoff, Ph.D. Department of Anatomy, University of Mississippi
Medical Center, Jackson, MS 39216-4505, USA.
J. W Mink, M.D., Ph.D. Department of Anatomy and Neurobiology,
Washington University Medical School, St. Louis, MO 63110, USA.
Ole P. Ottersen, M.D. Department of Anatomy, Institute of Basic
Medic.al Sciences, University of Oslo, N-0317 Oslo 3, Norway.
Ferdinando Rossi, Ph.D. Dipartimento di Anatomia e Fisiologia Umana,
Universita degli Studi di Torino, C.so Raffaello 30, 10125, Torino, Italy.
Francisco J. Rubia, Ph.D. Departamento de Fisiologia, Facultad de
Medicina, Universidad Complutense, 28040 Madrid, Spain.
 Contributors xiii

John 1. Simpson, Ph.D. Department of Physiology/Biophysics, New

York University Medical Center, New York, NY 10016, USA.
Constantino Sotelo, M.D., Ph.D. INSERM U-I06, Hopital de la Salpetriere,
75013 Paris, France.
Piergiorgio Strata, Prof. Dipartimento di Anatomia e Fisiologia Umana,
Universita degli Studi di Torino, C.so Raffaello 30, 10125, Torino, Italy.
Mutsuyuki Sugimori, Ph.D. Department of Physiology/Biophysics, New
York University Medical Center, New York, NY 10016, USA.
Joep Tan, Ph.D. Department of Anatomy, Faculty of Medicine, Erasmus
University, 3000 DR Rotterdam, The Netherlands.
Filippo Tempia, Ph.D. Dipartimento di Anatomia e Fisiologia Umana,
Universita degli Studi di Torino, C.so Raffaello 30, 10125 Torino, Italy.
W. Thomas Thach, M.D. Department of Anatomy and Neurobiology,
Washington University Medical School, St. Louis, MO 63110, USA.
Richard P. Tucker, Ph.D. Friedrich Miescher-Institut, 4002 Basel,
Switzerland.
Johannes Van der Steen, Ph.D. Department of Physiology, Faculty of
Medicine, Erasmus University, 3000 DR Rotterdam, The Netherlands.
Christopher Viereck, Ph.D. Friedrich Miescher-Institut, 4002 Basel,
Switzerland.
Marion Wassef, Ph.D. INSERM U-106, Hopital de la Salpetriere, 75013
Paris, France.
John P. Welsh, Ph.D. Department of Physiology/Biophysics, New York
University Medical Center, New York, NY 10016, USA.
Yosef Yarom, Ph.D. Institute of Life Sciences, Department of Neuro-
biology, Hebrew University, Jerusalem 91904, Israel.
 Part 1
Organization, Development, and Repair
of the Cerebellar Circuits

Since the pioneerings studies of Cajal, celebrated in this book, the organ-
ization of cerebellar circuitries has attracted many scholars who among
others, have tried to unravel most of the afferent and efferent connections
of this central region. However, despite all these efforts, as Alf Brodal
wrote in his last edition (1981) of his textbook of Neuroanatomy, "We
do not yet properly understand its function and its cooperation with other
parts of the brain." In fact, although the use of axonal tracing methods
during the last fifteen years or so has greatly improved the selectivity of
the anatomical analysis, the polysynaptic nature of most pathways involved
has hampered progress in fully understanding them.
Studies performed in recent years, owing to the convergence of anatom-
ical and physiological approaches, have greatly improved our present
knowledge on the functional organization of afferent and efferent cere-
bellar connections. The more we know of these pathways, the more they
appear to obey highly consistent patterns of organization.
One of the selected topics in this section concerns the organization of
the cerebellar projections originating from potine neurons, the principal
precerebellar region involved in transferring information to the cerebellum
about the current state of the cerebral cortex, particularly the motor cortex
of prime importance in mammals. The way this system is organized, at
least according to our present knowledge, raises the problem of the origin
of the excitatory input to the dentate nucleus. In fact, many anatomists
have failed to encounter collateral axons to the cerebellar nuclei in the
pontocerebellar projection originating from the basilar pontine nuclei
(BNP), suggesting that this system is not organized as the olivocerebellar
one which consistently sends collaterals to the deep nuclei. However, ponto-
cerebellar neurons are located not only at the BNP but also in the nucleus
reticularis tegmenti pontis (NRT), which undoubtedly project to the deep
nuclei, particularly to the dentate nucleus. If all these results are corrobo-
rated, they suggest that only about 20% of the whole pontocerebellar
system will provide excitatory inputs to the dentate nucleus. The cortico-
pontine projection, an input that emerges from almost every portion of
the cerebral mantle, is topographically organized. Only some restricted
regions, mostly the motor and somatosensory cortices, project to the
NRT. Thus, the very elaborated input from the cerebral cortex to the
2 The Cerebellar Circuits

cerebellum would regulate a nucleus without major excitatory drive. A

set of studies in the cortico-ponto-cerebello-thalamo-cortical loop by
Sasaki et al. reveals a very powerful return activation from the cerebellum
back to the cortex following cortical excitation. Thus, despite the anatom-
ical controversy, it seems appropriate to hypothesize that NRT is just
part of the pontine nuclear complex and that this subnucleus, as do the
others, gives collateral axons-organized with precise topography-to
the deep nuclei. It is clear that more anterograde tracing experiments are
needed to confirm or to invalidate this hypothesis.
Immunocytochemical methods to study potative neurotransmitters in
central synapses have been extremely useful in the search of possible candi-
dates for cerebellar synapses, particularly among amino acids with known
excitatory or inhibitory action. This volume reviews results obtained with
quantitative postembedding immunogold method to assess the ultrastruc-
turallocation of all these amino acids and to establish the pattern of their
distribution within the cortical cerebellar circuit. It is also emphasized
that this immunocytochemical approach can be an essential tool to analyze
synaptic release and glial uptake of amino acids.
The recent advances in screening molecular identities in neurons,
through the attainment of extensive monoclonal antibody libraries, have
provided unequivocal proof of the molecular heterogeneity of Purkinje
cells. A number of monoclonal antibodies, most of them oriented against
still not-completely characterized antigens, are expressed according to a
similar parasagittai band pattern, with subsets of Purkinje cells either
possessing or lacking the respective antigens. The cerebellar cortex is,
therefore, subdivided into precise longitudinal zones or compartments
based on their heterogeneous Purkinje cell population. Results reported
in this part point to the fact that the boundaries of the intrinsic cortical
compartments and those of the projectional maps, particularly the
olivocerebellar system, are congruent.
The correlation of these two maps in adult cerebellum indicates a
correspondence of developmental mechanisms. The organization of two
subpopulations of Purkinje cells into mutually exclusive compartments
could either result from postnatal synaptic connections with differential
afferent inputs or from the neuronal phenotype maturing independently
of afferent influences. The problem of the development of the Purkinje
cell heterogeneity is addressed, and a series of arguments are discussed in
favor of the essential role of these neurons in organizing projectional
maps during ontogenesis of the cerebellum. The matching of region-specific
chemical labels between incoming afferent fibers and heterogeneous sets
of Purkinje cells is the most appealing mechanism for the formation of
cerebellar maps.
This first part is also comprised of two other developmental studies on
(1) the role of cytoskeleton (especially microtubules) in the determinism of
neuronal polarity and the acquisition of neuronal shape and (2) the trophic
effect of excitatory amino acids on the survival of granule cells. These
two studies emphasize the point that the cerebellum is one of the most
idoneal paradigms for the analysis of the mechanisms governing the
sequential phases leading to the formation of central neuronal net-
works.
The Cerebellar Circuits 3

The last paper of this part addresses the problem of cerebellar develop-
ment and plasticity through the study of cerebellar repair by neural grafting
in murine heredo-degenerative ataxia. This experimental approach offers
the exceptional possibility of analyzing in vivo cellular interactions
between embryonic and adult neurons, allowing the synaptic integration
of the grafted neurons, leading to the subsequent restoration of the im-
paired cortico cerebellar circuit. These experiments also raise the possibil-
ity that embryonic Purkinje cells can induce in adult neural cells a new
type of plasticity; that of recreating a permissive microenvironment for
the integration of the grafted neurons.
1
Purkinje Cell Heterogeneity:
Its Role in Organizing the Topography
of the Cerebellar Cortex Connections
Marion Wassef, Pierre Angaut, Leonor Arsenio-Nunes,
Frank Bourrat, and Constantino Sotelo
The cerebellar cortex comprises a few cell types
and two main afferent systems arranged in a ste-
reotyped synaptic pattern that is repeated mono-
tonously throughout. The regularity of the
laminated structure of the cerebellar cortex per-
mits the identification of the main cell types, even
on conventionally stained sections, based on their
position and size. Contrasting with its regular
architecture, the cerebellar cortex is subdivided
into a mosaic of small functional zones defined
by the precise pattern of their afferent and efferent
connections. In this chapter, we report and dis-
cuss work from our laboratory aimed at under-
standing how such a precise organization of the
cerebellar projection maps is achieved during
development. Different lines of evidence are pre-
sented in favor of the hypothesis that, during
development, the cerebellar cortex develops an
intrinsic topographic map through its subdivi-
sion into small sets of biochemically different
Purkinje cells (PC). We propose that this PC
heterogeneity is subsequently recognized by
afferent fibers and thus underlies the topography
of the cerebellar cortex connections. Before pre-
senting our results, a brief description of the
early stages of the embryonic development of
the cerebellum in rats and mice is worthwhile.
A unique feature of cerebellar development is
that its neurons are generated by two neuro-
epithelia. Early in development, neurons com-
prising the projection neurons of the cerebellum
arise from the ventricular neuroepithelium.
Later on, a secondary neuroepithelium, the exter-
nal granular layer (EGL), produces the bulk of
cerebellar neurons that are local-circuit neurons.
The deep cerebellar nuclei (DCN), the PCs,
and the Golgi cells are generated by the ventri-
cular neuroepithelium in successive waves ex-
tending over 2 to 3 days for the projection neurons
and somewhat longer for the Golgi cells. The
DCN are produced first, peaking at Ell in the
mouse and E13 in the rat, followed by PC, peak-
ing at E12 in the mouse and E14 in the rat. Golgi
cells are produced between E12 and E15 in the
mouse and between E18 and P2 in the rat (Altman
and Bayer, 1978; Miale and Sidman, 1961).
Beginning from E16 in the rat, the external
granular layer arises from the "germinal tri-
gone," which is located at the periphery of the
cerebellar plate, close to the attachment of the
tela choroidea. The EGL spreads progressively
over the surface of the cerebellar plate, which
is eventually completely covered by E20 (Altman
and Bayer, 1978; see also Feirabend, 1983; and
Hanaway, 1967). The cerebellum is often de-
scribed as a late maturing structure because all
the interneurons of the cerebellar cortex, with the
exception of Golgi cells, are produced postna-
tally in small rodents. The onset of synaptoge-
nesis, as observed by physiological (Crepel, 1971;
Puro and Woodward, 1977a, b; Shimono et al.,
1976) and morphological (Altman, 1972a, b, c;
Larramendi, 1969) criteria, is also mainly
postnatal; it may be dated from P2-P3 in the
rat, although occasional synapses have been
observed in late embryos (West and del Cerro,
1976). To determine whether or not synapto-
genesis is related to the mechanisms underlying
the formation of projection maps, we have studied
the development of the olivocerebellar and
5
6 Marion Wassef et al.

spinocerebellar projections in rat pups, using
anterograde and retrograde axonal tracing
methods. We observed that, despite the imma-
turity of the cerebellum around birth, the afferent
fibers are already well organized and that their
broad distribution is indistinguishable from that
of adults, considering that the available methods
of investigation are rather crude. Therefore,
except perhaps in its finest details, the topogra-
phic organization of the cerebellar cortex afferents
seems to be independent from synapse formation.
birth (Arsenio-Nunes and Sotelo, 1985), indicat-
ing that both olivary and spinal afferent fibers
reach the cerebellum during fetal development.
Contrasting with the ubiquitous distribution of
olivary fibers in the white matter, at birth spinal
afferents are restricted to the territories that they
normally innervate in adult rats: the vermis
and intermediate cortex of the anterior lobe
(Fig. l.1A) and the copula pyramidis. Spinal
fibers begin to penetrate the cortical gray matter

Development of the
Olivo cerebellar Projection
The olivocerebellar fibers are already present,
arrested in the prospective white matter of the
cerebellum at birth (Sotelo et aI., 1984); a few
fibers begin at that time to invade the overlying
gray matter. Although the bulk of the olivary
fibers have not yet contacted their target PCs,
they are already distributed in their proper
terminal territories. As in adult rats, restricted
horseradish peroxidase (HRP) injections in the
cerebellar cortex result in the retrograde labeling
of neurons in restricted zones of the contralateral
inferior olive: the caudal half of the medial
accessory olive (MAO) projects to the vermal
cortex, the dorsal accessory olive (DAO) and
part of the rostral MAO to the intermediate
cortex, the principal olive to the hemispheric
cortex. At P5 a more precise analysis of the
topography of the olivocerebellar projection is
possible. The fibers have moved from the
prospective white matter toward the PC layer.
Although the peak of multiple innervation of
PCs by climbing fibers is reached at P5 (Crepel
et aI., 1976, 1981; Mariani and Changeux, 1981),
no later changes in the organizational pattern Figure 1.1. Organization of the spinocerebellar pro-
of the olivocerebellar projection have been jection in an adult normal rat (A) and in the agranular
observed. In P5 rats this pattern is identical to cerebella of an x-irridiated rat (B) and a staggerer
that observed at P15 after the one-to-one mutant mouse (C) injected at the same level of the
relationship of climbing fibers to PCs has been spinal cord (lower thoracic, upper lumbar) with
WGA-HRP. The sections are viewed under polarized
attained.
light. In the anterior lobe of the vermis of the control
rat the spinal fibers display a characteristic columnar
Development of the organization (A). This columnar distribution is pre-
Spinocerebellar Projection served in the irradiated rat (B) but severely altered
in the cerebellum of the staggerer mutant mouse (C)
Like olivary fibers, the spinal afferents are where the fibers are evenly distributed throughout the
present in the white matter of the cerebellum at vermal cortex. Bar = 200 flm in A and C, 100 flm in B.
1. Purkinje Cell Heterogeneity
by P3 in the anterior lobe. The mediolateral
variations in the density of the spinal fibers
result in a "protocolumnar organization"
(Arsenio-Nunes and Sotelo, 1985). At P5, the
fibers, as in adult rats, are organized in columns
but some labeled fibers still remain dispersed
between the columns. From P7 on, the adult
topography of the spinocerebellar projection is
achieved. Although some granule cells are present
in the internal granular layer before P5, their
number is very low. The observations about the
early organization of the spinocerebellar projec-
tion support two kinds of interpretations: either
the small number of early produced granule cells
is sufficient to organize the spinocerebellar
projection or this organization does not depend
on granule cells. This question has been addres-
sed by studying the organization of the spino-
cerebellar projection in different models of
agranular cerebella.
Pattern of the Spinocerebellar
Projection in Agranular Cerebella
The organization of the spinocerebellar projec-
tion was analyzed by anterograde axonal wheat
germ agglutinin HRP (WGA-HRP) tracing in
three types of adult agranular cerebellar cortices
induced either experimentally by postnatal x-ray
irradiation or occurring spontaneously in weaver
and staggerer mutant mice (Arsenio-Nunes et aI.,
1988). In x-irradiated rats (Fig. l.lB), as in
weaver mice, the granule cells are directly affected
and die early in development (Sotelo and
Changeux, 1974b), before migration and dif-
ferentiation. Nevertheless, the spinocerebellar
projection in the anterior vermis displays its
normal columnar pattern. Conversely, in stag-
gerer mutants, the spinocerebellar organization
is severely modified (Fig. 1.1 C), although granule
cell death occurs later, after migration and
differentiation, probably because of a PC de-
fect that prevents synaptogenesis and the subse-
quent synaptic stabilization of granule cells (Sotelo
and Changeux, 1974a). These data indicate that
specific synaptogenesis and even the presence of
granule cells are not essential for the establish-
ment of the normal spinocerebellar topography.
On the other hand, since PCs are the primary
target ofthe mutation in staggerer mice, in which
7
the spinocerebellar projection is disorganized, it
can be suggested that they are involved in the
topographical organization of this projection.
The preceding observations indicate that the
olivocerebellar projection is already well organ-
ized at birth. On the other hand, although the
spinocerebellar fibers have not yet adopted their
columnar pattern of projection at birth, it seems
that the cues necessary for their organization are
produced before birth. At least it is difficult to
interfere postnatally with the establishment of
the characteristic columnar pattern of this pro-
jection even by deleting the target neurons. This
prompted us to examine earlier events, seeking
for some early organizing mechanism that could
explain our postnatal observations about the
development of the olivocerebellar and spino-
cerebellar projections. Of particular interest was
the development of the projection neurons of the
cerebellum (i.e., the deep cerebellar neurons and
the PCs). We report below some observations
about the embryonic and early postnatal devel-
opment of PCs. We discuss how the early gene-
ration of subsets of biochemically different PCs
and their subsequent pattern of migration and
interdigitation in the cortex built up an intrinsic
topography of the cerebellar cortex. This marking
of the cortex through PC heterogeneity could
give cues to afferent fibers and allow them to reach
their proper site of termination.
Transient PC Heterogeneity
During Perinatal Development
Several proteins and glycoproteins have been
described that may be considered as PC markers
in the adult rat and mouse cerebellum, in the sense
that antibodies against these antigens stain all
the PCs and that, in the cerebellum, PCs are the
only immunoreactive cell type. By extension, the
term "PC marker" in the following description
refers indifferently to the antigens or their anti-
bodies. Among others, antibodies against cyclic
guanosine monophosphate (GMP)-dependent
protein kinase (cGK); (De Camilli et aI., 1984;
Lohmann et aI., 1981), vitamin D-dependent
calcium-binding protein (calbindin or CaBP)
(Jande et aI., 1981; Legrand et aI., 1983), Purkinje
cell-specific glycoprotein (PSG); (Langley et aI.,
1982; Reeber et aI., 1981), and PEP 19 (Ziai et aI.,
8 Marion Wassef et al.

1986) have been used to label specifically adult

PCs in the cerebellum. Apparently, apart from
being highly concentrated in PCs, these PC
markers have nothing in common. The onset of
PC immunoreactivity varies for each marker
(Wassef et aI., 1984, 1985, unpublished observa-
tions). The first immunoreactive PCs are observed
at E16 with anti-CaBP, E17 with anti-cGK, and
around E20 with anti-PSG and anti-PEP 19. In
addition to being the earliest PC marker, anti-
CaBP is also the one that gives the most detailed
picture of embryonic PCs. At E16, CaBP immu- cGK
noreactive PCs are still located against the
receding ventricular neuroepithelium. Most of
them are radially oriented and begin to extend
B f •

their axons dorsally. The CaBP immunoreactive

CaBP

Figure 1.3. Successive coronal sections through the

cerebellum of an E20 rat embryo stained for cGK (A)
B and CaBP (B). The fact that CaBP+, cGK- (tri-
angles) and CaBP-, cGK+ (stars) clusters of PC
coexist indicates that the immunoegativity of PCs for
a given antibody does not simply reflect a global delay
in their stage of development. Bar = 400 11m.

axons gather in the "intermediate fibrous layer"

Figure 1.2. Successive coronal sections from caudal
(top) to rostral (bottom) through the cerebellum of
an E18 rat embryo stained for CaBP showing the
succession of CaBP immunopositive (white stars)
and immunonegative (black stars) clusters of PCs.
Bar = 500 11m.
(Altman and Bayer, 1978). Beginning from day
17, PCs migrate dorsally to the cortical region
and it becomes apparent that all the PCs are not
CaBP immunoreactive (Fig. 1.2). For each of the
PC markers tested (CaBP, cGK, PSG, and PEP
19), PCs are arranged in alternating positive and
negative clusters (Figs. 1.2, 1.3, 1.4) that are sym-
metrically arranged in relation to the midline
(Fig. 1.4). With each antibody, the pattern is re-
producible at a given age and changes progres-
sively with the spreading and migration of PCs as
development proceeds. Interestingly, the pattern
of positive and negative clusters is different with
each antibody (Fig. 1.3), although some bound-
I. Purkinje Cell Heterogeneity
c PE P 19
Figure 1.4. Coronal sections at similar levels in the
rostral cerebellum of 1-day-old rat pups labeled for
cGK (A), CaBP (B), and PEP 19 (C). After birth, the
PCs are still organized in clusters characterized by a
high or low level of expression of each of these
markers. Note that the pattern of immunoreactivity
is different for each antibody. Bar = 1 mm.
aries are shared by several markers. This obser-
vation indicates that the PCs in the negative
clusters are not simply delayed in their overall
stage of maturation. In addition, it shows that
subsets of PCs adopt different timetables for the
expression of some of their characteristic proteins,
thus following different developmental pathways.
The organization of the cerebellar cortex in
alternating immunopositive and negative clus-
ters of PCs is still observed after birth (Fig. 1.4),
ruling out the possible existence of another cell
population mingled with PCs in embryos. It
must be stressed that the immunopositive and
immunonegative PC clusters are characterized
by marked differences in staining intensities. The
9
PCs ofthe negative clusters, however, do contain
lower amounts of the antigens, which increase
progressively as development proceeds until
around P5, when adjacent clusters become
indistinguishable.
The basic PC compartment (Wassef et a!.,
1985) is not the immunopositive or immuno-
negative cluster of PCs by itself but the inter-
section of such clusters where, by definition, all
the PCs are identical. The basic PC compartment
is characterized by a combination of proteins or
glycoproteins (markers) expressed at high or low
levels (A +, B -, C -, D +, ...); this combination
constitutes the "label" of the PC compartment.
Because the position in the cerebellar cortex of
each basic PC compartment is characteristic and
reproducible at a given age, the PC heterogeneity
makes up a reproducible map of the cerebellar
cortex. Even with a small number of markers,
the combinatory mechanism described above
can give a reasonably high number of different
labels. Because the basic PC compartments are
not directly amenable to analysis, it is difficult
to appreciate their sizes and shapes.
We have hypothesized that PC heterogeneity
serves as a labeling of the cortical structure and
is involved in the establishment of the cerebellar
projection maps. Obviously, as the antibodies
used to study PC development label all adult
PCs, the PC heterogeneity that we observe during
development is transient. Beginning from P5, the
differences in PC immunoreactivity with these
markers have faded. Nevertheless, the adult cere-
bellar cortex is still subdivided into sagittal strips
and patches of biochemically different PCs. This
heterogeneity of adult PCs can be evidenced
either by a differential sensitivity to mutations
affecting the postnatal survival of PCs or by
differences in reactivity using various histochemi-
calor immunocytochemical techniques.
Cell Heterogeneity in the
Adult Cerebellum
Cerebellar Mutations Disclose
Adult PC Heterogeneity in the Mouse
Several mutations have been described in the
mouse that affect the postnatal survival of PCs.
We have studied (Wassef et a!., 1987) three such
10 Marion Wassef et al.

mutations: nervous (NR), Purkinje cell degener-

ation (PCD), and tambaleante (TBL). In all three
mutations pathological changes are not observed
before the end of the second postnatal week, at
a time when PCs may be considered as almost
mature.
In NR (Landis, 1973; Sidman and Green,
1970), the first pathological change, an anomaly
in the size, shape, and location of PC mitochondria,
is observed throughout the cerebellar cortex at
P15. Apparently all the PCs are affected. However,
whereas most PCs undergo degenerative changes
and die before the end of the second month, a
small population (about 10%) recover and survive
throughout the whole life of the animal, although
some sporadic PC death may be observed
(Sotelo and Triller, 1979).
In PCD (Mullen et aI., 1976), PC loss is almost
complete: it begins during the third week of life
and then proceeds rapidly. By 4 months, fewer
than 120 PCs remain in the cerebellum (Wassef
et aI., 1986).
TBL is a recessive mutation, still not fully
characterized, which appeared spontaneously in
the CNRS breeding center of Orleans-La Source;
TBL seems to affect primarily PC survival
(Sotelo and Guenet, unpublished observations).
Figure 1.5. Coronal section through the vermal re-
The number of PCs is not reduced before about
gion of the cerebellum of a 4-month-old nervous
2 months, but between 2 and 3 months there is mutant mouse stained for CaBP by immunofluore-
a massive, but subtotal, degeneration of PCs. scence. Surviving PCs are grouped into longitudinal
Each of these mutations reveals a sub po pula- bands that are symmetrically arranged in relation to
tion of more resistant PCs. The pattern of these the midline. Bar = 400 flm.
"resistant" PCs was studied using CaBP immuno-
cytochemistry. In NR (Figs. 1.5, 1.6) and I-month-
old PCD (Fig. 1.6), surviving PCs are arranged
in clusters separated by areas of unstained
molecular layer. This topographic arrangement results as showing that each mutation unmasks
of PC survival results in a checkerboard pattern the underlying PC heterogeneity in a way that
of CaBP immunostaining in the cerebellar cortex depends both on the mutation and on the
of these mutants. In TBL, patches differing in physiological characteristics of PCs, the most
the density of surviving PCs are also observed. important implication probably being that the
At a given age, the pattern of surviving PCs is physiological heterogeneity of PCs is related to
reproducible in individuals bearing the same their position in the cerebellar cortex (topogra-
mutation. In all three mutations, the clusters of phically determined). The populations of "resis-
surviving PCs are oriented sagittally and they tant" PCs are partially overlapping, as was the
are symmetrically arranged in relation to the case for the immunoreactive clusters of PCs ob-
midline. Interestingly, the patterns of surviving served during development, an observation that
PCs differ for each mutation, indicating that we supports the same interpretation: the clusters of
are not simply dealing with a subpopulation of resistant PCs that are disclosed by the mutations
more robust PCs. We have interpreted these are not the basic PC compartments.
1. Purkinje Cell Heterogeneity 11

Level of PC Heterogeneity:
Single Cell or Cell Population?

We propose that the cerebellar cortex is built up

through the assembly of small heterogeneous
subsets of PCs. Each subset has a single signature,
its "label," constituted by a characteristic com-
bination of properties (the high or low expression
of a given set of markers) that individually are
shared with other PC subsets. The labeling of
PCs through a combination of different markers
offers interesting possibilities: a) a few different
markers may give rise, by combination, to a
variety of labels, or b) a hierarchy of the labels
is possible, allowing the progressive subdivision
of broad territories into smaller specialized zones.
The size of the smallest subdivisions, that we
termed "basic PC compartments," is, however,
unknown: do they comprise a single PC or a
subset of PCs?
The idea that each PC in the monolayer is
unique is supported by considerations about the
specificity of its developmental history and con-
nections. Apparently, it also has a strong anthro-
pomorphic background. The assumption that
individual target cells may be identified by the
afferent growth cones through a distinct bio-
chemical label is commonly referred to as Sperry's Figure 1.6. Coronal sections through the cerebellum
hypothesis (Sperry, 1944, 1963). Sperry specu- of a 3-month-old nervous (A: caudal; B rostral) and
lated that the conservation of the topographic I-month-old PC degeneration (C) mutant mice stain-
order from the retina onto the tectal surface ed for CaBP. Surviving PCs are arranged in sagittal
bands whose pattern is reproducible in individuals of
during the development or regeneration of optic
the same age bearing the same mutation. Compare
fibers in amphibians and fishes could be explained the pattern in the vermis in B with Fig. 1.5 taken ap-
by the existence of "an orderly cytochemical proximately at the same level from another nervous
mapping in terms of two or more gradients of mouse. The population of resistant PCs, however,
embryonic differentiation with their axes roughly is different in each mutation (compare A and C).
perpendicular. These separate gradients would Bar= Imm.
stamp each cell with its appropriate latitude and
longitude expressed in a kind of chemical code"
(Sperry, 1963). The graded property of the in marking out the cerebellar cortex and sub-
chemical code in Sperry's hypothesis is impor- dividing it into physiologically relevant zones.
tant to bring about a continuous ordering of the The labeling would allow afferent fibers to invade
projection. The usefulness of a discontinuous set their proper zone of termination. In this context,
of labels generated at the single cell level by a the basic compartments may be supposed to be
combination of unrelated markers is question- of variable sizes throughout the cortex but gen-
able: it seems difficult to imagine a matching erally not reduced to a single Pc.
mechanism for such a labeling. The role of the In theory, the organization of the cortex in
PC heterogeneity that we observe during develop- patches of biochemically different PCs could, in
ment and in mutants is, in our view, different. addition, be involved in the local ordering of
We propose that this heterogeneity is involved afferents. The common boundary between two
12
patches of different PCs may be used in the
generation of gradients of positional informa-
tion, as has been proposed for the organization
of subfields during development (Meinhardt,
1982). As discussed by Meinhardt (1982, 1983),
we need only to assume that two patches of PCs
cooperate for the production of a label, present
at their surface or in the extracellular matrix.
This required cooperation makes the synthesis
of the label only possible at the boundary. By
diffusion and decay, a gradient of positional
information is formed, with the highest concen-
tration of label at the limit between two PC
patches. If the afferent fibers are supposed to
have some mechanism allowing them to maintain
the neighboring relationship of their neurons of
origin, this further refinement of the cortical
labeling could allow the establishment of a pre-
cise topographic order of the cerebellar cortex
afferent fibers even before the beginning of synap-
togenesis (Von der Malsburg and Wills haw,
1977).
Histochemical and Immunohistochemical
Evidence of PC Heterogeneity in the
Adult Cerebellar Cortex
Several histochemical and immunohistochemical
techniques provide histological evidence of the
subdivision of the cerebellar cortex, and parti-
cularly its molecular layer, into sagittal bands
differing in their reactivity. Thus, variations
in the staining intensity for the enzymes 5'-
nucleotidase in the mouse cerebellum (Marani
and Voogd, 1973; Scott, 1964) and acetylcholin-
esterase in the cat and monkey cerebellum (Brown
and Graybiel, 1983; Hess and Voogd, 1986;
Marani and Voogd, 1977) have been described".
Binding experiments using radioactive ligands
for muscarinic acetylcholine receptors (Neustadt
et ai., 1988) also disclose a sagittal banding pat-
tern of the molecular layer. In these experiments
the cellular localization of the enzymes or the
receptors is not always clear.
By contrast, several antibodies against peptides
or enzymes (antimotilin: Chan-Palay et ai., 1981;
Nilaver et ai., 1982; see also Lange et ai., 1986;
anti-glutamate decarboxylase: Chan-Palay et ai.,
1981; anti-cystein sulfinic acid decarboxylase:
Chan-Palay et ai., 1982) as well as monoclonal
Marion Wasser et al.
antibodies directed against unidentified epitopes
(Bl: Ingram et ai., 1985; mabQl13 or zebrin 1:
Hawkes et ai., 1985; Hawkes and Leclerc, 1987)
react with molecules that are clearly localized to
a subset of PCs. The populations of immuno-
reactive PCs observed with each antibody are
characteristically organized in sagittal strips that
are symmetrically arranged in relation to the
midline and whose pattern is reproducible in a
given species.
Relationship Between Heterogeneity
and the Pattern of Connections
of the Cerebellar Cortex
In order to demonstrate that the PC heterogeneity
is directly involved in the segregation of afferent
fibers and thus underlies the topography of the
cerebellar projection maps, much new infor-
mation about early cerebellar development is
needed. For example, the timing of arrival of
early afferents into the cerebellar plate and their
position in relation to the PC clusters are still
unknown. Even the comparison between the
pattern of afferents and the pattern of the PC
immunoreactive clusters observed transiently
after birth is technically uneasy. It seemed
worthwhile to investigate first the relationship
between the adult pattern of PC heterogeneity,
as revealed by the zebrin 1 innunoreactivity, and
the pattern of projection of one of the main
cerebellar afferent systems. The climbing fiber
projection seemed the most appropriate for this
study since a) the termination of climbing fibers
on the dendritic tree of PCs makes the compari-
son between the pattern of projection and the
pattern of PC immunoreactivity easy, b) the
location of the terminal axonic field of the
climbing fiber in the molecular layer allows its
unambiguous identification even if the tracer
injection is not restricted to the inferior olive
and involves neighboring structures projecting
in the granular layer as mossy fibers, and c) the
olivocerebellar projection could share the same
principles of organization, in parasagittal strips,
as the zebrin 1 PC bands.
Hawkes and collaborators (Gravel et ai.,
1987) have compared the pattern of the climbing
fiber projection with that of PC immunoreacti-
vity using the monoclonal antibody Ql13, which
1. Purkinje Cell Heterogeneity
labels a subset of PCs (zebrin 1 +) in the cerebel-
lum of adult rats. Two days after an injection of
WGA-HRP in the inferior olive of adult rats,
alternate cerebellar sections were treated either
with the tetramethyl benzidine (TMB) method
to reveal the anterogradely labeled climbing
fibers, or for zebrin 1 immunocytochemistry.
These authors found that "the mabQ113 + /
mabQ113 - boundaries and the bands of climb-
ing fibers seen by using anterograde tracer typi-
cally coincide and that it is likely that the climb-
ing fiber projection to the cerebellar cortex and
the distribution of the two PC phenotypes share
a common compartmental organization." These
results were of particular interest; however, several
considerations weaken the conclusions of Gravel
et al. (1987). First, the comparison between the
two patterns relies on the observation of succes-
sive sections, one of which has shrunk in the
acidic solutions used to reveal the WGA-HRP
anterograde tracer. In this condition it is difficult
to appreciate whether there is a coincidence
between the bands oflabeled climbing fibers and
the zebrin 1 + or zebrin 1 - PC bands rather
than a simple similarity between two longitudi-
nally oriented patterns of labeling in the molec-
ular layer. Second, the authors observed that
the bands of labeled climbing fibers coincide
generally with either a zebrin 1 + or a zebrin
1- band of PCs. This is surprising because it is
unlikely that a random injection in the inferior
olive could repeatedly result in the complete
labeling of determined cortical bands, although
some zebrin 1 cortical bands may be completely
filled with labeled climbing fibers locally.
Because of the great interest of these results for
us, in collaboration with Hawkes we repeated
the experiments of Gravel et aI. (1987) using tri-
tiated leucine as anterograde axonal tracer from
the inferior olive. This allowed us to reveal both
the bands of anterogradely labeled climbing fibers
and those of immunoreactive PCs on the same
section. In addition, we used this material for a
statistical analysis aiming at disclosing if the
observed coincidences between the discontinuities
in the olivocerebellar projections and the shifts
in PC immunoreactivity could occur by chance.
Before reporting our results, a brief account of
the rationale of such an experiment is worth-
while.
13
Random Tracer Injections in the
Inferior 0 live Disclose Intrinsic
Subdivisions of the
Olivocerebellar Projection
Some properties of the olivo cerebellar projec-
tion are well established: a) the projection covers
the whole cerebellar cortex (Campbell and
Armstrong, 1983a, b) climbing fibers from a cir-
cumscribed region of the inferior olive terminate
in the cerebellar cortex in narrow sagittal zones
that extend through several lobules (Armstrong
et aI., 1974; Groenewegen and Voogd, 1977;
Groenewegen et al., 1979). In general, a small
random injection in the inferior olive results in
the labeling of several sagittal strips of climbing
fibers (Figs. 1.7 through 1.11), c) in some parts
of the inferior olive, in particular the rostral
DAO, a single olivary axon may supply branches
to two or more nonadjacent sagittal zones in the
anterior lobe (Armstrong et al., 1973; Ekerot and
Larson, 1982), d) in both the anteroposterior and
the mediolateral axes a band of cerebellar cortex
a, b, c, d, may originate from a band of olivary
neurons whose organization is d, b, a, c (Azizi and
Woodward, 1987). Figure 1.7B is a schematic
drawing that considers some of the above-
mentioned proper ties of the olivo cerebellar
projection. Each of the olivary zones (a, b, c, d)
terminates respectively in, and fills completely,
the a', b', c', and d' sagittal strips of the cerebellar
cortex. It is assumed that the local topography
of the projection of a onto a', b onto b', and so
forth are conserved. There is a rostrocaudal shift
in the projection maps between a and b, and an
inversion between c and d. As shown in Figure
1.7B, a random tracer injection represented by
stippling in the inferior olive results in the label-
ing of sagittal bands of climbing fibers (stippled
in the cerebellar cortex). In several areas (indi-
cated by big dots) the bands of labeled climbing
fibers abut against the boundaries of the a', b', c',
and d' sagittal zones. These boundaries are char-
acteristic of the topography of the olivocerebel-
lar projection and predate the actual injection.
Thus, a random injection can uncover intrinsic
boundaries of the olivocerebellar projection
(local discontinuities in this projection). Another
illustration of this property is given in Fig. 1.8,
which is slightly modified from Groenewegen
14 Marion Wassef et al.

result in the outlining of intrinsic boundaries of

the olivo cerebellar projection. This notion is
fundamental for the analysis and interpretation
of the experiments of Gravel et ai. (1987) and
our own, which tested the possibility that the
CEREBELLAR PC heterogeneity evidenced by zebrin 1 differen-
CORTEX
tial immunolabeling coincides with intrinsic sub-
B divisions of the olivocerebellar projection.
Nine Wistar rats were used for this study. One
or two injections of 50 to 100 nl of tritiated
INFERIOR OLIVE

a b c d

Figure 1.7. Schematic representation of some of the

features of the topography of the olivocerebellar
projection. A tracer injection in the inferior olive
results in the anterograde labeling of sagittal climbing
fiber bands extending over several lobules in the
cerebellar cortex (A). A map of the olivocerebellar
projection is represented in B. The projections of the
contiguous territories of the inferior a, b, c, dare DAO MAO PO
organized in sagittal bands in the cerebellar cortex,
respectively a', b', c', d'. Due to local discontinuities in
the topography of the olivocerebellar projection (a
rostrocaudal shift between the projections of a and b
and an inversion of the terminal fields of c and d), a
tracer injection (stippling in the inferior olive) results
in the labelling of bands of climbing fibers (stippling
in the cerebellar cortex) that are partially delimited
(big dots) by the intrinsic boundaries (between a', b',c',
and d') of the olivocerebellar projection. Figure 1.8. The topologic properties of the olivo-
cerebellar projection are illustrated in a real injec-
tion case (stippled on a flattened representation of
the inferior olive, bottom) in the inferior olive of a
et al. (1979). The pattern of the climbing fiber
cat (cat H8854; redrawn with permission from
projection labeled in the cerebellar cortex of a
Groenewegen et aI., 1979). It is clear that the limits
cat (case H8854 of Groenewegen et aI., 1979) of the climbing fiber bands labeled by this injection
after an injection in the inferior olive was may frequently coincide with the boundaries of the
reported on the map. It is clear in this case that A... D sagittal zones. (Adapted with permission from
the rostrocaudal as well as mediolateral dis- Groenewegen et aI., 1979. Copyright by Wiley-Liss, a
continuities in the olivocerebellar projection Division of John Wiley and Sons, Inc.)
I. Purkinje Cell Heterogeneity 15

Climbing Fibers Labeling

The injections were large, and resulted in two
kinds of labeling in the cerebellar cortex. First,
in some cortical areas the climbing fiber labeling
was extensive, and the ARG labeling intensity
varied along sagittal cortical bands. Second, in
other regions, the labeling was fractionated and
restricted to several sagittal bands of molecular
layer extending over several folia separated by
unlabeled regions (Figs. 1.9, 1.10, 1.11). The pat-
terns of ARG labeling were closely similar in

Figure 1.9. Coronal sections through the cerebellar

cortex of adult rats injected with 3H amino acids in
the inferior olive. The sections were treated for zebrin
1 immunoreactivity and ARG. The ARG-labeled
climbing fibers are visualized in darkfield (white dots)
in A and C and in brightfield (black dots) in B. A
sagittal climbing fiber band contained in a zebrin
1 area and extending over three folia abuts against a
zebrin 1 PC band (A). Either located in a zerbin 1- (B)
or in a zebrin 1 + (C) PC band, the ARG-Iabeled
climbing fiber bands often end exactly at the shift in
PC immunoreactivity. Bar = 400 fl.m in A, 50 fl.m in B
and C.

leucine (5 to 10 j.LCi, 50 Ci/mM) were placed

unilaterally or bilaterally in the inferior olive
using a ventral approach. Two days later, the
animals were fixed by aldehyde perfusion. Frozen
sections through the cerebellar region were cut
in the coronal or horizontal plane and processed
successively for zebrin 1 labeling by immuno-
cytochemistry and tritiated leucine anterograde
axonal tracing by autoradiography (ARG). One
out of 6 sections was processed for autoradio-
graphy without prior immunolabeling.
Figure 1.10. Coronal section through the caudal
vermis of an adult rat injected with 3H amino acids
in the inferior olive, same treatment as in Fig. 1.9. The
section is viewed under dark field (A), brightfield (B),
and a combination of both (C). Note the high number
of coincidences (arrows) between the limits of the
ARG-labeled bands of climbing fibers and the zebrin
1 PC bands. However, some ARG-Iabeled bands cross
one (squares) or more (triangles) limits of the zebrin
1 PC bands. Bar = 500 fl.m.
16
Figure 1.11. Coronal section through the cerebellum
of another adult rat injection with 3H amino acids in
the inferior olive. The section is taken at the same
level illustrated in Fig. 1.10. The same zebrin 1+ PC
band was involved by both injections but to different
extents. The arrows point to boundaries that are
common to the ARG-labeled climbing fiber bands
and the zebrin 1 PC bands. Bar = 500 J!m.
alternate sections whether or not the sections
were treated for immunocytochemistry before
autoradiography.
Relationship Between the Bands of
Labeled Climbing Fibers and the
Bands of Zebrin 1 + and Zebrin 1 - PCs
in Rats Injected with Tritiated Leucine
in the Inferior Olive
As expected, labeled climbing fibers were located
in zebrin 1+ as well as in zebrin 1- molecular
layer strips. When observed in darkfield, the
ARG labeling was usually brighter in zebrin 1-
areas, where it appeared white, whereas -in the
Marion Wassef et al.
zebrin 1+ regions it acquired a reddish reflection
from the diaminobenzidine (DAB) reaction
product. This difference was less marked on
micrographs. However, in order to eliminate a
possible influence of the DAB precipitate on the
intensity of the ARG labeling, the statistical
analysis reported below was restricted to those
areas where the climbing fiber labeling was frac-
tionated. In addition, we supposed that the in-
trinsic discontinuities of the olivocerebellar
projection were probably involved in the forma-
tion of this fractionated projection (see Figs. 1.7B
and 1.8) and more likely to be evidenced in this
region.
In areas of extensive climbing fiber labeling,
marked variations in climbing fiber labeling
intensities were observed. Confirming the obser-
vation of Gravel et al. (1987), in many instances
these variations coincide with shifts in zebrin 1
immunoreactivity. As mentioned previously, this
region was not extensively studied. Our study
focused on the zones of the cerebellar cortex
where the labeling was fractionated. When the
relationship between the climbing fiber and the
zebrin 1 labeled bands were analyzed, several
patterns were observed as expected:
1. The ARG-Iabeled band crosses one or two
zebrin 1 zones and its boundaries are not related
to the zebrin 1 immunoreactivity shifts (triangles
in Fig. 1.10). In most cases the limits of these
ARG-Iabeled bands are not sharp, but this is not
always the case.
2. The ARG-Iabeled band crosses one or more
zebrin 1 zones and its boundaries may coincide
on one or both sides with a shift in PC immuno-
reactivity (squares in Fig. 1.10).
3. The ARG-Iabeled band may be entirely en-
closed within the limits of a zebrin 1 band sharing
no, one, or two boundaries with it (arrows in
Figs. 1.9, 1.10, 1.11).
Several observations about the correspondence
between the labeled climbing fiber bands and
the zebrin 1 PC bands were made. In many
instances a row of labeled climbing fibers stops
exactly on the last PC of a zebrin 1 band (Figs.
1.9B, C; arrows in Figs. 1.9, 1.10, 1.11). An exact
coincidence between the ARG and zebrin 1 bands
is observed more frequently in some regions,
particularly in the vermis (Figs. 1.9, 1.10, 1.11)
I. Purkinje Cell Heterogeneity 17

and the intermediate cortex. It is unlikely that together with landmarks on the sections were
this observation is due to a peculiarity of our drawn on a transparent paper superimposed on
injection technique, because our observations in the darkfield photographs. The shifts in zebrin
the caudal vermis (Figs. 1.10, 1.11) are similar 1 immunoreactivity were reported on the same
to those of Gravel et al. (1987), who used a dorsaldrawing positioned on the darkfield photographs
approach. Rather, it may be proposed that the and the coincidences observed between both
regions of the inferior olive projecting inside or kinds oflabeling were checked at higher magnifi-
between the ARG-Iabeled bands are relatively cation under the microscope. The lengths of
distant and rarely affected together by the tracer labeled and unlabeled molecular layer were
injection. Figures 1.10 and 1.11 show the same measured on enlargements of these drawings
region of the caudal vermis in two cases of in- using a Hewlett-Packard microcomputer inter-
ferior olive injections resulting respectively in faced with a graphic tablet.
the total and partial filling of the same zebrin 1+ A total length of 137.8 mm of molecular layer
bands. In both cases there is a striking coinci- was screened, containing 77 ARG-Iabeled bands
dence between the border of the ARG-Iabeled and 137 shifts in zebrin 1 immunoreactivity. The
climbing fiber band and the limit of a zebrin 1 length of ARG-Iabeled molecular layer was
PC band. 38.4 mm (28% of total) and the number of ARG
In several instances, in successive sections of band borders was 152 (in two cases the limit of
the same animal, a band of ARG labeling may the band was too ill-defined). The number of
be observed first inside a zebrin 1 band, then PC shifts in immunoreactivity occurring inside
abutting against its limit with both boundaries an ARG-Iabeled band was 17 and the number
remaining coincident in several sections before of exact coincidences was 43.
either crossing the zebrin 1 boundary or becom- We have compared the observed number of
ing located again inside the zebrin 1 band. This shifts occurring in the ARG-Iabeled molecular
pattern is similar to the one depicted in Fig. 1.7.layer to the number expected under the null
hypothesis of independence between the pattern
Are the Coincidences Observed of zebrin 1 immunoreactivity and the ARG
Statistically Significant? labeling. If the ARG labeling and zebrin 1
immunoreactivity were independent, given an
In order to decide whether there is a significant ARG labeling of 28% of the total length of
correlation between the pattern of the zebrin 1 molecular layer, one would expect 38.4 (28% of
labeled bands and the topography of the olivo- 137 shifts) shifts in zebrin 1 immnoreactivity to
cerebellar projection, we measured several para- fall inside the ARG-Iabeled bands. Comparing
meters in regions where the ARG labeling was the observed number (17) to this expected num-
fractionated. These regions were selected as ber using a chi-square statistic leads to a p value
follows. The sections were screened in darkfield of .0001.
at low magnification (x 10 or x 6.3) and areas Assuming the distance from center to center
where the climbing fiber labeling was intense, between two adjacent PCs to be 30 pm (31 ± 2;
sharply delimited, and fractionated were selected. Altman and Winfree, 1977), the number of PCs
A single field was selected on a given section. It aligned within 137.8 mm of molecular layer is
was photographed under darkfield, brightfield, about 4600. The expected number of coincidences
and a combination of both. All the ARG-Iabeled between shifts in PC immunoreactivity (137) and
bands on the micrographs were considered. It limits of ARG-Iabeled bands (152) is 5. The
was assumed that the areas on each photograph comparison of this expected number using a
were large enough - at least 3.6 mm x 2.4 mm- Poisson approximation leads to a p value of less
to reduce a: bias in sampling. Three injected rats than lO-12.
were rejected from the study because the labeling These results invalidate the null hypothesis of
was too extensive and diffuse; the observations independence and indicate that the topography
reported below are derived from the six remain- of the olivocerebellar projection is highly cor-
ing animals. The limits ofthe ARG-Iabeled bands related with the pattern of the zebrin 1 PC bands.
18
Figure 1.12. E13 cerebellar anlage grafted into a
cavity in the cerebral cortex over the choroid plexus
of an adult host and allowed to develop for 2 months.
The section was double stained for zebrin 1 (DAB)
and CaBP (FITC) and viewed under a combination
of transmitted light and FITC optics. The zebrin 1 +
(black) and zebrin 1- (white) PCs are arranged in
clusters, indicating that the differential expression of
the zebrin 1 epitope by PCs is not under the control
of the olivary afferents. Bar = 200 j1m.
In addition, they suggest that contiguous sub-
divisions of the inferior olive often project to
nonadjacent cortical areas and that the topo-
graphy of the olivo cerebellar map is rather
complex.
Is the Pattern of Zebrin 1
Immunoreactivity Under the Direct
Control of Climbing Fibers?
Because the zebrin 1 epitope is not detected before
P6 (Leclerc et aI., 1988), its expression could be
Marion Wassef et al.
under the control of climbing fibers, which would
explain the good correlation observed between
zebrin 1 immunoreactivity and the topography
of the olivo cerebellar projection. To eliminate
this possibility, in collaboration with Alvarado-
Mallart and Hawkes, we have isolated rat cere-
bellar anlage at E13, before the olivary afferents
have reached the cerebellar plate (unpublished
observations), and grafted them in a hole made
by aspiration in the cerebral cortex of adult
hosts. The grafts were analyzed 2 months later
by double labeling for CaBP and zebrin 1 immu-
noreactivity. As reported previously (Alvarado-
Mallart and Sotelo, 1982; Das, 1973), these cere-
bellar transplants develop a quasinormal lami-
nated cerebellar structure. In the cortical region
of the transplant (Fig. 1.12), PCs are arranged
in alternating zebrin 1+ and zerbin 1- patches,
indicating that the organization of PCs in clusters
differing in their zerbin 1 immunoreactivity does
not necessitate the presence of climbing fibers
and is probably not under their control.
Conclusion
We propose that the development of the cere-
bellum involves an early mechanism of self-
organization of its cortical topography. A map
of the cerebellar cortex is progressively built up
by the migration and spreading of several cate-
gories of biochemically different PCs. Although
the proper establishment of the reproducible
adult pattern of sagittal zonation of PC subsets
certainly depends on cellular interactions during
development, the generation of biochemically
different categories of PCs is apparently an
intrinsic program of the neuroepithelium of the
cerebellum.
Several observations support the hypothesis
that the zonation of the cerebellar cortex defined
by PC heterogeneity is involved in the organiza-
tion of the cortical connections. The PC hetero-
geneity appears earlier than the ingrowth of
the cerebellar afferents into the cerebellar plate
during embryonic development; it could provide
a complex marking of the cerebellar cortex
through a combinatory mechanism. The study
of the spinocerebellar projection in mutant mice
reveals that their characteristic columnar pattern
of projection is less sensitive to an early deletion
1. Purkinje Cell Heterogeneity
of theit: target (granule cells) than to a genetic
PC defect, which suggests a role for PCs in the
patterning of this projection. In the adult rat
cerebellum, there is a close correlation between
the shifts in zebrin 1 immunoreactivity and the
local discontinuities in the olivocerebellar pro-
jection.
Several points, however, merit further investi-
gation: how is PC heterogeneity generated? Is
PC heterogeneity reflected on their surface? What
are the timing and pattern of ingrowth of the
afIerents into the cerebellar plate? How are the
large clusters of PCs observed during early
development subdivided reproducibly, giving
rise eventually to the adult monolayer and its
characteristic zonation? These questions are
currently under investigation.
Acknowledgments. We wish to thank C. Hill for
the statistical analysis of our measures, R.M.
Alvarado-Mallart for teaching us to the graft
technique, R. Rouse for critical reading of
the manuscript, P. Greengard, R. Hawkes,
J. Morgan, M. Thomasset, and J.-P. Zanetta for
the gift of antibodies, B. Cholley for technical
assistance, and D. Le Cren for photographic
work.
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2
Zebrins: Molecular Markers
of Compartmentation in the Cerebellum
Richard Hawkes, Gino Brochu, Louise Dore, Claude Gravel, and Nicole Leclerc
Ordered projections in the brain are established
in several stages. Initially, the formation of an
afferent pathway depends on white matter inter-
actions, such as contact guidance along genetically
determined pathways and selective axon fasci-
culation, to guide neurites to the correct target
fields. Subsequently, target cell recognition by
afferent growth cones and competition between
growth cones for targets (and between targets for
inputs) serve to eliminate superfluous or incorrect
projections and may refine the topography. Many
regions, including the neocortex, the superior col-
liculus, the striatum, and the dorsal column nuclei,
are functionally organized in the form of patches
or stripes that correspond to the discrete segrega-
tion of the afferent or efferent axons. The same
appears to be true in the cerebellum. Studies using
the retrograde-anterograde axonal transport of
tracers, electro physiological recording, somato-
topic mapping, and molecular mapping have all
revealed a parasagittal bandlike topographical
organization of the cerebellar cortex and its afferent
and efferent connections. We are using pattern
formation in the cerebellar cortex as a model to
explore the rules that give rise to topographically
ordered projections. In this chapter we concen-
trate on molecular markers of cerebellar com part-
mentation in the adult, and consider in particular:
1. how many independent topographical maps
art! present in the cerebellum and in what ways
. caliber afferent fibers. For example, in the ferret
are the different maps interrelated?
2. how are the maps constructed and correlated
during development?
The afferent and efferent connections of the
22
mammalian cerebellar cortex are organized into
parasagittal zones. A parasagittal zonation was
shown first for the Purkinje cell efferent projection
to the cerebellar nuclei and the lateral vestibular
nuclei (e.g., Chambers and Sprague, 1955a, b;
Courville and Diakiw, 1976; Haines et aI., 1982;
Jansen and Brodal, 1940, 1942; Voogd and Bigare,
1980). Subsequently, a similar afferent parcellation
was shown to be present in some mossy fiber
(MF) pathways (Chan-Palay et aI., 1977; Scheibel,
1977; Voogd, 1969; Yaginuma and Matsushita,
1986) and in the climbing fiber (CF) projection
from the inferior olives (Armstrong et aI., 1974,
1982; Beyedet aI., 1982; Brodal, 1976; Brodal et aI.,
1975; Courville, 1975; Courville and Faraco-
Cantin, 1978; Eisenman, 1981, 1984; Eisenman
et aI., 1983; Groenewegen and Voogd, 1977;
Groenewegen et aI., 1979; Oscarsson, 1969, 1980;
Oscarsson and Sj6lund, 1977; Van Gilder and
O'Leary, 1970; Voogd, 1964, 1967, 1969). The
sagittal compartmentation of the afferent and
efferent connections of the cerebellum have a
histological equivalent in the myeloarchitecture
of the white matter. By using the Haggqvist me-
thod to stain myelin, Voogd (1969) was able to
identify numerous longitudinal axon compart-
ments, based largely on differences in the diam-
eters of the fibers. In particular, the large caliber-
Purkinje cell axons are fasciculated together to
form independent clusters, interposed by smaller
anterior lobe, five abrupt changes in the axon
diameters were identified in each hemicerebellum,
thereby defining six parallel compartments: A,
adjacent to the midline, and the B, Cl, C2, C3,
2. Zebrins
and D compartments disposed laterally. An equi-
valent compartmentation was identified in the
same way in the hedgehog, the cat, the tree shrew
(Tupaia), the slow lori (Nycticebus), and the
monkey (M acaca).
These data are clearly central to the overall
problem of cerebellar architecture, but as they
have been reviewed extensively in the recent past
(e.g., Bloedel and Courville, 1981), they will not
be considered further here except to correlate
patterns of afferent termination with the intrinsic
cerebellar compartmentation. Instead, the dis-
cussion will concentrate on molecular markers
of cerebellar compartmentation.
Figure 2.1. Four views of the cerebellar cortex in the
rat stained for AChE. The plane of focus is at the level
of the most superficial synaptic glomeruli and hence
parts of the tissue are always out of focus. A and B
show the patchy distribution of AChE in the vermis,
in lobule IV in A and lobule VIII in B. In both cases
there is a patch of elevated activity that straddles the
midline (P 1+) and two patches to either side (P2 +,
23
Intrinsic Markers of
Cerebellar Compartments
5' - Nucleotidase
The first evidence that the cerebellar cortex was
nonuniform biochemically came from the studies
of Scott (1963, 1964) on the cerebellar distribution
of the enzyme 5' -nucleotidase (S'N) in the mouse.
In the cerebellum, S'N is concentrated primarily
in the molecular layer but it is also found in asso-
ciation with the Purkinje cell perikarya, Golgi
cells, and some axons in the granular layer and
white matter tracts. Scott (1963) found a rostro-
P3 +). The patches encompass both the granular layer
(G L) and the white matter (WM) but not the molecular
layer (ML). C and D show similar patchy staining in
the hemispheres, in C corresponding to P6 + of the crus
II, and in D showing the P5+ patch in crus I. Patches
were identified by reference to the zebrin I distribution
on adjacent sections (not shown). Scale bar = 200 11m.
(Reprinted with permission from Boegman et aI., 1988.)
24 Richard Hawkes et al.

ca.udal gradient in enzyme activity, with strong activity granular layer of the granular layer
staining in the nodulus, intermediate staining in appears patchy in frontal section, and the dis-
the uvula, pyramis, tuber, and declive, and weak tribution of these patches corresponds to the
staining anteriorly in the culmen, centralis, and arrangement of some antigenic compartments
lingula. Microdensitometry revealed a twofold (Boegman et aI., 1988, and below).
difference in intensity from anterior to posterior
lobe. However, in horizontal and frontal sections Glutamic Acid Decarboxylase
the distribution of the enzyme revealed a wholly
Much literature exists to support the notion that
unexpected pattern of longitudinal bands of acti-
gamma aminobutyric acid (GABA) is the princi-
vity. One band was astride the midline of the
pal. neurotransmitter of cerebellar Purkinje cells,
cerebellar cortex and the others were symmetri-
so It was a great surprise when Chan-Palay et al.
cally disposed to either side. The maximum num-
(1981) reported heterogeneity in the distribution
ber of bands was seen in horizontal sections where
of glutamic acid decarboxylase (GAD) immuno-
the cerebellum was at its widest: in addition to the
reactivity in adult rat, mouse, and monkey. The
~idline band, six others were seen radiating, fan-
GAD+ Purkinje cells had a characteristic mor-
lIke, from the caudal surface in each hemicerebel-
phology, being slightly smaller and with smaller
lum. A more detailed analysis of the 5'N distri-
nuclei. Broad parasagittal zones could be dis-
bution in the adult mouse is presented in Marani
~erned, interposed by unstained compartments;
(1982a, b).
III all, only about half the Purkinje cells were
GAD immunoreactive.
Acetylcholinesterase
Another histochemical demonstration of cere- Moti/in
bellar compartmentation has been made with
In the same article that first described GAD hetero-
acetylcholinesterase (AChE) staining of young
gen~i~y, ~ nonuniform distribution ofthe peptide
and adult cats (Brown and Graybiel, 1983; Marani,
motIlIn III Purkinje cells was reported (Chan-
1982a; Marani and Voogd, 1977; Raman-Moliner,
Palay et aI., 1981). Immunoreactive Purkinje cells
1972). For example, Marani and Voogd (1977)
were most frequent in the flocculus, paraflocculus,
have described a pattern of five positive and six
and the lateral hemispheres. By contrast, stained
interposed negative bands in the molecular layer
profiles were rare in the vermis, appearing either
of young cats. No staining was seen in the white
as scattered individuals or as contributors to three
matter, the Purkinje cell somata, or the granule
very fine para sagittal bands in lobules I to IV. In
cells. No structural basis for the staining was
lobules V and VII additional microzones were
identified. Preliminary evidence suggests that a
identified. However, these data are at variance
similar AChE segregation can be demonstrated
with those of Lange et aI. (1986), who could not
in monkeys (Hess and Voogd, 1986; Ingram et aI.,
stain the cerebellum with seven other antimotilin
1985). The differential staining in the molecular
antisera, and obtained only a rather uniform stain-
layer diminished with age and was not seen beyond
ing with yet another antiserum. It may be that
4 months postnatal. Similarly, in the granular
the region.al heterogeneity in the reported staining
layer and the white matter of the adult rat cere-
pattern ~Ith ~ntimotilin is due to a cross-reacting
?ellar cortex there is a patchy AChE staining that
contamlllant III the polyclonal antiserum, or that
IS associated primarily with the synaptic glomeruli
there are motilinlike molecules in the cerebellum
(Fig. 2.1) (Boegman et aI., 1988). There are obvious
that are recognized selectively by different sera,
differences from region to region within the gra-
and expressed differentially by different cell types
nular layer, associated with the numerical density
(see also Beinfeld and Korchak, 1985).
of strongly immunoreactive glomeruli. Bands are
clear both in the anterior (Fig. 2.1A) and posterior
Cysteine Sulfinic Acid Decarboxylase
lobe vermis (Fig. 2.1 B). Similar prominent patches
of strongly stained glomeruli are seen in crus II Immunocytochemical studies of the distribution
(Fig. 2.1C) and in crus I (Fig. 2.1 D). The AChE ofthe enzyme cysteine sulfinic acid decarboxylase
2. Zebrins
(CSADCase)-an enzyme in the taurine synthesis
pathway-revealed that the antigenicity was
confined to a subset of Purkinje cells. Further-
more, autoradiography with eH]-taurine revealed
that many Purkinje cells also accumulate taurine
(Chan- Palay et aI., 1982). It is not clear that these
two subsets are identical. The distribution of the
CSADCase immunoreactive cells has been ex-
plored in both mice and rabbits (Chan-Palay
et aI., 1982). Overall, the distributions were similar
between species. Most imunoreactive profiles were
seen in lobules I and X of the vermis, and in the
paraflocculus and flocculus. In all cases, micro-
bands of immunoreactive neurons were disposed
symmetrically about the midline and interposed
by nonreactive compartments. In the anterior
lobe, there are seven reactive bands, one at the
midline and three pairs to either side; in the poster-
ior lobe, the bands are less distinct, and the pre-
sence of bands in the hemispheres is difficult to
distinguish except in lobules VI and VII, where
at least two are present. Immunocytochemistry
with antibodies to taurine itself has complicated
the issue. Several groups have demonstrated tau-
rinelike immunoreactivity in Purkinje cells, but
in these cases all Purkinje cells were immuno-
reactive (Campistron et aI., 1986; Madsen et aI.,
1985; Tomida and Kimura, 1987). More recently,
Magnussen et ai. (1988) demonstrated bands of
taurine-immunoreactive Purkinje cells under light
fixation conditions that, with greater tissue cross-
linking, tended to disappear. These bands are also
CSADCase-immunoreactive. The physical basis
of the fixation sensitivity remains unclear.
Zebrin I
In a specific attempt to identify molecules whose
distributions reflect or encode cerebellar topo-
graphy, an extensive monoclonal antibody library
was constructed against rat cerebellar polypeptide
antigens, and was screened by using immuno-
cytochemistry for epitope distributions that are
nonuniform across the cerebellum. One such anti-
body is anti-zebrin I (mabQl13). Anti-zebrin I
recognizes an epitope, zebrin I, that appears as a
single polypeptide band on Western blots of rat
cerebellar cortex, with an apparent molecular
weight of 120 kDa (Hawkes et aI., 1985). Because
of the striped appearance of the immunopero-
25
xidase-stained cerebellum, the antigen was named
"zebrin I." Its function is unknown. In sections
of cerebellum immunohistochemically stained
with anti-zebrin I, the immunoreactivity is con-
fined exclusively to the Purkinje cells, where it is
distributed throughout the somata, the dendrites,
and the dendritic spines. The Purkinje cell axons,
axon collaterals, and terminals in the cerebellar
nuclei are also immunoreactive (Gravel et aI.,
1986; Hawkes and Leclerc, 1986,1989). The other
cell types in the cerebellum are immunonegative,
although elsewhere in the central nervous system
there is a weak glial immunoreactivity (Plioplys
and Hawkes, 1986, 1988). Electron microscopy
shows that the immunoperoxidase reaction pro-
duct is intracellular and cytoplasmic. In regions
of the Purkinje cell that are rich in microtubules,
such as the primary dendrites, the reaction
product appears to be microtubule-associated;
however, regions of the cytoplasm devoid of
micro tubules are equally stained so this associ-
ation is probably adventitious. The interesting
feature of zebrin I is that it is expressed only in a
subset of Purkinje cells. In the cerebellum as a
whole, about one third of the Purkinje cells are
zebrin 1+. The proportion of zebrin I + to zebrin
1- Purkinje cells varies from lobule to lobule. For
example, in the anterior lobe vermis, only 25% of
the Purkinje cells are zebrin I + whereas c~udal to
the primary fissure 40% are zebrin 1+, and in the
most posterior regions more than 90% of the
profiles in anyone section may be zebrin I + and
there is a tendency for the zebrin I - bands to
disappear completely.
The immunoreactive Purkinje cells are clustered
together to form a family of parasagittal bands
running roughly rostrocaudally throughout the
cerebellar cortex. Zebrin I + bands alternate with
similar bands of zebrin I - cells. The alternation
of immunoreactive and unreactive bands is found
in both the vermis and in the hemispheres. The
expression of the zebrin I epitope seems not to be
all-or-nothing and, in general, bands tend to be-
come weaker and less well defined as they pass
into the anterior lobe. The more lateral bands
tend to be less strongly stained than the more
medial. Subsequently, four other zebrins have
been identified (zebrin II-V).
The parasagittal zonation revealed by zebrin I
is symmetrical about the midline and is repro-
26
du,cible from individual to individual both in the
number and general arrangement of the bands
and in much of the finer detail (Fig. 2.2). The
bands ofzebrin 1+ cells have been called P+, and
a e
d
Figure 2.2. Illustration of the reproducibility of the
zebrin I staining pattern in the rat. Horizontal sections,
each 50 11m thick, were taken at level interaural4.5 mm
(according to the atlas of Paxinos and Watson, 1982)
from eight individuals and immunoperoxidase stained
with anti-zebrin I. Bands PI + through P7 + are identi-
fied in panel c. The pattern of immunoreactivity is
similar in all eight individuals. Three sources of inter-
Richard Hawkes et al.
zebrin I - bands called P -. In each hemicerebel-
lum, rows of Purkinje cells are linked from lobule
to lobule to create a midline band (PI +) and six
lateral p+ bands (P2+ to P7 +) in each hemicere-
individual differences can be seen: the variable location
of P4 + in lobules VIII and IX of the posterior vermis;
the variations in P5 +- P6 + in crus II (e.g., arrowhead
in panel b); the presence of satellite bands. Scale bar =
2.0mm. (Reprinted with permission from Hawkes and
Leclerc, 1987. Copyright 1987, Wiley-Liss, a Division
of John Wiley & Sons, Inc.)
2. Zebrins 27

Vllb

VIII
CRUS Iia
IXb
CRUS lib
IXa

Figure 2.3. a: Whole mount of the rat cerebellum of
an adult rat immunohistochemically stained with anti-
zebrin I as seen from the posterior. The bands are
clearly visible in both the vermis and the hemispheres.
b: The distribution of immunopositive elements in the
whole mount has been mapped. The parasagittal band
organization is clear in the vermis but in the hemisphere
the distribution is also consistent with a checkerboard
of antigenic patches. (Reprinted with permission from
Hawkes and Leclerc, 1987. Copyright 1987 by Wiley-
Liss, a Division of John Wiley & Sons, Inc.)
28
be.llum. The bands of zebrin I - cells have been
mimed after the immediately medial P + band.
The pattern of bands can also be seen in whole-
mount preparations of the cerebellum that have
been permeabilized with cycles of freezing and
thawing and then immunostained with anti-
zebrin I (Fig. 2.3).
The seven major p+ bands that we have identi-
fied in each hemisphere are as follows:
1. PI + extends the entire length of the vermis
from lobules I to X and sits adjacent to the
midline (Hawkes and Leclerc, 1987). That is to
say, PI + is split by the cerebellar midline as
revealed by the olivocerebellar projection
(Gravel et aI., 1987) and no P- band separates
the PI + bands in left and right hemisphere.
The PI + band is the narrowest of the P + family
and is often represented, in a given section, by
only one or two zebrin 1+ Purkinje cells.
2. P2 + is the widest vermal band and extends
throughout all lobules. The band broadens
gradually as it passes from anterior to post-
erior in the vermis.
3. P3 + probably also runs throughout the vermis
but the immunoreactivity becomes noticeably
weaker in the anterior lobe and it is often diffi-
cult to follow P3 + into lobules II and I.
4. P4 + runs along the border between the ver-
mis and the hemispheres and can be followed
throughout the cortex in favorable cases. As
for P3 +, it tends to fade away in the more
anterior lobules. Typically, P4 + is split in crus
IIa and crus lIb into a major median band
(P4a +) and a minor lateral sub-band (P4b +).
This bifurcation cannot be followed farther
anteriorly so either P4 + contains a small island
of zebrin I - cells (P4a -) or, in accordance with
the tendency of bands to fade toward the ante-
rior, the P4b + band extends farther but cannot
be identified. Because P4 + follows the vermian
margin in lobules IX and X, the P4 - band
stops caudally in the copula pyramidis.
The negative bands in the vermis become
very narrow in lobule VI, and separate com-
partments are often difficult to resolve.
S. PS + is confined to the hemispheres where it
is found in all lobules. Inspection suggests an
ambiguity as to the behavior of PS + in the
posterior cortex. Following our classification,
Richard Hawkes et al.
PS + stops at the border oflobule VIII and the
uvula. However, an alternative is to consider
that P4 + and PS + in fact fuse and continue
together in lobules IX and X. Like P4 +, PS +
also shows a subdivision within crus II. In this
case, the minor band is the median PSa + and
the major is PSb + more laterally (also iso-
lating a local PSa -). In more ventral sections,
the division of PS + can be followed into the
paramedian lobule. As with P4b +, PSa + does
not appear to continue into the anterior lobe
of the hemisphere.
6. P6 + runs throughout the hemisphere and is
unbranched.
7. P7+ also runs throughout the hemisphere and
is unbranched. There is a tendency for the
immunoreactivity to weaken in the more lateral
bands so that P7 + is often difficult to trace
with confidence.
Most Purkinje cells in the flocculus and para-
flocculus are zebrin 1+. Differences in staining
intensity may reflect some compartmentation but
their three-dimensional organization has not been
reconstructed. Likewise, in some animals there is
a suggestion of an even more lateral P8 + band
skirting the periphery of the hemisphere but the
weakness of the immunoreactivity in the most
lateral part of the cerebellar cortex has made this
impossible to verify.
The above classification of Purkinje cells into
parasagittal bands is natural in the vermis, but
in the hemispheres the matter is less evident, and
it may be that the distribution of the zebrin I
epitope in the hemispheres is better described as
a series of patches. For example, considering the
PS+ -P6+ region, it may be that PS+ and P6+
stop at the border of crus II and the paramedian
lobule, or rather continue as a zebrin I - territory,
and that, in terms of natural architecture, the
anterior extension of P6 + from the copula pyra-
midis and the paramedian lobule into crus II
might actually be PS -. The resolution of this
question must await studies that correlate the
distribution ofthe zebrin I epitope with the hodo-
logy of the cerebellar cortex.
A frequent source of interindividual differences
are so-called satellite bands. These are defined as
narrow, positively immunoreactive bands of cells,
often only one cell wide, lying adjacent to one of
2. Zebrins
the major bands. In contrast to bands such as
P4b + and P5a +, satellite bands are not constant
from individual to individual nor are they neces-
sarily bilaterally symmetrical. They do, however,
extend substantially in the dorsoventral plane
and thus are not simply occasional zebrin 1+
Purkinje cells isolated within zebrin I - territory.
Analogous zebrin 1- satellite bands may also
exist but would be much harder to detect.
Zebrin I in the squirrel monkey cerebellum is
confined to the Purkinje cells as in rodents (Leclerc
et aI., I 990a). Not all Purkinje cells are immuno-
reactive, and both in the vermis and the hemi-
spheres stretches of zebrin 1+ Purkinje cells
alternate with zebrin I - zones. Clusters of anti-
genic Purkinje cells are arranged in parasagittal
bands running throughout the vermis with a nar-
row band straddling the midline (PI +) and two
others running laterally to either side (P2 + and
P3 +). The P + bands are interposed by similar P-
bands (Fig. 2.4). This numbering scheme follows
Figure 2.4. A frontal section through the cerebellar
cortex ofthe squirrel monkey, immunoperoxidase stain-
ed for zebrin I. Clear zebrin I + and zebrin 1- parasa-
gittal compartments are seen in the vermi5. Although
29
that used for the rat cerebellum (Hawkes and
Leclerc, 1987). Both the number and the position
of the individual bands is highly reproducible
from individual to individual. The situation in the
hemispheres is more complex, due probably to
the elaborate lobulation. However, both zebrin
I + and zebrin 1- Purkinje cells are present, and
preliminary studies indicate that there are four
lateral bands of P + cells as in the rat. Both zebrin
I + and zebrin 1- Purkinje cells are present in the
human cerebellum, but clear parasagittal bands
could not be identified (Plioplys et aI., 1985).
Zebrin II
A second zebrin was identified by a monoclonal
antibody raised against the cerebellum of a weak-
1y electric fish (Apteronotus). The antigen (zebrin
II) is a 36-kDa polypeptide of unknown function.
It is confined exclusively to a subset of the Purkinje
cells, and has an identical distribution in the rat
both zebrin I + and zebrin I - patches are also obvious
in the hemispheres, their precise organization is less
clear.
30
Figure 2.5. A frontal section through the cerebellar
cortex of a young adult rat (P30), immunoperoxidase
stained for zebrin II. The distribution of zebrin 11+
cerebellum to zebrin I (Fig. 2.5) (Brochu et aI.,
1990). It differs from zebrin I in its species distri-
bution. Whereas zebrin I immunoreactivity is
confined to some mammals, zebrin II is expressed
in numerous species, including fish, birds, and
mammals. For example, in Apteronotus the corpus
and valvula of the cerebellum are zebrin 11+,
whereas the lobus caudalis is zebrin II -. Similarly,
zebrin II reveals parasagittal bands in all mam-
mals so far tested: a representative frontal section
from the adult opossum is illustrated in Figure 2.6.
We have at least three other monoclonal anti-
bodies that recognize subsets of Purkinje cells
(zebrins III- V), but these remain to be character-
ized.
Bl
A monoclonal antibody to an undefined epitope
present in rat embryonic forebrain membranes
(antigen Bl) (Ingram et aI., 1985) has revealed a
Richard Hawkes et al.
Purkinje cells is identical to that revealed by usin!:,
anti-zebrin I.
novel heterogeneity among Purkinje cells in the
primate cerebellum. There is a broad pattern of
parasagittal bands of Purkinje cells either B 1 + or
B 1 -. The epitope differs from the zebrins in being
expressed also by various other classes of neurons,
including neurons in the cerebellar nuclei, the
motor cortex, and the spinal cord. A nonuniform
distribution has also been found in the rat.
Synaptophysin
Classically, the cerebellar granular layer is con-
sidered to have no architectonic or chemical
boundaries, and most markers reveal no signs of
compartmentation. However, this appears to be
an oversimplification. For example, detailed
quantitative studies of Golgi cells in the rat have
identified significant differences in cell density
from region to region, with most found in the
posterior lobe vermis, and fewest in the anterior
lobe hemisphere (Lange, 1982). Furthermore, our
2. Zebrins
Figure 2.6. A frontal section through the cerebellum
of an adult opossum (M onodelphis), immunoperoxidase
stained with anti-zebrin II. As in other species tested,
studies with a monoclonal antibody to the synap-
tic vesicle antigen synaptophysin reveal clear
differences between lobules in the extent of the
infraganglionic plexus, which is prominent in
some lobules and almost absent in others (un-
published data). Synaptophysin immunoreactivity
also reveals parasagittal compartments.
By using an antisynaptophysin antibody
(mabQ 155), a pattern of parasagittal bands was
demonstrated in the mouse cerebellar cortex that
encompasses both the granular and the molecular
layers (Fig. 2.7)(Hawkes et aI., 1985; Leclerc et aI.,
1990a). Unlike zebrin I, the synaptophysin bands
are not as sharply delineated as those of zebrin
I, and reflect quantitative levels of staining
intensity rather than all-or-none differences.
Synaptophysin is a polypeptide associated with
synaptic vesicles, and the mabQ155 epitope has
been demonstrated in all prominent classes of
cerebellar and cortical synapse (Leclerc et aI.,
31
only the Purkinje cells are immunoreactive, and zeb-
rin II + cells are clustered together to form a family of
parasagittal compartments.
1990a; Rice and Hawkes, in preparation). Wheth-
er regional differences in immunoreactivity are
due to the level of antigen per vesicle, number of
vesicles per synapse, or synaptic density remains
uncertain.
Cytochrome Oxidase
A banded distribution of cytochrome oxidase
(CO) activity is present in the monkey cerebellum
(Hess and Voogd, 1986; Leclerc et a!., 1990b).
Alternating compartments of high and low CO
activity are detected in the white matter and gran-
ular layer of the vermis, but not the hemispheres.
These compartments correspond to those identi-
fied by using AChE histochemistry. A similar
situation pertains to the rat. Both the molecular
and the granular layers of the rat cerebellar cortex
contain CO activity (Leclerc et a!., 1990b). In the
hemispheres, the activity is distributed uniformly,
32
Figure 2.7. Adult rat cerebellum immunoperoxidase
stained with monoclonal antisynaptophysin. A: Light
micrograph of a 50 lim thick sagittal section through
the cerebellar cortex. Dense punctate deposits corres-
ponding to the distribution of synapses are seen in the
molecular layer, surrounding the Purkinje cell bodies,
and in the granular layer. B: Electron micrograph of a
region of the molecular layer showing labeling of sy-
naptic vesicles in the presynaptic terminus of a parallel
fiber. No reaction product is seen in the postsynaptic
Purkinje cell dendritic spine. C: Horizontal section
through the cerebellum of an adult mouse showing a
series of bands in the vermis of the cerebellar cortex.
Banding is seen in both granular and molecular layers.
Scale bars = 20 ~m (A), 250 ~m (B), and 100 pm (C).
(Reprinted with permission from Hawkes et aI., 1985.)
but in the vermis differences in staining intensity
reveal alternating patches oflow and high activity
that include both granular and molecular layers.
In the molecular layer, the staining is associated
predominantly with the Purkinje cells. The den-
dritic arbors display different levels of reactivity
to yield alternating patches. In the granular layer,
the low and high activity patches align with those
Richard Hawkes et al.
in the molecular layer. The high CO patches are
joined rostrocaudally to form three parasagittal
bands in each hemivermis.
Are All Molecular
Maps Congruent?
It is crucial to establish exactly how many maps
we are dealing with in the cerebellum. Published
evidence is contentious, with several studies claim-
ing that compartment markers incompletely
overlap whereas others describe complete con-
gruence.
In the rat, we have compared the correlation
between zebrin I bands and other band markers:
1. Zebrin I bands of Purkinje cells are completely
congruent with those revealed by staining for
zebrin II. Preliminary studies suggest that this
is also the case for zebrins III through V.
2. Zebrin 1+ Purkinje cells have been compared
directly with the patches of AChE activity seen
in the granular layer. There is good general
agreement that the AChE + patches underlie
the zebrin I + compartments but the degree of
precision of the match is unclear (Boegman
et aI., 1988). In frontal sections of the cerebellar
vermis, AChE histochemistry reveals a patchy
distribution of activity in the granular layer
and the white matter tracts that corresponds
in position to the distribution of zebrin 1+/
zebrin 1- Purkinje cell compartments. The
fact that AChE patches are seen only in the
vermis indicates that although they correlate
with zebrin I staining, they are not an obligate
correlate. In the hemispheres, where zebrin I
patches are prominent, for example in crus II,
the AChE activity in the granular layer is dis-
tributed uniformly. The staining pattern in
adult rats is clearly different from that reported
in young cats (Marani and Voogd, 1977;
Ramon-Moliner, 1972) and cannot have the
same structural basis. Nevertheless, studies in
both cats and rats concur that bands and
patches are only seen in the vermis.
3. Zebrin 1+ Purkinje cell compartments in the
mouse are congruent with the 5'N bands in the
molecular layer, both in the vermis and in the
hemispheres (Eisenman and Hawkes, 1990).
2. Zebrins 33

4. Zebliin 1+ compartments in the vermis have More recently, a monoclonal antibody, P-

been compared directly in rat and monkey PA TH, has been developed that recognizes an
with the compartments revealed by using CO acetylated epitope expressed by a variety of cere-
histochemistry. In the rat, comparison of the bellar gangliosides. When P-PATH is used to
CO distribution with adjacent sections stained stain the rodent cerebellum, a complex pattern of
for zebrin I reveals that the CO-rich bands compartmentation is revealed in the molecular
correspond precisely to the zebrin I - compart- layer which is closely complementary to that re-
ments and the CO-weak bands to zebrin 1+ vealed by using antizebrins (Leclerc et aI., 1990c).
compartments. Thus, in the anterior lobe of P-PATH is thus the first consistent positive
the vermis, we can distinguish three narrow marker for the "zebrin-negative" Purkinje cell
immunoreactive bands, PI + on the midline compartments. However, the demarcation is not
and two more lateral bands (P2 +, P3 +) on quite that clear-cut, as it appears that some com-
each side. The CO compartmentation shares partments, most notably P3 +, are double-labeled
the same structural boundaries as the antigenic both by antizebrin and P-PATH. The details of
zonation, with high CO in Pl- and PT and the relationship are still to be determined.
low CO in PI +, P2+, and P3+. The same These data taken together suggest that there
spatial correlation holds for the posterior ver-are at least two different sorts of compartment
mis and again, high CO levels are associated markers in the adult. On the one hand, there are
with the zebrin I - compartments. The finding antigens such as zebrin 1/11, that are expressed by
that high CO activity is associated with the rata subset of one cell type, in this case Purkinje cells.
zebrin I - compartments is the first report of On the other hand, others are expressed in a more
a positive marker for this set. complex fashion. Thus, the CSADCase bands
The primate CO compartmentation shares involve not only the Purkinje cells, but also basket
the same structural boundaries as the zebrin I and stellate cells in the same compartment
zonation. However, in the squirrel monkey the (Chan-Palay et aI., 1982). In this case, it seems
CO-rich bands correspond to the zebrin 1·+ that something within the compartment environ-
compartments and conversely the CO-weak ment is conducive towards marker expression by
bands underlie the zebrin I - compartments. many cell types. The same situation also applies
Evidently, care is needed when making com- to differences in CO levels between compartments.
parisons about homologous compartments A plausible model to explain this behavior is that
between species, based on their molecular compartmentation is defined fundamentally by
pherrotype. the type of Purkinje cell (i.e., zebrin I + or zebrin
5. Hess and Voogd (1986) show that AChE and I - phenotypes): the differential expression by
CO bands correspond in the vermis of the other cell types in the compartment being secon-
macaque. dary, and dependent on local interactions. Thus,
for example, the presence of bands of 5'N activity
However, in the case of other markers, the in the mouse molecular layer seems to depend on
situation is not so straightforward. Thus, com- the Purkinje cells remaining intact (Hess and
parisons of the heterogeneity revealed by staining Hess, 1986). Alternatively, it is possible that the
for GAD, motilin, and CSADCase suggest that expression of some molecular markers is related
although they overlap, the different markers are to the dynamic metabolic state of the cell rather
not codistributed. Similarly, according to Ingram than to its permanent phenotype, a situation that
et ai. (1985), the B 1 antigen is not codistributed could presage a distinction between topographi-
with the other compartmentation marker they cally defined cerebellar compartments and func-
tested (AChE), and its distribution does not tional units.
resemble those described for motilin, GAD, or
CSADCase. However, our direct comparison of
Bland zebrin I immunoreactivities in adjacent
sections of squirrel monkey vermis showed a
complete correspondence (unpublished data).
34
The Relationship Between
Compartmentation and Connectivity
Climbing Fibers: Complete Correspondence
It is imperative to determine how the intrinsic
chemistry of the cerebellar cortex relates to the
innervation patterns. Anterograde tracer injec-
tions into the inferior olivary complex generate
discrete bands of climbing fiber (CF) terminals in
the cerebellum. This occurs because adjacent
regions in the inferior olives do not necessarily
project to adjacent regions of the cerebellar
cortex. (For example, projections to the uvula
and pyramis of rat show that contiguous para-
sagittal bands in the cerebellar cortex can receive
afferents from noncontiguous groups of olivary
cells (Eisenman, 1981, 1984; Eisenman and
Goracchi, 1983).
Direct comparison shows that CF compart-
ments and the expression of zebrin I seem to
Richard Hawkes et al.
respect a common compartmentation: both are
arranged into parasagittally organized bands or
patches in the cerebellar cortex; the two patterns
are reproducible; there is a high level of concor-
dance between the cortical localization of transi-
tion zones delineating discrete olivocerebellar
projection territories and the location of transition
zones between zebrin I + and zebrin I - cortical
regions (Fig. 2.8) (Gravel et aI., 1987). It remains
to be determined whether every cluster of zebrin
I + and zebrin 1- Purkinje cells receives afferent
input from an individual, specific subgroup of
olivary cells.
Mossy Fibers: Incomplete Correspondence
We have concentrated on low thoracic- high lum-
bar (LTHL) spinocerebellar projections to com-
pare mossy fiber (MF) terminal fields to zebrin I
compartments in the rat (Gravel and Hawkes,
Figure 2.8. A comparison of
bands of climbing fibers (A)
with bands of zebrin I in
lobule VIII of the posterior
vermis (8) in the rat as seen
in serial horizontal sections
100 f.lm apart. Labeled CF
terminate both on zebrin 1+
and zebrin 1- Purkinje cells
(A). Contralateral to the
injection site (left), the P2 +
zebrin I + band receives a
discrete WGA-HRP labeled
CF input due to tracer
diffusion across the midline:
the olivocerebellar
projection in the adult rat
is purely ipsilateral. The
arrows in A and 8 point to
the same blood vessel.
Rostral is toward the top
and the midline is indicated
by a dotted line. Scale
bar = 200 f.lm. (Adapted with
permission from Gravel
et aI., 1987. Copyright 1987
by Wiley-Liss, a Division of
John Wiley & Sons, Inc.)
2. Zebrins 35

1990). Several studies in mammals have shown
that the target area for spinocerebellar terminals
comprises the anterior lobe (mainly lobules II - V)
and lobule VIII of the posterior lobe and that
within the receiving areas the rosettes are clus-
tered into discrete terminal fields that run sagit-
tally in the granular layer ofthe vermal and inter-
mediate cortex (Arsenio-Nunes and Sotelo, 1985;
Hazlett et ai., 1971; Robertson et ai., 1983; Voogd,
1964, 1969; Yaginuma and Matsushita, 1986). In
the vermis of the anterior lobe, the number of
sagittal L THL terminal fields per hemicerebellum
goes from three to four as they extend caudally
from lobule II to lobules III, IV, and V. In both
the anterior and posterior lobes, the medial group
ofterminals appears divided in two at the midline,
particularly in lobules V and VIII. This may be
because this group consists of two contiguous
longitudinal bands directly adjacent to the mid-
line. The observation that the central area of
termination for LTHL terminals is split by the
midline would also be consistent with the my-
eloarchitectonic compartmentation in the cere-
bellum of the mouse (Marani, 1982a), rat (Voogd
et ai., 1985), and other mammals (Voogd, 1969),
and also with the organization of corticonu-
clear projections (Armstrong and Schild, 1978;
Goodman et ai., 1963), olivocerebellar projections

P3 P2 P1 P2 P3
2.0
~ c:J

Figure 2.9. A comparison of Pur-

kinje cell compartments (darkly
shaded rectangles) and mossy fiber 1.5
terminal fields (pale rectangles), after

1
a WGA-HRP anterograde tracer
injection into the LTHL of the spi-
nal cord. Alternate sections were
1.0
processed for anterograde transport
ofWGA-HRP and zebrin I immu-
noreactivity. The vermis of lobule
d
II has been schematically unfolded,
flattened, and viewed from dorsal. 0.5
The region at the tip of the lobule
(around coordinate 0) was not re-
constructed. The dorsal (d) and
ventral (v) aspects are indicated, and o
the scales are in mm, either from
the rostral extent of the cerebellum
(vertical) or from the midline (hori-
zontal). The PI + - P3 + Purkinje cell
0.5
compartments are clear both dor-
sally and ventrally. LTHL terminal

1
fields are differently disposed in the
two faces. For example, dorsally,
there is a parasagittal terminal field 1.0
running beneath P2 +, whereas
ventrally it is displaced medially into
Pl-. Similarly, the LTHL compart-
ment beneath P3 + dorsally, is locat- 1.5 v
ed beneath P2 - ventrally. This has
the effect of subdividing the P + jP-
parasagittal compartments into
short patches with changes in MF 2.0
innervation patterns occurring at L-
____- L______ ~L- ___ --J
----.J
the tip of the lobule. -1.0 0.5 o 0.5 1.0
36 Richard Hawkes et al.

(Campbell and Armstrong, 1983a, b), and zebrin how the ostensibly uniform cerebellar cortex
I territories (Gravel et aI., 1987). In the posterior might encode complex somatotopic maps. The
lobe the two more medial MF clusters on each further subdivision of zebrin compartments by
side are clearly separated by a midline gap, as is MF terminal fields increases the resolution of the
the case in the dorsal face of sublobule VIlla in topography to a point such that anatomical com-
the cat (Yaginuma and Matsushita, 1987). partment widths become compatible with the
A crucial question is, do the MF terminal fields widths of physiological compartments in the
respect the Purkinje cell (PC) compartments? In somatotopic map.
a general sense, since both PC and MF are orga- How longitudinal bands are subdivided to give
nized longitudinally there is inevitably some the patchy mosaic characteristic of the cerebellar
measure of correspondence. However, unlike the somatotopy remains unclear. The obvious means
olivocerebellar projection (OCP), where there is by which longitudinal compartments are sub-
a direct synaptic interaction and precise compart- divided mediolaterally is the natural cerebellar
ment alignment (Gravel et aI., 1987), the mossy foliation. Electrophysiological studies of somato-
fiber-Purkinje cell (MF-PC) interaction is in- sensory projections to the cerebellum have shown
direct (via granule cells), and may be architectu- that each folium and folial complex receives
rally less precise. The problem is compounded inputs from mixtures of peripheral sources and
further by the interlobular and intralobular dif- submodal types, suggesting that each might be a
ferences and by uncertainty as to the trajectories unique functional unit (Joseph et aI., 1978; Kassel
of the granule cell axons. et aI., 1984;Shambeset aI., 1978a, b; Welker et al.,
The limits of the LTHL terminal fields corre- 1987; Welker and Shambes, 1985). For example,
late well with the boundaries of some, but not all, in lobule VIII of Galago, sublobules a, b, and c
zebrin I compartments. For example, in the have receptive fields for the lower neck-upper
anterior lobe the medial LTHL terminal field back region, whereas sublobule d has middle-
occupies the granular layer beneath both PI + back representations (Welker et aI., 1988). The
and the medial third of PI - and so apparently cerebellar lobulation provides a natural means
does not respect the PI +IPI - interface. Likewise, by which afferent inputs can be directed to one
the first lateral pair of LTHL terminal fields sub- or another cortical territory: at the base Qf each
stantially and consistently extend beyond P2 + sulcus, the white matter tracts must bifurcate.
medially into the lateral half of PI - but never Hence, there is a natural transition at the base of
extends across the PI +IP2 - interface laterally, each sulcus by which the granule cells on one side
although in most cases it does abut against either are driven by the MF ventrally and those on the
its lateral or medial edge (e.g., lobule II dorsal) other side by the MF located dorsally. However,
(Fig. 2.9). The organization of LTHL terminal most somatotopic boundaries are not at the base
fields in the anterior lobe results in the gross of the sulci, and for these other mechanisms must
partitioning of the granular layer under PI - into besought.
a central region devoid of labeled terminals, and The MF terminal fields do not only subdivide
medial and lateral receiving areas to either side Purkinje cell compartments sagittally, they also
(except for the ventral part oflobule II where the divide them mediolaterally. An example from
reverse is the case-see below). This is clearly dif- lobule II is shown in Fig. 2.9. Anterograde tracer
ferent from the olivocerebellar projection where has been injected into the lower thoracic-higher
a precise alignment of boundaries was consistently lumbar spinal cord of the rat, and the labeled
found, and zebrin I compartments are never sub- spinocerebellar axons mapped in the cerebellar
divided by afferent terminal fields (Gravel et aI., vermis. In the dorsal surface of lobule II the
1987). LTHL terminal fields are centered more or less
beneath the P + compartments; in contrast, on the
M ediolateral Compartmentation due ventral face they lie in P- compartments, with
to M F Terminal Fields the exception of the midline, where MF and P+
The identification of parasagittal bands of affer- compartments are always congruent. In the
ents and Purkinje cells serves in parr to explain posterior lobe, the LTHL terminal fields in lobule
2. Zebrins
Figure 2.10. Anti-zebrin I labels the caudal portion of
the fastigial nuclei preferentially. Three horizontal sec-
tions (A, B, C) through a fastigial nucleus in the rat
immunoperoxidase stained by using anti-zebrin I to
show the rostrocaudal gradient of immunoreactivity.
The dorsolateral protuberance (dp) is always uniformly
positive. At higher magnifications (D), the individual
37
anti-zebrin 1+ synaptic boutons are seen. The target
neurons themselves are not immunoreactive. Scale
bar = 200 11m (A-C) and 20 11m (D). (Adapted with per-
mission from Hawkes and Leclerc, 1986. Copyright
1986 by Wiley-Liss, a Division of John Wiley & Sons,
Inc.)
38
VIII show the absence of labeled glomeruli
beneath the P2 + compartment on the dorsal sur-
face. As a result, the innervation of the same
compartment can clearly be different between
dorsal and ventral faces of a lobule. Together with
the natural separation that occurs at the depths
of the sulci, the MF distribution allows parasagit-
tal compartments to be subdivided mediolater-
ally into patches with different patterns of afferent
innervation.
In theory, the differences between the dorsal
and ventral aspects of lobule II could be due to
the differential cortical growth. If the ventral
cortex expanded more than the dorsal, and this
expansion did not affect the MF terminal posi-
tions, the result would be as is actually found, the
relative displacement of the MF terminal fields
medially from the p+ to the P- compartments
(i.e., from P2 + to PI - and from P3 + to P2 -).
However, this is probably not the case. In fact,
the spacing of the PC compartments does not
differ between dorsal and ventral, as would be
predicted by the differential expansion hypothe-
sis, but rather it is the MF terminal fields that
come closer together on the ventral face.
Corticonuclear Projection
In addition to the cerebellar afferents, the projec-
tion of the Purkinje cells to the cerebellar nuclei
and lateral vestibular nuclei is organized into
parasagittal compartments (reviewed in Haines
et aI., 1982), and these compartments correspond
to those in the olivocerebellar projection. The
topography ofthe corticonuclear projection with
respect to Purkinje cell compartmentation is
currently under investigation. However, immuno-
cytochemical studies of the fastigial nucleus with
anti-zebrin I reveal the presence of discrete ter-
minal fields for zebrin I + and zebrin 1- Purkinje
cells (Fig.2.10) (Hawkes and Leclerc, 1986).
Because zebrin I is found in the axons and axon
terminals of zebrin 1+ Pu'rkinje cells, individual
synaptic boutons are readily identifiable in the
cerebellar nuclei (Fig. 2.1OD). For example, zeb-
rin I immunocytochemical studies of the projec-
tion to the ipsilateral fastigial (medial) nucleus
have shown that the PI + Purkinje cells terminate
preferentially in the caudal pole and the Pl-
Purkinje cells terminate in the rostral pole (Fig.
2.10) (Hawkes and Leclerc, 1986).
Richard Hawkes et al.
Are There Higher Levels
of Organization than
the Compartment?
Purkinje cell axons have two modes of termina-
tion within the cerebellum. They are the sole
efferent projection from the cerebellar cortex to
the deep nuclei, but they also participate in corti-
cal local circuits by means of recurrent collaterals
that arborize within the cerebellar cortex. There
is reason to think that the mediolateral extent of
Purkinje cell axon collaterals is influenced by the
parasagittal zonation of the cerebellar cortex. In
rodents (Chan-Palay, 1971; O'Leary et aI., 1968;
Palay and Chan-Palay, 1974) and in the cat
(Bishop, 1982) the recurrent axon collaterals of
Purkinje cells spread extensively in the parasagit-
tal plane but have only limited longitudinal spread
within the folium. Bishop (1982) has noted for the
cat that the lateral extent of the collateral plexus
is similar to the dimensions of the sagittal zones
of the cerebellar cortex as defined by the topo-
graphy of the afferent axon terminals. Given that
neighboring Purkinje cells are a frequent synaptic
target for axon collateral terminals (Chan-Palay,
1971), it has been suggested that Purkinje cell
collaterals may define zonal boundaries by func-
tionally linking topographically related Purkinje
cells (Bishop, 1982).
The strict demarcation of vermal zebrin I + 11-
para sagittal zones seen at the level of the somata
and dendritic trees is only partly maintained by
the recurrent axon collaterals (Hawkes and Leclerc,
1989). Collaterals invade into neighboring terri-
tory by up to five Purkinje cell widths in the'
anterior lobe, thereby innervating 20% or so of the
width of the P- bands. In the posterior lobe of
the vermis all the P - bands are occupied (Fig. 2.11).
Hence, there is no simple segregation of collaterals
within their antigenically homologous bands. On
the other hand, the density of stained collateral
profiles does drop dramatically at the P + IP-
boundaries. If we assume that the density of the
zebrin 1+ collaterals behaves in similar fashion,
then the infraganglionic plexus of lobule IV
changes from more than 90% zebrin 1+ beneath
a zebrin 1+ Purkinje cell soma at a p+ IP- bound-
ary to less than 10% zebrin I + at the neighboring
zebrin I - cell.
2. Zebrins 39

Figure 2.11. Two examples of recurrent collaterals In both examples, immunoreactive collaterals extend
(arrowheads) of zebrin 1+ Purkinje cell axon in lobule deep into the zebrin I - territory of P2 - . Scale bar =
IX of the vermis in the rat (the P2 - compartment, with 50/!m. (Reprinted with permission from Hawkes and
P2 + medially to the left and P3 + laterally to the right). Leclerc, 1989.)
40
The functional significance of the recurrent
axon collaterals of Purkinje cells is obscure. It is
evident that the physiological role of such recur-
rent axon collateral in the integrated action of the
cerebellum will depend on the target neuron it
inhibits. The major target for recurrent axon col-
laterals seems to be other Purkinje cells and thus,
their role outside the band of origin might be to
inhibit firing in the neighboring medial compart-
ment. This could serve to sharpen the physiologi-
cal boundaries and limit the spread ofMF- granule
cell activation via the parallel fibers. However,
several other targets have been identified for the
recurrent collaterals, including basket cells, Golgi
cells, and Lugaro cells. With this in mind, it is
clear that the consequences of collateral input will
depend on which targets are implicated. Taken
together with the observation that Golgi cells
receiving Purkinje cell collateral input are much
more frequent in the hemispheres than in the
vermis, this suggests the possibility that collaterals
play different physiological roles in different re-
gions of the cerebellum, perhaps even in different
bands.
The observation that zebrin 1+ collaterals from
P2 + oflobule IV preferentially invade P - terri-
tory medially (P 1 -) over laterally (pr), and thus
differentiate medial from lateral band boundaries,
may reflect a still higher level of cerebellar func-
tional organization than the zebrin I compart-
ment. On the other hand, the explanation of the
polarized collateral distribution may be embryo-
logical rather than functional. During cerebellar
development, Purkinje cells develop earliest in
the vermis, thus creating a mediolateral gradient
of maturity in Purkinje cells ofthe neonate. Thus,
when synapses are formed there could be an excess
of mature targets medially, biasing the mature
collateral distribution. This hypothesis cannot be
tested directly as bands of zebrin 1+ Purkinje cells
are not apparent until 15 to 20 days postnatal
(Leclerc et aI., 1988), at which stage the collateral
distribution resembles that in the adult. The only
previous indication that medial and lateral boun-
daries of parasagittal bands may be different
comes from studies of the S'N distribution in
mice that showed that the sagittal bands have
sharp lateral but blurred medial borders (Marani,
1982a).
Richard Hawkes et al.
The Cerebellar Module
The above considerations suggest that a modular
description ofthe cerebellum may be appropriate
(Fig. 2.12). There are about 280,000 Purkinje cells
in the adult rat cerebellum (Hillman and Chen,
1981). If we take the number of para sagittal com-
partments as 32 (as defined by using zebrin im-
munocytochemistry: this is an underestimate, in
part because the molecular complexity may be
higher, and in part because the MF terminal fields
split antigenic compartments), and the number of
lobules dividing the vermis and hemispheres
mediolaterally as, on average, 10 (lobules are
important because they represent opportunities
for axon tract bifurcation and thereby for the
differential distribution of afferents to different
modules), then we obtain some 320 compart-
ments per cerebellum. Each subcompartment may
be further split by differences in MF terminal
aUerents
11
cerebellar
COl1eo
1
cerebellar
nuclei
1m]
1
euerents
Figure 2.12. The cerebellar module. Left: The rat hemi-
cerebellum is schematically drawn. There are eight
zebrin I + and eight zebrin I - para sagittal compart-
ments. In this model, each compartment is divided
mediolaterally by natural foliation and by shifts in the
MF terminal fields to yield 320 individual modules.
Right: One such module is expanded: it comprises
some 400 individual Purkinje cells that project onto
20 target neurons in the terminal field of the cerebellar
nuclei (and lateral vestibular nuclei).
2. Zebrins
fields from dorsal to ventral faces, to yield some
600-odd discrete modules, each averaging 400 to
500 Purkinje cells. The efferent output from each
module is via the corticonuclear or corticovesti-
bular projections. Both these pathways are highly
convergent. The precise Purkinje cell/nuclear cell
ratio in rat is not known, but taking it to be 26,
as in the cat (Palkovits et aI., 1977), then each
cerebellar cortex module would project to some
20 to 40 cells in the cerebellar nuclei. The degree
of overlap between corticonuclear terminal fields
is unknown.
The diversity of afferent sources to the cerebellar
cortex is easily sufficient to assure that each such
module would receive a unique pattern of inner-
vation. Furthermore, even if modules received
identical inputs they might well terminate dif-
ferently, either overlapping in different combina-
tions to generate unique composite terminal fields
or terminating uniquely on terminal fields with
different patterns of extracerebellar connectivity.
Species Variation Suggests
that Compartment Number
Is Conserved Across Phylogeny
Cerebellar antigenic compartmentation can be
demonstrated in species as phylogenetically
separate as fish and primate. Even within the
mammals, the cerebellum of the squirrel monkey
contains many-fold more Purkinje cells than there
are in the rat, and the human cerebellum is
that much larger again. How is this expansion
achieved? Two possibilities can be contrasted:
either the cortical surface increases by the addition
of new compartments or it is through the expan-
sion in volume of a constant set of compartments.
The former mechanism seems to be employed for
example in the frontal cortex and striatum, where
modular columns are of roughly similar sizes in
many species and expansion occurs by the addi-
tion offurther modules. However, comparison of
rat and monkey cerebellar cortices stained for
zebrin I suggests that cerebellar expansion occurs
not by the addition of new compartments but by
the expansion of the existing ones. At least in the
vermis, the zebrin I compartmentation is rather
similar in the two species, with clear PI +, P2 +,
and P3 + bands identified in both, each set with
similar relative sizes. In the hemisphere, the cere-
41
bellum of the monkey has evidently undergone a
massive expansion in surface area but we see no
evidence of large numbers of new alternating
zebrin I + and zebrin I - compartments. Rather,
the individual zebrin 1+ compartments seem to
have increased in width such that very long conti-
nuous stretches of zebrin 1+ Purkinje cells are
seen compared to the rat. This idea receives
support from comparison of the corticonuclear
zonation of primates and rodents. In both the rat
and the squirrel monkey, the vermal A and B zones
are both narrow, but in the para vermis and hemi-
spheres squirrel monkey of the squirrel monkey,
there is a significant relative broadening of the
Cl, C2, C3, and D zones (Haines et aI., 1982).
Although no detailed correlation has been made
between corticonuclear zones and antigenic com-
partments, this is obviously consistent with what
we see here with zebrin I, with relatively narrow
vermal bands in both species and almost all the
relative expansion in the monkey cerebellar cor-
tex concentrated in the zebrin 1+ compartments
of the paravermis· and hemispheres.
Events Leading to the Establishment
of Cerebellar Connectivity
The Development of Intracerebellar
Compartments: Perinatal Markers
A selection of immunological markers are now
available to monitor the maturation of Purkinje
cells in rodents. The earliest antigen to be expres-
sed is cyclic guanosine 3': 5' phosphate-dependent
protein kinase (cPK) that appears for the first
time at embryo day 17 in a subset of Purkinje
cells (Wassef and Sotelo, 1984) (the Purkinje cell
terminal mitoses are between embryo days 12 and
15: Altman, 1972). Differential expression by a
subset of Purkinje cells is continued until birth.
In the first 5 days postnatally antigen expression
gradually extends to include all Purkinje cells,
and differential expression is not seen in the adult.
Several other perinatal compartment antigens
have also been described. Antisera to vitamin D-
dependent calcium-binding protein (CaBP) and
Purkinje cell-specific glycoprotein (PSG) both
selectively recognize transient perinatal Purkinje
cell populations (Wassef et aI., 1985). As was the
case for cPK expression, the heterogeneity seems
42 Richard Hawkes et al.

A
 2. Zebrins
to reflect timing differences in the onset of expres-
sion, and disappears during the first few postnatal
days.
Zebrin J/JJ
Both zebrin I and zebrin II follow an identical
developmental timetable in the rat, and can be
discussed together (zebrins 111- V have not yet
been investigated). We have identified three stages
in the expression ofthe zebrin I epitope, an initial
phase in which all Purkinje cells are zebrin 1-, an
intermediate stage in which all become zebrin 1+,
and the final, adult stage of selective expression
by parasagittal bands of cells (Leclerc et aI., 1988).
The late expression of zebrin I during corticoge-
nesis precludes it from playing a direct role in the
initial encoding of positional information since
some of the para sagittal zonation ofthe cerebellar
cortex is already established at birth (e.g., Sotelo
et aI., 1984). A similar developmental profile is
not observed in other brain regions where zebrin
I - immunoreactivity is primarily glial (Plioplys
and Hawkes, 1986, 1988), and differential staining
is present from the earliest time of expression
(e.g., the habenular complex: Plioplys and Hawkes,
1987).
Zebrin 1/11 induction begins at the median
posterior vermis at P6. By P7, clusters of im-
munoreactive Purkinje cells are clearly evident in
the posterior lobe vermis (Fig. 2.l3A, B, D), but
~,-----------------------------------------------------------------
Figure 2.13. The distribution of zebrin I immunore-
activity in the rat cerebellar cortex at P7 and P12 seen
in horizontal sections. A: Specific Purkinje cell staining
in the vermis oflobule VIII at P7. The immunoreactive
cells in this section are organized into three major
clusters and in each case, there is a suggestion of a
subcompartmentation within the clusters. B: The poste-
rior lobe vermis at P7 contains numerous clusters of
zebrin I + and zebrin 1- Purkinje cells. The reaction
product in the white matter is a mixture of specific
Purkinje cell axon immunoreactivity and nonspecific
staining. C: In the anterior lobe vermis there is still
no specific immunoreactivity at P7. Some nonspecific
staining, due to the high anti-zebrin I levels used, is
seen in the immature granular layer (IGL) and the
Bergman glial cell bodies of the Purkinje cell layer
(PcL). No reaction product is deposited in the external
granular layer (EGL). D: A higher power view oflobule
43
the anterior lobe vermis (Fig. 2.13C) and the hem-
ispheres are unreactive. Expression radiates both
anteriorly and laterally so that, about 5 days
after the onset, all Purkinje cells are zebrin 1+
(Fig. 2.13E, F, G). The spread of zebrin expression
is not smooth, but rather seems to involve several
clearly defined stages, with sharply defined medio-
lateral and rostrocaudal boundaries: for example,
the first signs of expression during development,
in the most caudal lobules, stop abruptly midway
along lobule VIII. The transient banding pattern
has disappeared by P12, but differences in stain-
ing intensity indicate the positions of the future
p+ IP- compartments. The relationship between
the early and mature compartmentation is un-
clear. The factors that control the induction and
spread of zebrin I expression in the Purkinje cell
population are unknown.
In the opossum, the expression of zebrin II
during development closely resembles that in the
rat. M onodelphis is born after only 14 days in
utero, and remains in the maternal pouch for
several weeks thereafter. When rat and opossum
zebrin II expression timetables are compared, the
correspondence is remarkable: the first immuno-
reactivity appears at P20 (equivalent to rat P8),
uniform expression by all Purkinje cells is seen at
P28, and zebrin II + III - bands become obvious
at about P35. The final maturation of the band
array appears to be more protracted than in rat,
and the adult appearance is only achieved after
VIII at P7 to emphasize the presence of a substructure
within the major Purkinje cell compartments. E: At
P12 the axons can be reliably followed through the
internal granular layer (arrowheads). At the midline
(dotted line), the corticonuclear projection appears to
be exclusively ipsilateral as in the adult, and we never
see immunoreactive axon profiles projecting to the
contralateral cerebellum. F: Although all Purkinje cells
are zebrin I + at P12, in the vermis the putative nascent
bands of Purkinje cells that will become zebrin I + in
the adult are distinguished by their higher levels of
immunoreactivity (arrowheads). G: Nascent bands are
also apparent in the hemispheres at P12. Shown here
is the putative P6 + band (arrowheads) in the immature
crus II. Scale bars = 300 /lm (A, B, F) and 100 /lm (C, D,
E, G). (Modified with permission from Leclerc et a!.,
1988. Copyright 1988 by Wiley-Liss, a Division of
John Wiley & Sons, Inc.)
44
P56 (Dore et aI., 1990). In the chicken, the dev-
elopmental timetable has not yet been fully deter-
mined, but clear zebrin 11+111 - compartments
are seen in the vermis by 7 days posthatching
(Brochu and Howkes, unpublished observations).
There remain obvious gaps in our knowledge
concerning the timetable of Purkinje cell bio-
chemical compartmentation in the rat. Three
stages have been identified: perinatal (e.g., cPK
immunoreactivity: Wassef and Sotelo, 1984;
Wassef et aI., 1985), the early zebrin 1+ Izebrin 1-
clusters in the posterior lobe vermis (P6-P9), and
the mature display of bands and patches (from
PIS). How do these three relate to one another?
Unfortunately, the three patterns never coexist:
the early clusters revealed by using anti-zebrin I
do not appear until the cPK is distributed uni-
formly, and then they are swamped by the phase
of global zebrin I expression at around P12. A
direct correlation could be made via the topo-
graphy of the olivocerebellar projection or by
using CO histochemistry, but this has not been
done and it is not even certain that such a correla-
tion is possible. Previous studies of chemical com-
partmentation during perinatal development
concluded that multiple overlapping families of
Purkinje cells were present, and all markers do
not respect common boundaries.
Afferent Input Does not Regulate
Zebrin I Expression
Given the close interrelationships between afferent
compartments and intrinsic cerebellar cortical
markers (above), it is tempting to speculate that
these are not independent. For example, a topo-
graphically ordered afferent input, such as the
one from the inferior olive, could take control of
the differentiation pathway of the homogeneous
target Purkinje cell field. This is superficially plau-
sible for three reasons: a) there is a strong correla-
tion between the positions of the cortical bands
in the adult olivocerebellar projection and the
antigenic bands revealed by anti-zebrin I (Gravel
et aI., 1987), b) the maturation ofthe CF projection
parallels the development of differential zebrin I
expression (Crepel, 1971, 1982; Crepel et aI., 1976;
Mariani and Changeux, 1981), and c) the CF com-
partments are present in the white matter before
synaptic contact with the Purkinje cells (Crepel,
Richard Hawkes et al.
1971; Puro and Woodward, 1977). A test for this
is to lesion selected afferent inputs and then look
for alterations in the pattern ofzebrin I com part-
mentation. Lesion experiments do not support a
role for afferent input in the regulation of the
Purkinje cell zebrin I phenotype. No gross changes
have ever been observed in the adult cerebellar
bands as a result of selective lesioning in the adult.
Thus, the number of bands, their relative sizes,
and their relative positions were unaffected, except
where abnormal lobulation made the latter un-
avoidable. The range oflesions was not exhaustive,
but we have eliminated CF bilaterally and selec-
tive MF bilaterally and unilaterally. Likewise,
no changes in the parasagittal zonation of the
cerebellum were found after the elimination of
hindlimb input bilaterally and vibrissal input
unilaterally. This was true both chronically (after
30 days) and acutely (after 3 days). Hence, there
is no evidence that tonic afferent input, either
MF or CF, is required to maintain the selective
expression of the zebrin I epitope.
It is perhaps not surprising that afferent input
does not regulate the zebrin I phenotype of com-
mitted, differentiated Purkinje cells. It is more
important that the same holds for lesions in the
afferent cerebellar projections of the newborn rat.
Unilateral olivary lesions, either electrolytically
or by pedunculectomy, unilateral lesion of the
trigeminal and gigantocellular nuclei, bilateral
sectioning of the dorsal and ventral spinocerebel-
lar tracts, and unilateral lesion of the infraorbital
nerve did not grossly modify the adult zebrin I
band distribution. In the case of the vibrissal
input, similar lesions have been shown to result
in a reorganization of thalamocortical afferents
(Killackey and Belford, 1980) and abolition of the
whisker barrel fields in the mouse somatosensory
cortex (Van der Loos and Woolsey, 1973); thus,
some cortices can respond anatomically to such
peripheral lesions. The general conclusion is clear:
there is no evidence of plasticity in the cerebellar
map comparable with that in the somatosensory
cortex. Furthermore, neonatal lesions had no effect
on the timing or pattern of the initial induction
of zebrin I during development.
Lesion studies in the neonate preclude a role
for afferent inputs in the regulation of zebrin
expression postnatally, but a prenatal role in com-
mitment was still possible. To explore this, cere-
2. Zebrins
bellar anlagen were dissected from embryos at
E12 through E15, that is, before any contact with
afferents, and transplanted ectopically into adult
hosts (Wassef et ai., 1990). In the first series of
experiments the grafts were placed into the an-
terior chamber of the eye, and in the second series
into cavities prepared in the neocortex. Grafts
were allowed to mature and then were immuno-
peroxidase stained for zebrin I immunoreactivity.
Zebrin I was expressed by grafted Purkinje cells
in cortico and in oculo. Double-labeling experi-
ments confirm that both the zebrin + and the
zebrin - phenotypes are present. Therefore, it
seems probable that afferent input does not play
a role in the determination of the zebrin phenotype
of Purkinje cells.
If afferent regulation is not the source ofPurkinje
cell compartmentation, then where does it come
from? One possibility is that bands of zebrin 1+
and zebrin 1- Purkinje cells arise by clonal ex-
pansion from a small number of committed pre-
cursor cells. For example in chimeric mice, a
fine-grained mosaicism of nonrandom clonal
compartments has been described in the mature
cerebellar cortex (Oster-Granite and Gearhart,
1982). How these clonal compartments are re-
lated to the zebrin I compartmentation is still
unknown, but this line of reasoning could explain
the similarities in compartmentation between
different cerebellums from chick to primate. If all
Purkinje cells arise from a small number of early
precursors, each already committed to (but not
yet expressing) its zebrin I phenotype, then such
a ground plan might well be very stable through-
out evolution: early embryonic patterns of develop-
ment are difficult to change as their ramifications
for later development tend to be wide, and hence
the consequences of change tend to be fatai. How-
ever, differences in the extent of clonal expansion
can be accommodated more easily, and would
result in different species having a common set of
cerebellar compartments, but of widely differing
sIzes.
Acetylcholinesterase
There are few other studies of chemical band deve-
lopment. As regards AChE, Marani and Voogd
(1977) have noted that in cats, bands of elevated
activity in the molecular layer are present in neo-
45
nates during a narrow developmental window
that opens at 7 weeks and closes by 16 weeks
postnatally. In the rat, the maturation of the
AChE patches and the bands of zebrin 1+ Izebrin
1- Purkinje cells occurs at much the same time
(Boegman et ai., 1988). In both cases, staining in
the vermis appears in the second postnatal week
but, whereas zebrin I immunoreactivity is uni-
formly distributed by P12 and forms bands only
thereafter (Leclerc et ai., 1988), the AChE staining
of the granular layer is patchy from the first. The
crudely similar postnatal developmental timetables
ofzebrin I and AChE cannot be taken as evidence
of a close causal link between the two because
during this period both the afferents and the cere-
bellar neurons and glia undergo major differen-
tiation and adopt their mature appearances.
Furthermore, both the AChE and the zebrin I
compartments develop relatively late in cerebel-
lar maturation, and follow well after both Purkinje
cell and CF compartments appear to be estab-
lished (Sotelo et ai., 1984; Wassef and Sotelo, 1984).
It is interesting that the patchy distribution asso-
ciated with the granular layer and the white matter
is apparent before the formation of synaptic glo-
meruli. Given the location of the AChE activity,
a more likely temporal correlation can be made
with the development offunctional MF input and
the maturation of the granular layer. The MF
axons in the cerebellum terminate on granule cell
dendrites in complex synaptic glomeruli that
are generated primarily between PI 0 and P20,
but the MF growth cones are already present in
the cerebellum at birth (e.g., Arsenio-Nunes and
Sotelo, 1985; Mason and Gregory, 1984) and wait
on the arrival of granule cells that migrate from
their germinal external granular layer. Although
adult AChE staining is associated with the glo-
meruli, there is no activity in the external granular
layer where the granule cells are formed, further
suggesting a primary role for the MF afferents in
the creation of AChE patches.
Cytochrome Oxidase
In the rat, CO patches are already present at
birth. Because zebrin Illl are not expressed before
P6, a direct comparison between CO and zebrin
I is not possible perinatally. However, from the
general disposition of the clusters, the relationship
46
between zebrin I and CO phenotypes does not
appear to change during maturation. Because
CO can be used as a compartment marker from
birth, it should be possible to use it in conjunction
with zebrin I and/or cPK immunoreactivity to
correlate prenatal with postnatal compartmenta-
tion.
Matching Connections
to Compartments
Both the Purkinje cell compartments (Wassef and
Sotelo, 1984; Wassef et aI., 1985) and at least
rudimentary MF compartments (Arsenio-Nunes
and Sotelo, 1985) are present in the cerebellar
white matter at birth, before the onset of granule
cell migration, and hence before normal synapto-
genesis. The natural conclusion is that nonsynap-
tic interactions in the axon tracts are responsible
for at least the gross features of axon compart-
mentation. This is reinforced by the extensive
studies of myeloan;hitecture (Marani, 1982; Voogd,
1964, 1969; Voogd et aI., 1985) showing the pre-
sence of alternating, parasagittal axonal compart-
ments in the mature white matter that presumably
reflect the migratory histories of the afferent and
efferent growth cones.
Climbing Fibers
Although compartments are seen in the olivocere-
bellar projection from birth and thus predate the
zebrin compartments, the evidence suggests no
dependence of cortical compartmentation on
afferent input either in the adult or during post-
natal development. Rather, it seems that the
maturation ofthe cerebellum requires the precise
matching of topographically organized afferent
inputs to predefined cerebellar modules. At what
locus is the matching achieved? Two models can
be suggested (Fig. 2.14). In the first (e.g., Sotelo,
1987), matching occurs directly, by afferent growth
cones recognizing markers expressed on the
Purkinje cells (Fig. 2.141). Once the appropriate
axon fascicle is identified CF branches run both
to the cerebellar cortex and to the corticonuclear
target territory. For the CF zonation, recognition
would ha ve to be through nonsynaptic mechanisms
since it is clear that bands reminiscent of those in
the adult are present in the olivocerebellar pro-
Richard Hawkes et al.
.
~ ~.L-'-~.- -'~c. ,Lt
::
t, :
---,I cc
1 1 [j 1CN
1
II
Figure 2.14. A schematic drawing to contrast two mo-
dels by which olivocerebellar topography might be
attained. The adult situation is shown in the upper
box. Purkinje cell modules in the cerebellar cortex
(CC) terminate in precise target zones within the cere-
bellar nuclei (CN) and lateral vestibular nuclei. Afferents
from discrete regions in the inferior olivary complex
(IOC) terminate topographically both on the Purkinje
cell compartment and on the CN terminal field. Reci-
procal connections from the CN are also probably
present. This topography might be achieved in one of
two ways. In the first (I), olivocerebellar afferents contact
specific Purkinje cell axons and then send branches to
the cerebellar cortex and the cerebellar nuclei. In the
second (II), both Purkinje cell and olivocerebellar sub-
sets terminate independently in the nuclear target zone,
and then the climbing fibers follow the Purkinje cell
axons into the cerebellar cortex. The difference bet-
ween the two models lies in the locus of the positional
information: in I, it is in the Purkinje cells; in II, it lies
in the cerebellar nuclei.
jection at birth (Crepel, 1982; Sotelo et aI., 1984),
several days before the first CF synapses are de-
tected (Crepel, 1971; Puro and Woodward, 1977).
This model is a plausible one, and is consistent
with long-established views that the Purkinje cell
is the prime organizer of cerebellar morphology.
An alternative model is that matching occurs
indirectly because both the Purkinje cells and the
afferents recognize a common third party (Fig.
2.1411). One candidate to match cerebellar cortex
afferent and efferent topographies are the cere-
bellar nuclei (Boegman et aI., 1988). There is some
evidence that the olivonuclear projection is topo-
2. Zebrins
graphically ordered such that an olivary neuron
contacts both a Purkinje cell and that cell's target
field within the cerebellar nuclei. Thus, Purkinje
cell efferents and olivary afferents could be brought
together because they share a common target
territory in the cerebellar nuclei. Subsequently,
the olivary input to the nucleus could branch to
form the much larger projection to the cerebellar
cortex, perhaps by fasciculation along the Purkinje
cell axons. This view would reverse the usual
nomenclature by treating the olivonuclear pro-
jection as the primary afferent pathway to the
Figure 2.15. A cut-away view of the cerebellar vermis
to illustrate a possible model by which MF afferents
might differentially innervate dorsal and ventral faces
of a lobule. The two kinds of MF are laminated within
the white matter. Thus, when they come to identify
synaptic partners, the more dorsal MFs naturally in-
47
cerebellum and the CF input to the cerebellar
cortex as a secondary collateral path. Such a view
is consistent with physiological interpretations of
cerebellar organization, and suggests an alter-
native view of cerebellar afferent development.
The normal description of olivocerebellar organi-
zation is that CF efferents from the inferior olivary
complex project topographically to the Purkinje
cell layer and send collateral branches to appro-
priate targets in the cerebellar nuclei. This may
have introduced hidden semantic bias into our
thinking about cerebellar development. The olivo-
nervate the granular layer dorsally, and those more
ventral, the granular layer ventrally. The differences in
terminal field position arise through chemically or
physiologically based decision making during synap-
togenesis.
48
cerebellar projection is already present and orga-
nized into compartments before synaptic contact
with the Purkinje cell targets (Sotelo et aI., 1984).
Given the relative maturity of the cerebellar nuc-
lei at birth, it may be that the primary projection
is olivonuclear rather than olivocortical and that
it is the cortical projection that is the secondary
collateral. From this perspective the olivocerebel-
lar topography would be generated by the olivo-
cerebellar and corticonuclear projections both
first recognizing common nuclear target zones.
The same sequence might apply also to the matu-
ration of the various MF inputs.
Mossy Fibers
The matching ofMF terminal fields to appropriate
Purkinje cell compartments is more complex, be-
cause there is no direct synaptic interaction bet-
ween MF and PC in the adult. Once within the
target area, the differences in the terminal field
distributions might arise either through physio-
logical or molecular mechanisms. For example,
the circuitry could be sculpted by competitive
interactions. The protracted maturation of the
MF-GC circuitry during the first postnatal month,
and probably beyond, could provide an ideal
substrate for experience/activity-dependent re-
finement of cerebellar circuitry. For example, dif-
ferent MF inputs might preferentially couple to
Purkinje cells that receive an appropriate olivo-
cerebellar input. During postnatal maturation
synapses with inappropriate physiological pro-
perties would be eliminated.
Alternatively, molecular cues could operate.
Although regional differences within granule cells
have not been demonstrated, they may be present
either intrinsically or due to induction by the
overlying Purkinje cells. There are also temporary
synaptic interactions between MF growth cones
and Purkinje cells that could serve to aid align-
ment. However, any model based on Purkinje cell
organization has to explain the facts that MF
terminal fields are frequently spanning zebrin 1+
and zebrin r compartments, and often appear
to be distributed differently on different faces of
the same lobule. In this context, how might this
differential distribution of LTHL terminals bet-
ween dorsal and ventral aspects be achieved?
There are two aspects to the problem: to distribute
Richard Hawkes et al.
different MF afferents to either the dorsal or the
ventral half of the lobule, and then to identify
appropriate synaptic partners. For the first part,
we suggest that a dorsoventral lamination ofaxons
within the white matter leads naturally to a dor-
soventral bias in the distribution of their terminals
within the granular layer (Fig. 2.15). Extending
this segregation into the individual folia could
result in individual longitudinal myeloarchitec-
tural compartments that are also subdivided into
dorsal and ventral fascicles. The MF growth cones
are already situated in the white matter at birth,
before the arrival of their granule cell targets. As
the granule cells descend, during the first three
postnatal weeks or so, the MF growth cones
invade the granular layer and form the synaptic
glomeruli. With an intrinsic lamination in the
white matter, the more dorsal axons will naturally
innervate the dorsal aspect of the lobule and vice
versa.
What the Cerebellum Can
Tell us About Cartography
In the nervous system of vertebrates connectivity
between functionally coupled groups of neurons
in different brain regions is defined topographi-
cally. This is obvious in the brain stem, for example,
where neurons are clustered into discrete nuclei,
but even in ostensibly uniform structures there
appears to be a compartmental, modular organi-
zation. Well known examples of such organization
would include the cortex, the striatum, and the
cerebellum. This need not be the case a priori: it
would be easy to imagine an organization in
which a target would consist of a homogeneous
mix of two cell classes, each of which would be
contacted by a different population of inputs.
Indeed, this may well often be the case, as exempli-
fied by the segregation ofCF inputs to the Purkinje
cells and the MF inputs to the granule cells in the
cerebellum. However important this segregation,
it probably involves different interactions than
those involved in the establishment of topogra-
phically specific projections.
The fact that spatial segregation is predominant
probably reflects the restricted repertoire of cell
recognition molecules, and the need to reuse them
in multiple contexts. What is the significance of
compartmentation? One reason might be embryo-
2. Zebrins 49

logical - to achieve the precise correlation of
afferent and efferent projections. By alternating
clusters of different chemospecific cells the target
presents a set of sharply differentiated internal
boundaries to the afferent growth cones. For
example, once pathway guidance and axon fasci-
culation have brought the CF to approximately
the correct position, precise terminal fields are
identified thanks to the steplike borders.
We have used the cerebellum and its afferent
projections as a model to explore map formation
in the central nervous system (Fig. 2.16). In order
to characterize the cerebellar maps, it will be
useful to define a number ofterms. To begin, there
is the definition of the map itself. The projection
map is the distribution of afferent terminals from
a projection field onto a target field . For the
present purposes, a map is characterized by a
point-to-point correspondence between the pro-
jection and target fields. All such projections can
be characterized in two ways. First, a projection
may be convergent or divergent (Fig. 2.161). In
a convergent projection, the projection field is
larger than the target field-an example in the
cerebellum is the corticonuclear projection, where
there are up to 10 times more Purkinje cells in
the cerebellar cortex than there are target neurons
in the cerebellar and lateral vestibular nuclei. In
a divergent projection, the projection field is smal-
ler than the target field . One example is the olivo-
cerebellar projection, where each olivary cell
innervates up to 10 Purkinje cells. From a purely
topographical perspective, a divergent projection
is merely empty magnification. However, it might
be the basis for topographically identical input
fields with dissimilar outputs- that is, branching-
or it might permit the convergence of different
combinations of inputs that can be combined
functionally. Of course, divergent projections have
other functional advantages; for example, they
permit signal amplification, improve the signal-
to-noise ratio during message transmission, and
allow the same afferent data to be processed in
different ways.
Second, projections may be overlapping-with
a consequent loss of resolution - or nonover-

A A A A A A

(DJ
A A A A A A
A A A
A B B B B B B
B B
e e
B B B ~ B B B B B B
e e e
e e e e e e
e e e e e e

A A A A A A A A A
AB AB AB
II B B B B B B B B B
Be Be Be
e e e c e c c e c

Figure 2.16. Illustration of different

[]
kinds of maps found in the cerebellum. A A A A A A
In I, the projection field can terminate BBB
either convergently (smaller target: e.g., III B B B B B B eee
corti-conuclear) or divergently (target c e e c e e
larger than projection field, e.g., olivo-
cerebellar). In II, a projection can be
nonoverlapping (e.g., olivocerebellar) or
overlapping (corticonuclea r, MF). In A A A A A A A A A
III, the field is completely or incompletely
filled. In IV, the mapping is continuous
IV B B B B B B e e e
or discontinuous (e.g., olivocerebellar). e e e e e e B B B
50
lapping. For the olivocerebellar projection, com-
partments seem to be discrete and nonoverlapping.
This seems to be equally true in the neonate as
in the adult. For MF and for the corticonuclear
projections, there is probably some overlap (Fig.
2.1611).
A "field" could be defined in a number of ways;
for example, as a group of cells that share a
common target field with no functionally signifi-
cant heterogeneity. This definition present prob-
lems-fields are defined by their connectivity and
so the argument is circular. It does, however,
serve to focus attention on the central question
here: can fields be defined independently of their
connectivity? In the case of the cerebellum, the
answer is clearly, yes. Future target fields can
already be identified in the embryo as early as
E 16, well before contact with the afferents. Like-
wise, the differential expression of zebrins by
Purkinje cell subpopulations is independent of
afferent input (Leclerc et ai., 1988).
The map may be completely filled or incomplete
(Fig. 2.16III). This may be an issue of definition
only, as fields are defined by their terminals, for
example, patches of granular cells that do not
receive spinocerebellar inputs can simply be treat-
ed as outside the field. The issue is important only
if these gaps are filled at some time during develop-
ment: there is no evidence of this either for the
CF or the MF terminals, although it clearly is the
case in other brain regions (e.g., the corticocortical
projection via the corpus callosum).
A map may be continuous or discontinuous
(Fig. 2.16IV). If a continuous translation in the
projection from field to neighboring field maps
to a smooth displacement in the target, then the
map is continuous. If the corresponding transla-
tion in the terminal fields is saltatory, the projec-
tion is discontinuous. It is striking that both MF
and CF projections are discontinuous. The reason
for this is unknown, but discontinuous projections
have one evident advantage-sharp internal
boundaries. From the point of view of target
recognition, these can serve to assure that growth
cones that reach approximately the correct target
territory by white matter interactions are forced
to terminate within a small, precise area, simply
because the adjacent areas are incompatible in
some way. This then will prevent the statistical
degradation ("blurring") of the topography that
Richard Hawkes et al.
would present problems if the topography were
continuous, and provides a ready mechanism for
error elimination and secondary, perhaps func-
tional, refinement.
The number oftopographically identical parallel
pathways within a field-to-field projection can be
considered as a measure of the topographical
redundancy. In the adult animal, this number is
presumably related to the density of information
passing along the pathway. However, during devel-
opment other considerations come into play. It
is a characteristic of much of the nervous system
development that ordered patterns are sculpted
from initially homogeneous arrays by selective
elimination of supernumerary components (e.g.,
Changeux and Danchin, 1976; Law and Constan-
tine-Paton, 1980; O'Leary et ai., 1986), and several
theoretical models of map formation predict the
source-specific banding of afferent terminals when
fibers interact competitively (Constantine-Paton,
1982; von de Malsburg and Willshaw, 1976).
One of the better known examples is found in
the cerebellum-the elimination of excess CF
synapses during the maturation of the olivocere-
bellar pathway (Crepel et ai., 1976; Mariani and
Changeux, 1981). These might serve either to
ensure topographic mapping despite poor aim
(i.e., improve topographical fidelity) or to ensure
complete filling. Elimination occurs both in the
CF and the MF projections. In the case of the
olivocerebellar projection, the available data do
not support an important role for collateral elim-
ination in the refinement of topography (Crepel,
1982; Sotelo et ai., 1984). However, target filling
is clearly a crucial problem for projections such
as the olivocerebellar, where the target receives
only one input in the adult; thus, the most plausible
role for excess CF collaterals is to ensure that
each Purkinje cell receives at least one CF input.
As far as the MF are concerned, the matter is
less clear. For example, the circuitry could be
sculpted by competitive interactions. The pro-
tracted maturation of the mossy fiber-granule
cell circuitry during the first postnatal month,
and probably beyond, could provide an ideal
substrate for experience/activity-dependent re-
finement of cerebellar circuitry. For example, dif-
ferent spinocerebellar inputs might preferentially
synapse on Purkinje cells that receive an appro-
priate olivocerebellar input. During postnatal
2. Zebrins
maturation synapses with inappropriate physio-
logical properties would be eliminated. Similarly,
an exuberance of granule cell dendrites could be
an anatomical substrate for the refinement of the
MF pathways (e.g., Morest, 1969; Ramon y Cajal,
1911 ).
Developmentally, the question is whether the
projection fields and the target fields are naturally
compartmentalized before the establishment of a
map. In the cerebellum it appears that they are,
and precise topography is achieved by mapping
an ordered input onto an ordered target. What
is the advantage of such a matching strategy over
a method involving transynaptic determination?
The most obvious gain is that system maturation
need not proceed linearly. In a neuronal chain in
which target topography passes from member to
member, transynaptic determination requires that
development occur serially and proceed in the
descending direction. The converse, where the
target topography is independently determined,
permits elements of the chain to mature indepen-
dently. Such a mechanism also avoids a second
problem with linear determination-that errors
high in the chain will disrupt the topographic
organization at all lower levels. Thus, in the
cerebellum, the descending efferent pathways can
mature earlier than the cerebellum itself without
compromising anatomical topography.
Acknowledgements. Thanks are due to Marc
Colonnier who constructed the various artwork,
to Suzanne Bilodeau for typing numerous versions
of the text, and Mike Lannoo and Andre Parent
for their comments. The experimental work was
dependent on the technical assistance of Line
Thivierge, Jamel Rafrafi, and Rachel Sasseville,
and was supported by grants from the Medical
Research Council of Canada, the F.R.S.Q., the
F.C.A.R., and the N.I.H.
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3
Cerebellar Granule Cells and the
Neurobiology of Excitatory Amino Acids
Robert Bala.zs, Nicola Hack, and Ole S.
The discovery that the development in the nervous
system involves the loss of a significant number
of nerve cells in many structures has given one of
the deep insights into mechanisms underlying the
development of neuronal organization and of the
maintenance of nerve cells (for reviews cf. Cowan
et aI., 1984; Hamburger and Oppenheimer, 1982;
Levi-Montalcini, 1982). It seems that during their
differentiation nerve cells become dependent on
environmental influences for survival. Most of
the evidence concerning specific survival require-
ments relates to target-derived trophic factors,
which are available in limited supply; the best
characterized example is nerve growth factor
(NGF). In addition, it is believed that afferent
nerve fibers also exert trophic influences on nerve
cells, but hitherto these have received less atten-
tion than the target-derived effects (Cunningham,
1982). Our recent studies on cerebellar granule
cells have implicated an afferent system, the mossy
fibers, as a source oftrophic influence at a critical
stage of the maturation of these cells (e.g., Balazs
et aI., 1988a, b; Gallo et aI., 1987a). Here we pre-
sent observations indicating that this influence is
mediated through the stimulatioJl of receptors for
excitatory amino acids (EAA) on the postsynaptic
granule cells and put forward evidence suggesting
that such trophic effects are not unique to the
cerebellar granule cells.
Cerebellar Granule Cells
in Culture
We discovered the trophic effect of EAA while
studying cerebellar granule cells in culture. With
certain manipulations of the culture conditions it
56
J~rgensen
is possible to obtain a preparation from dissociat-
ed cerebella ofearly postnatal mice or rats [usually
postnatal day (P) 6-8], which predominantly
comprises interneurones (about 95%), primarily
granule cells (e.g., Thangnipon et aI., 1983). Ini-
tially, most of the cells surviving the procedure
of tissue dissociation and the transfer to in vitro
conditions are immature granule neurones. Many
ofthese cells would continue proliferating in vivo,
but in vitro they are unable to do so and are
prematurely induced to differentiate. This is
characterized by neurite emission soon after plat-
ing, the production of a fine network of fibers,
and the migration of neurones along the fibers to
form clumps. During cultivation the aggregates
increase progressively in size and the fibers fasci-
culate to form large, more or less interconnecting,
bundles (for the appearance of a "mature" culture
see, e.g., Figs. 3.1a and 3.2a). Biochemical indices
show that maturation starts more or less im-
mediately after seeding (Meier et al., 1984) but, in
terms of most parameters examined, it is slow in
the first few days followed by a rapid phase of
development, reaching a plateau by 7 to 8 days
in vitro (DIV) (e.g., Gallo et aI., 1987a). Matura-
tion is therefore faster in culture than in situ: this
is due to the fact that in vivo cerebellar develop-
ment is prqtracted because the genesis of nerve
cells takes place during a relatively long period,
the first three postnatal weeks. Thus, although
certain differences in the state of maturation of
granule cells have been noted (Trenker et aI., 1984),
the relatively homogeneous compositions of these
cultures can facilitate the detection of transient
and maturation stage-dependent effects, which
could easily be masked in vivo.
3. Granule Cells and Excitatory Amino Acids
Figure 3.1. Survival of granule cells in culture is depen-
dent on chronic K + -induced depolarization, whose
effect is mediated through a stimulation of Ca2+ in-
flux. Cells obtained from P6-8 rat cerebella were grown
in 10% fetal calf serum containing BME supplemented
with 2 mM glutamine and 100 Jlgjml gentamicin on
polylysine-coated Falcon dishes for 8 DIV. Glial cell
proliferation was inhibited by adding cytosine
arabinoside (10 JlM) at about 19 hours after seeding.
When cells were grown in "low" K + -containing
medium (5-15 mM) survival was compromised usually
by the end of the first week (15 mM K + in b). In con-
trast, cells survived well in "high" K + -containing
medium (25 mM K + in a). Elevated K + could be
Survival Requirements
of Cerebellar Granule Cells:
Chronic Depolarization
It is characteristic of these cultures that in con-
ventional serum-containing medium nerve cells
57
replaced by calcium agonists: the effect of the
dihydropyridine, (+ )-S-202 791, added to a sister cul-
ture of bin K15 is shown in c and d (1 x 10- 6 and
5 x 10-8 M, respectively). Other dihydropyridine
agonists, such as CGP 28392 and BAY K 8644, had
comparable effects, BAY K 8644 being the most potent
(lowest effective concentration 1 x 10-8 M). Addition
to the cultures in K25 of Ca2+ antagonists [lOmM
Mg2 + , 10 JlM D600 or the dihydropyridines nifedipine
and (- )-(R)-202 791 at 10- 7 M] resulted in cell death
as in the "low" K + -containing medium (appearance
of cultures similar to that in b, thus not shown
separately). Scale bar = 100 Jlm. (Reprinted with
permission from Gallo et aI., 1987a.)
die- usually quite abruptly toward the end of the
first week-unless the extracellular K + concen-
tration is elevated (> 20 mM) (Lasher and Zagon,
1972; Thangnipon et al., 1983). It is noteworthy
that initially the appearance of the cells and their
biochemical maturation are similar in "low" and
58
"high" K +-containing medium (in our studies
5-15mM K +, K5-15, and 25mM K +, K25, res-
pectively)(Gallo et aI., 1987a). With respect to the
possible mechanism underlying the survival-
promoting effect of chronic depolarization, two
observations were rather telling. We found that
the dependence of granule cells on K +-induced
depolarization develops within a narrow window
of time, between 2 and 4 DIV. Furthermore, we
obtained evidence that the effect of high K + on
cell survival is mediated through transmembrane
Ca 2 + influx. This could be demonstrated con-
vincingly after the discovery that granule cells are
endowed with voltage-sensitive Ca 2 + channels
(VSCC), at which dihydropyridine (DHP) calcium
effectors are as potent as in muscle cells, not only
in terms of binding, but also in eliciting functional
changes such as stimulation of Ca2+ entry and
transmitter release (Kingsbury and Balazs, 1987).
Significantly, DHP calcium agonists were able to
replace chronic depolarization in promoting cell
survival in "low" K + medium, while calcium an-
tagonists blocked the effect of high K + (Fig. 3.1).
Assuming that chronic depolarization in vitro
mimics critical physiological events in vivo, the
crucial question relates to developmental events
that have similar characteristics as those on which
granule cell survival depends in culture. As men-
tioned before, a great proportion of the initially
surviving nerve cells from the dissociated cere-
bellum are immature granule cells, which in vitro
start to differentiate immediately. In vivo, the
postmitotic granule cells take about 2 days to
reach the internal granular layer (IGL), while their
differentiation has already been in progress from
the time they had completed the last division
(Altman, 1982). This timing is therefore similar to
that of the development of the K +-dependence
of granule cells in culture. In vivo, at the end of
migration a unique event takes place in the life
of granule cells: they receive the first afferent
input. This is from the mossy fibers, many of
which seem to be glutamatergic (Somogyi et aI.,
1986). Granule cells, in turn, are endowed with
glutamate (Glu) receptors including the N -methyl-
D-aspartate (NMDA)-preferring subtype (Cull-
Candy et aI., 1989), which is linked to an ion
channel permeable not only to monovalent ions
but also to Ca 2 + (Mayer and Westbrook, 1987).
We have therefore put forward the hypothesis
that K +-induced depolarization in vitro mimics
Robert Balazs, Nicola Hack, and Ole s. J~rgensen
the trophic effect of the first innervation of granule
cells in vivo, which is mediated through Ca 2 +
influx linked to the stimulation ofNMDA recep-
tors (Balazs and Jy)rgensen, 1987; Balazs et aI.,
1988a, b; Gallo et aI., 1987a).
Survival Requirements
of Cerebellar Granule Cells:
Effect ofNMDA
The hypothesis was tested by studying the effect
ofNMDA on the survival of granule cells (Balazs
et aI., 1988a, b). It was observed that NMDA treat-
ment can indeed replace high K + in securing the
survival of these cells (Fig. 3.2). Quantitative eva-
luation is presented in Fig. 3.3, which also shows
that the effect depends on the concentration of
NMDA. Under the conditions reproduced in
Fig. 3.3, cell number and DNA content approach-
ed, although was usually less than, the value ob-
tained in cultures chronically depolarized with
25 mM K + (K25). Rescue of cells by NMDA was
also influenced by the degree of depolarization,
within a range at which depolarization on its own
does not substantially improve cell survival: thus,
at 5 to 15 mM K T both the efficacy and the po-
tency of NMDA increased as a function of the
K + concentration (Fig. 3.4, see also Fig. 3.3b). In
the experiment reproduced in Fig. 3.4 the half-
maximal effective concentration of NMDA was
about 50,uM at 5 mM K + (K5), 40,uM at 10 mM
K+ (KI0), and 20,uM at 15mM K+ (KI5). The
latter value is similar to the affinity ofNMDA in
binding to cerebral membranes (Olverman et aI.,
1984). These characteristics of the survival-
promoting effect of NMDA are consistent with
the voltage-dependence of the ion conductance
through the NMDA receptor-linked channel
(Mayer and Westbrook, 1987).
Observations displayed in Fig. 3.3b also in-
dicated that NMDA primarily rescued nerve cells.
The amounts of proteins that are known to be
greatly enriched in neurones, such as N-CAM,
D3-protein, and synaptin (Bock et aI., 1982;
Jy)rgensen, 1983; Nybroe et aI., 1985), showed a
similar dependence both on NMDA concentra-
tion and on the degree of membrane depolariza-
tion, as did cell survival. On the other hand, the
amount of glutamine synthetase, which was esti-
mated as a marker of astrocytes, the most common
nonneuronal cell in the cultures, was very low
3. Granule Cells and Excitatory Amino Acids
Figure 3.2. Granule cell survival in "low" K + -
medium (lOmM K +; KlO) is promoted by NMDA.
Seven DIV cultures in K25 (a) are well preserved,
whereas those in KlO show massive degeneration (b).
This was prevented by supplementing KlO with
NMDA (140.uM;c), whose effect was blocked by the
59
antagonist D-APV (140.uM; d). Neither APV (e) nor
NMDA (f) (140 .uM) had obvious effects on cultures in
K25. Scale bar = 100 .urn. (Reprinted with permission;
Bahizs et aI., 1988a. Copyright by Pergamon Press
PLC)
60 Robert Balazs, Nicola Hack, and Ole S. J!6rgensen

DNA CELL NUMBER PROTEIN

24 240 240
0

20 200 200
~

~
E,..

!~
~ 160 160
ii
~ 120 ! 120
! !
.
t t

t i f
Q.

BO eo
8 +
40 40

o ~ 70 140 o ~ 70 140 o ~ 70 140

NMDA(~M)

b
N-CAM D3-PROTEIN SYNAPTIN
120

100

eo

60

40

20

APV 0 35 70 140 APV 0 35 70 140 APV 0 35 70 140

NMDA(~M)

Figure 3.3. Effect of NMDA on the rescue of granule
cells is concentration-dependent. a: Cells were grown
in 6-em dishes for 8 days in KlO in the presence of
NMDA without (0) or with equimolar concentration
of the antagonist D-APV (e). Controls cultured in
K25 (0). b: NMDA promotes the survival of
neurones. This was assessed by measuring proteins,
which are known to be markedly concentrated in
nerve cells. Cells were cultured in to-em dishes for
7-8 days in KlO (0) or Kt5 (e). Some cultures in
KlO or Kt5 also received 280pM D,L-APV (,6, or
A) either without NMDA or with t40pM NMDA.
Values represent total amounts ofrespective proteins
per dish, expressed as a percentage of the estimate in
K25. As a marker of astroglia the amount of glutamine
synthetase was also determined: this was very low
under all conditions tested and was not significantly
affected by NMDA or high K + (not shown). ANOVA
showed a significant stimulating effect of NMDA for
all estimates in a and b (p < .05, except for synaptin
in KlO). Note that APV not only blocked the effect
of NMDA, but also reduced the limited cell survival
seen in "low" K + cultures. Furthermore, NMDA had
no effect on nerve cell survival in K25. (Reprinted with
permission from Balazs et aI., 1988b. Copyright by
Pergamon Press PLC.)

and did not show significant changes upon supple-
mentation of the cultures with NMDA.
The effect of NMDA was blocked by selective
NMDA receptor antagonists. Figures 3.2 and 3.3
show that the effect of NMDA is completely
blocked by equimolar concentrations of the com-
petitive antagonist D-2-amino-5-ph"sphonova-
leric acid (APV). The IC so for APV in the presence
of 140 JlM NMDA is about 28 JlM, whereas the
most potent inhibitor is MK-801 (IC so about
50nM) (Balazs et aI., 1989). MK-801 is a non-
competitive antagonist, believed to block the
NMDA receptor-linked ion channel (Wong et aI.,
1988): this compound is known to have a very
3. Granule Cells and Excitatory Amino Acids 61

0.5
a

1. ;
0.4

o 0.3
g';;.

0.2

0.1
140 280 420 560
NHOA (""1

b c
80 40

~
:E
"".....
..
2;
0

60 20

40 J----.L.-----:-'10L---~15
10 15
K+ (1floI) K+ (1floI)

Figure 3.4. Granule cell survival depends both on the
concentration ofNMDA and on the degree of depola-
rization of granule cells. Cultures grown in the absence
or the presence ofNMDA (at concentrations indicated)
in K5 (A), KI0 (.), K15 (e), and K25 (.). At 7 DlV
the reduction of a tetrazolium salt (MTT) to a colored
formazan product, which takes place only in viable
cells, was measured at 570nm (Manthorpe et aI., 1986).
a: Cell survival was a function of the concentration
of both NMDA and K +. However, at each [K +Je
survival reached a plateau, while the slope of the
increase in survival depended on the concentration of
K +. This information is displayed in b, giving the
maximal survival at the various concentrations of K +
as a percentage of survival in K25 (efficacy) and in c
exhibiting the concentration of NMDA effecting
half-maximal survival at the various [K +Je (potency).
(Reprinted with permission from Balazs et aI., 1988b.
Copyright by Pergamon Press PLC.)
62 Robert Balazs, Nicola Hack, and Ole S. J\ilrgensen

Table 3.1. Glutamate in the culture medium is res- the limited cell survival that is detectable in the
ponsible for the survival of a proportion of granule absence of NMDA in "low" K + media (Fig. 3.3
cells. and Table 3.1). These findings indicate, therefore,
40/lM Alanine amino- that substances present in granule cell cultures
APV transferase have a trophic influence, which is mediated through
NMDA receptor stimulation. Although the cul-
% of control l00±3 79±2 73±2 ture medium does not contain added Glu, it is
Cerebellar granule cells in "low" K + -medium (K5) in the
known that this amino acid is released by granule
presence of the NMDA antagonist D·APV or 10 U alanine cells into the medium (e.g., Kingsbury et aI., 1988).
aminotransferase + 10 mM pyruvate or in the absence of To test whether Glu was responsible for the limit-
additions (control). At 8 DIV, cell survival was assessed by ed cell survival in "low" K +, the medium was
the reduction of a tetrazolium salt (MTT assay; see legend substituted with alanine aminotransferase and
to Fig. 3.4).
Data from Balazs et aI., 1989.
pyruvate to remove Glu continuously from the
extracellular fluid. This treatment resulted in a
similar loss of cells as was obtained with APV
(Table 3.1).
high affinity for the NMDA receptor ionophore Electrophysiological responses to Glu appear
complex in binding assays using brain membrane to be mediated by at least three classes of pharma-
preparations (Wong et aI., 1986). cologically defined receptors (Watkins and Evans,
In contrast to the selective NMDA receptor 1981). These are named after the agonist that pre-
blockers, other EAA antagonists, including ferentially excites them: NMDA, lX-amino-3-hy-
gamma-D-glutamylaminomethyl sulphonic acids droxy-5-methyl-4-isoxazole-propionate (AMPA)
(GAMS) and kynurenic acid (KYN), at a relative- or quisqualate (QA) and kainate (KA). In recent
ly high concentration (200.uM), affected only studies we examined whether receptors other than
slightly, if at all, cell survival promoted by 140 .uM the NMDA preferring subtype are involved in the
NMDA. Findings on KYN are interesting, in trophic effect ofEAA (Balazs et aI., 1990a). It was
view of observations, that this substance, in addi- observed that KA exerts a significant and complex
tion to being a wide spectrum EAA receptor effect in cerebellar cultures (Table 3.2). At low
antagonist, also displaces glycine from an allosteric- concentrations (up to 50.uM) KA promoted cell
stimulating site on the NMDA receptor- survival, resulting in an approximately 50% in-
ionophore complex although its potency is low crease in comparison with cells grown in K5 at
(Asher and Nowak, 1987). However, in recent the maximum, 25 to 50.uM. This is less than
studies we have found that 7-chlorokynurenate obtained with NMDA under optimal conditions
(a more powerful antagonist at this site) is a (see Fig. 3.3) or with 25 mM K + (about two- or
potent blocker of NMDA effects in cultured threefold increase in cell numbers). In contrast to
granule cells (with Annelies Resink and Van der NMDA, the effect of KA, tested at 50.uM, was
Valk, unpublished observations). Thus, consider- not voltage-dependent, the degree of stimulation
ing the pharmacology of the effect, it seems that being similar in a 5 or 10 mM K + -containing
NMDA influences granule cell survival through medium. The selective nature of the effect is in-
a direct stimulation of the NMDA receptor- dicated by reports that claim that KA at the con-
ionophore complex. centration promoting the survival of granule cells
(50 .uM) is toxic to inhibitory interneurones in the
culture (e.g., Novelli et aI., 1987). Elevation ofKA
Involvement of Glutamate concentrations above 50.uM resulted at first in
Receptors Other Than the the cessation of the positive effect on cell survival,
NMDA-Preferring Subtype whereas at relatively high concentrations cells
in the Trophic Effect become vulnerable to KA (Table 3.2). .
It is known that KA can evoke Glu release from
of Excitatory Amino Acids cultured cerebellar granule cells (Gallo et aI.,
NMDA antagonists inhibited not only the 1987b). Thus, the effect of KA on cell survival
trophic effect of NMDA, but also compromised may have been secondary to the stimulation of
3. Granule Cells and Excitatory Amino Acids 63

Table 3.2. Granule cell survival is promoted or compromised depending on the

concentration of kainic acid

Cell survival (as a percentage of OD s7o given by the MTT assay in K5)
KA (JIM) 12.5 25 50 100 800

% of control loo±2 120±9 155 ± 7" 154 ± 7" 113±7

Granule cells were grown in K5 in the absence or the presence ofkainic acid (KA) at the concentrations
indicated, for about one week. Cell survival was assessed by means of formazan production by live
cells (MIT assay; see legend to Fig. 3.4). Data were analyzed by ANOVA. The effect of KA was
significant (p < .00001): the significance of the differences compared with control (no added KA) were
assessed using the Newman-Keuls' test.
a p = .01.
b p = .05.

(Data from Balazs et aI., 1990a.)

NMDA receptors by Glu released by KA from
granule cells. This possibility was examined by
determining the influence of KA on cell survival
after selectively blocking the NMDA receptors.
Table 3.3 shows that MK-801, which on its own
compromises cell survival, potentiated rather than
counteracted the cell survival promoting effect of
KA (the competitive antagonist APV had the
same effect)(BaUtzs et aI., 1990a, b). Furthermore,
whereas the effect of KA was found not to be
voltage-dependent, the potentiation of the KA
rescue by NMDA receptor antagonists was
voltage-dependent (Balazs et aI., 1990b).
One of the possible interpretations of these
unexpected observations was that 50 JiM KA not
only rescues cells, but is also responsible for bring-
ing about conditions causing cell death, which is
mediated through an EAA receptor that can be
blocked by NMDA antagonists. If so, the addition
of an NMDA agonist should counteract the
potentiation of the effect of KA by a competitive
NMDA antagonist. Indeed, quinolinic acid,
which is a very weak NMDA agonist in the cere-
bellum (Stone and Burton, 1988), counteracted in
a concentration-dependent manner the poten-
tiation ofKA effect by APV (Balazs et al., 1990b).
On the other hand, NMDA added together with
KA was never found to be toxic under our experi-
mental conditions. Thus, it seems that in the
presence ofKA an NMDA-like receptor becomes
manifested, which is activated by Glu in the culture
medium, and mediates cytotoxicity. Since at this
receptor NMDA is not an agonist, while NMDA
antagonists exert a potent and voltage-dependent
effect, this receptor seems to be distinct from the
conventional ionotropic receptor subtypes. A
receptor with similar properties has also been
described in cerebral cortical cultures on the basis
ofEAA-stimulated 45Ca 2+ influx (Frandsen et aI.,
1989). Furthermore, evidence consistent with the
existence ofNMDA receptor subtypes in different
parts of the brain has also been obtained recently
(Monaghan et aI., 1989); based on molecular bio-
logical information, it is more than likely that the
heterogeneity of ionotropic Glu receptors is much
Table 3.3. NMDA receptors are not involved in the
promotion of cell survival by kainic acid
Cell survival (as a percentage of LDH
activity in control cultures)
Kainate + +
MK-801 + +
% of control lOO±3 124±3" 84±2" 167±2a ,b
Granule cells were grown in K5 in the absence or the presence
of 5 JIM MK-801 and of 50 JIM kainate. At 8 DIV, cells were
harvested and the activity of lactate dehydrogenase, which
was proved to correlate with cell numbers, was determined
as a marker of cell survival. Activities per 35 mm dish were
expressed as a percentage of the mean value obtained in
the control (cells grown in K5 without any further additions;
activity 6,88 ± .19 Jimol lactate formed per hour; n = 6).
One-way ANOVA showed overall significance (p < .001).
" Conditions that were significantly different from K5.
b MK-801 significantly potentiated the effect of kainate
(Newman-Keuls test, p = .05).
Data from Balazs et ai., 1990a.
64 Robert Balazs, Nicola Hack, and Ole S. J9Irgensen

greater than indicated by the pharmacological this approach will not similarly facilitate a better
characterization (e.g., Bettler et at, 1990). understanding of afferent-mediated trophic in-
The initial effect of KA is, however, exerted via fluences. However, the hypothesis assigning a tro-
a conventional KA receptor. DNQX (6,7-dini- phic influence to alTerent inputs, mediated through
troquinoxaline-2,3-dione), which is a relatively the stimulation of postsynaptic EAA receptors,
selective KA/AMPA antagonist (Honore et at, awaits substantiation by in vivo studies.
1988), blocked in a concentration-dependent
manner the effect of KA on cell survival. Never- Maturational Stage Dependence
theless, the trophic effect of KA seems to be me- of Effects of Excitatory Amino Acids
diated indirectly: the KA rescue, as well as the
The maturational stage dependence of the re-
potentiation ofthis effect by NMDA antagonists,
sponse of cultured granule cells to NMDA was
are blocked by calcium antagonists, including the
indicated by the observation that the survival
DHP nifedipine (NF), which also counteract the
requirement for NMDA was manifested, as in the
trophic influence of high K +, but not that of
case of chronic depolarization, within the narrow
NMDA (Balazs et at, 1990b). Thus, the stimula-
timespan of 2 and 4 DIV (Balazs et at, 1988b).
tion ofthe KA receptor results, through increased
The question was not addressed in the studies
Na + conductance, in membrane depolarization,
described above whether or not the maturation
which activates VSCC, and it is the increased
of granule cells can reach a stage in culture when
Ca 2 + influx that serves as the intracellular signal
EAA become toxic rather than trophic factors.
for the trophic influence.
However, preliminary studies have indicated that
Finally, our recent investigations have shown
this· is the case: exposure of the cultures to a
that in addition to NMDA and KA, treatment
relatively high concentration of Glu (0.5-1 mM)
with QA or AMPA can also exert a trophic in-
at 2 DIV had no adverse effect, but Glu at much
fluence on cerebellar granule cells (Balazs et at,
lower concentrations killed the cells when added
1991). For some time we have overlooked this
to mature cultures at about 8 DIV (A. Resink and
effect, because it is relatively small (maximal
R. Balazs, in preparation). Furthermore, extensive
stimulation about 25%) and it is detectable only
investigations by Costa and coworkers have
at a very narrow range of concentration (0.5-2 JlM
clearly demonstrated that "mature" granule cells
for QA and 5-10 JlM AMPA). The pharmacology
do degenerate after Glu exposure in culture (e.g.,
of the effect is rather complex. QA/AMPA rescue
Manev et at, 1990). These observations on the
is blocked both by NF and by NMDA antago-
development with maturation of vulnerability to
nists; thus, it is mediated by Ca2+ influx both
Glu are similar to findings on nerve cells other
through VSCC activated by QA/AMP A-induced
than granule cells (e.g., Choi et at, 1987; Frandsen
membrane depolarization and through the open-
and Schousboe, 1987; Rothman, 1983).
ing of NMDA receptor-linked channels by Glu,
The maturational stage dependence is high-
which is known to be released from granule cells
lighted by changes in the responsiveness of granule
by QA/AMPA (Gallo et at, 1987b).
cells to NMDA in the cerebellum during develop-
ment. Detailed investigations of Garthwaite and
colleagues have shown that in cerebellar slices
Conclusions and Implications NMDA can induce granule cell degeneration, but
The observations described in this chapter have that the effect is age-dependent (e.g., Garthwaite
referred to investigations on cultured nerve cells. and Garthwaite, 1986a, b; Garthwaite et at, 1987).
In vitro studies have provided important experi- Combining the information on the toxic action
mental avenues for the elucidation of the role of of NMDA in vivo and in cerebellar slices with
target-derived factors on the survival and dif- the trophic action of NMDA in granule cell cul-
ferentiation of nerve cells (Thoenen, 1991; Varon tures, we propose the following scenario for EAA
et at, 1988) and there is no reason to suppose that receptor-mediated events in the developing cere-
3. Granule Cells and Excitatory Amino Acids
DEVELOPING
CEREBELLUM
Effect of
NMDA
a • • • •
• • • •
EGL
~ ~ ~
~ (g)(g)(g)
ML
a
P
+ • • • •
0 0 0 ® IGL
0 0 0 0
Figure 3.5. Scenario of excitatory amino acid receptor-
mediated events in the cerebellum. In the immature
cerebellum, differentiating granule cells (0), but not
the replicating or migrating cells (. or .), are
responsive to NMDA: cells, which have just completed
migration and receive the first innervation by mossy
fibers (heavily stippled symbols), are stabilized in their
survival by EAA receptor stimulation, whereas at a
bellum (Fig. 3.5): Replicating and migrating
granule cells are not responsive to NMDA, which
is toxic only to differentiating granule cells, al-
though those in the upper part of the IGL are not
adversely affected (Garthwaite and Garthwaite,
1986a). The latter cells have just completed migra-
tion, which takes about the same time as does the
development in culture of granule cell depend-
ence on either high K + or NMDA (see above).
According to our hypothesis, these trophic effects
in vitro mimic the influence mediated in vivo
through the first innervation that these cells
receive, primarily from the mossy fibers. The
short-term slice experiments of Garthwaite and
Garthwaite (1986a) cannot provide information
on whether mossy fibers exert a trophic influence
on this cohort of granule cells via postsynaptic
EAA receptors. The observations of Rakic and
Sidman (1973), however, are consistent with such
a role for mossy fibers. In a mutant mouse, the
"weaver" (wv), the genetic defect is primarily
manifested in the cerebellum after granule cell
65
MATURE
CEREBELLUM
Effect of
NMDA
ML
(g)(g)(g) P
a
000
o 0 0
0
0 IGL
o 0 0 0
further stage of maturation they become vulnerable
to NMDA (lightly stippled symbols). In the fully
mature cerebellum, the vulnerability of granule cells
to NMDA is low (0). EGL, external granular layer;
ML, molecular layer; P, Purkinje cell layer; IGL,
internal granular layer. Effect of NMDA: 0, limited;
+, survival promoting; -, toxic.
replication has been completed. Most ofthe post-
mitotic granule cells die soon after their genera-
tion, with the exception of those that are in an
ectopic position but are innervated by aberrant
mossy fibers.
The stage of maturation at which NMDA has
a trophic influence on granule cells is superseded
by a stage in which cells are vulnerable to NMDA
(Garthwaite and Garthwaite, 1986a). It seems
that both the trophic and the toxic effects of
NMDA are mediated through the same mech-
anism, that is, increased Ca 2 + influx (Balazs et aI.,
1988b; Balazset aI., 1992; Choi, 1987; Garthwaite
and Garthwaite, 1986a; Rothman and Olney,
1987). It is an intriguing but still unanswered
question as to what is the mechanism(s) that
underlies the maturational stage-dependent life-
or-death response of the cell to the same signal.
In this respect, differences in the effectiveness of
intracellular regulatory mechanisms, which serve
to maintain free Ca2 + levels within nontoxic limits,
deserve investigation.
66
NMDA TCP
c lH
Figure 3.6. Regional distribution of binding sites
associated with the NMDA receptor recognition site
(left half of brain) and the NMDA receptor-linked ion
channel (right half) in the rat brain. The receptor was
labeled with 200 nM eH]Glu in the presence of 1 11M
QA, whereas the ion channel on adjacent sections with
20 nM 3H-N-(1-[2thienyl]cyclohexyl)3,4-piperidine
eH-TCP). Note that throughout the brain there is
excellent correlation in the binding of ligands
Finally, with maturation completed, the re-
sponsiveness of granule cells to NMDA is greatly
reduced (Garthwaite et aI., 1987). It would deserve
investigating whether or not this is related to the
unusual kinetic properties of ligand binding to
the NMDA receptor-linked ion channel in the
adult cerebellum (Maragos et al., 1988) (Fig. 3.6).
The Survival-Promoting Influence
of Excitatory Amino Acids, As
Their Toxic Effect, Is Not Restricted
to the Cerebellar Granule Cells
It has long been known that EAA can exert toxic
effects on nerve cells throughout the brain (e.g.,
Meldrum and Garthwaite, 1990). Results are forth-
Robert Balazs, Nicola Hack, and Ole S. Jj6rgensen
detecting either the recognition site or the ion channel
of the NMDA receptor in different structures, except
in the adult cerebellum, where TCP binding was
negligible. However, there is TCP binding in the
cerebellum, but the affinity is lower than in other brain
parts. All sections are from the left hemisphere, but
the TCP autoradiograms were reversed. (Reprinted
with permission from Maragos et aI., 1988.)
coming that indicate that EAA can also promote
the survival of nerve cells other than the cerebellar
granule cells. Thus, Brenneman et al. (1990) have
reported that NMDA receptor blockade can,
depending on concentration, either promote or
suppress neuronal survival in dissociated spinal
cord cultures. Since these cultures contain a
mixed population of nerve cells, it is possible that
the trophic or toxic effects reflect distinct matura-
tional stages of the affected cells. Furthermore,
Ruijter and Baker (1990) have described condi-
tions that are characterized by NMDA receptor
stimulation exerting either a trophic or a toxic
effect on nerve cells in organ cultures of cerebral
cortex.
3. Granule Cells and Excitatory Amino Acids 67

Trophiic Effects of Excitatory period of development within the kitten visual

Amino Acids Beyond Mere cortex in response to monocular deprivation
Survival of Nerve Cells (Kleinschmidt et aI., 1987; Rauschecker and Hahn,
1987; for review see Collingridge and Singer, 1990)
New observations indicate that EAA can exert
whereas, in the optic tectum of surgically pro-
trophic influences not only in terms of cell sur-
duced three-eyed tadpoles, APV caused desegrega-
vival, but also of advancement of maturation and
tion of the terminals of the retinal ganglion cells
of neurite outgrowth, also affecting nerve cells
(Cline et aI., 1987).
other than the cerebellar granule cells in the de-
The effect of NMDA on nerve cells is known
veloping brain (see various papers in connection
to be voltage-dependent (e.g., Mayer and West-
with both the trophic effe~t of EAA and their
brook, 1987). This property seems to be critical
involvement in plastic changes in the nervous
concerning the role of NMDA receptors both in
system in Balazs, 1990). For example, Pearce et ai.
the plastic changes and in the trophic effects of
(1987) have noted that NMDA receptor stimula-
EAA within the nervous system. This implies that
tion promotes neurite emission from cultured
Ca 2 + conductance through the NMDA receptor-
cerebellar granule neurones, whereas Moran and
linked ion channel, which is instrumental in the
Patel (1989) described an advancement of the
experience-dependent modulation of synaptic
biochemical maturation ofthese cells. Moreover,
connections, is subject to regulation by coopera-
it has also been noted that the trophic effect is
tive interaction with other afferent inputs. Inputs
cell type-specific: in subcortical cultures NMDA
may include afferents operating with EAA, which
appeared to promote the maturation of GABA-
also .stimulate postsynaptic receptors other than
and Glu-ergic, but not the cholinergic cells (Patel
the NMDA-preferring subtype.
et aI., 1990).
The involvement of EAA receptors (including
Glu can also influence the outgrowth of neurites
metabotropic receptors according to recent ob-
of hippocampal pyramidal cells, differentially
servations) in lasting alterations of synaptic trans-
affecting dendrites and axons in a dose-dependent
mission in adult brain structures, exemplified by
manner, and thus modulating the development of
long-term potentiation in the hippocampus or
neuronal shape (Mattson et aI., 1988). However,
the cerebral cortex and long-term depression in
it should be noted that in contrast to cerebellar
the cerebellum, is well documented (for reviews
granule cells, hippocampal neurones respond to
see Collingridge and Singer, 1990; Ito, 1989).
Glu by a reduction of dendritic growth rate and,
Furthermore, NMDA receptors, in particular,
although Ca 2 + seems to serve as the second mes-
seem to play an important role in certain forms
senger for both cell types, the receptors involved
of learning in both immature and adult animals
are the KA/QA preferring subtype. Morphologi-
(Lincoln et aI., 1988; Morris et aI., 1986).
cal maturation of cerebral cortical neurones has
also been claimed to be advanced in culture after Mechanisms Underlying Trophic
exposure to low concentrations of Glu (Aruffo Irifluences of Excitatory Amino Acid
et aI., 1987). on Developing Nerve Cells
There is evidence indicating that the toxic effect
Plastic Changes Induced
of EAA is mediated through an increase in the
by Excitatory Amino Acids
entry of extracellular calcium into granule cells
It seems that processes mediated by EAA are also (Garthwaite and Garthwaite, 1986b). Our obser-
involved in plastic changes in both the developing vations show that Ca 2 + influx also plays a critical
and the adult central nervous system, and that role in the trophic effects' of EAA: NMDA rescue
NMDA receptors again playa critical (although is inhibited by blocking the NMDA receptor-
presumably not exclusive) role. It has been shown linked ion channel, and thus the stimulated cal-
that during development these receptors influ- cium conductance (Balazs et aI., 1989), whereas
ence the fine-tuning of synaptic positions. Thus, the positive influence of non-NMDA receptor
NMDA receptor blockade prevents the ocular agonists (or high K +) on cell survival is com-
dominance shift normally seen during a restricted promised by reducing the availability of extra-
68
cellular calcium through the depolarization-
activated VSCC (Balazs et al., 1990a, b, 1992;
Gallo et aI., 1987a). It seems, furthermore, that
the DHP-sensitive L-type of VSCC plays an im-
portant role in cell rescue by high K + or non-
NMDA receptor agonists, although DHP block-
ade of these channels inhibits only a fraction of
the K + depolarization-induced 45Ca 2 + uptake
into granule cells (Carboni and Wojcik, 1988;
Kingsbury and Balazs, 1987). These findings imply
that concerning the trophic influence, the site of
Ca 2 + entry is also relevant. In this respect, it
should be noted that the cell survival promoting
effect of NMDA was resistant to NF, so that
Ca2+ entering not only through the DHP-sen-
sitive channels, but also through the NMDA
receptor-linked channels, can be effective.
It is now well established that exposure to EAA
or high K + elicits in granule cells, as in other
nerve cell types, an increase in the concentration
of free cytoplasmic Ca 2 +, which may involve,
however, depending on the agonist, quite complex
interactions (Courtney et aI., 1990). Furthermore,
our recent observations have extended previous
findings (Gallo et aI., 1987a) indicating that after
the elevation of [Ca2+L calmodulin-mediated
reactions are critically involved in the cascade,
leading ultimately to cell survival (Balazs et aI.,
1992). Ongoing investigations suggest that the
next step involves protein phosphorylation me-
diated by the Ca 2 + -CaM-dependent protein
kinase II (CaM kinase II) (Balazs et aI., 1992). It
should be noted here that the toxic effect of EAA
on granule cells also seems to involve protein
phosphorylation, but in this instance the mediator
is protein kinase C (see, e.g., Manev et aI., 1990).
CaM kinase has certain properties that can
facilitate its role in mediating the trophic effect of
EAA. The activation by Ca 2 + results in the auto-
phosporylation of the kinase, which leads to the
release of the enzyme from the membrane-cyto-
skeleton complex and renders CaM kinase II
independent from Ca 2 + (Miller and Kennedy,
1986; Saitoh and Schwartz, 1985). As a conse-
quence, the kinase may reach sites where protein
phosphorylation may elicit long-term effects and
the enzyme can even function under conditions
when regulatory mechanisms keep the overall
free Ca2+ levels low. Furthermore, it seems that
prolonged increases in [Ca2+1 are required in
order to convert a significant proportion of the
Robert Balazs, Nicola Hack, and Ole S. J~rgensen
kinase into the phosphorylated form: this may
occur after l~ng or repeated bursts of electrical
activity simulated by the experimental conditions
in our studies in which cells are chronically ex-
posed to high K + or EAA.
Treatment of diverse cell types with agents
influencing growth and differentiation results in
the rapid and transient expression of a set of early
response genes, which include the proto-oncogene
c-fos (for review see Morgan and Curran, 1991).
c-fos is coding for a nuclear protein that, in com-
bination with products of other early response
genes such as lun, is involved in the regulation
of gene expression. Glutamate or NMDA was
found to induce the expression of c-fos in granule
cell cultures (Didier et aI., 1989; Szekely et aI.,
1989). However, at the present time we do not yet
know whether the EAA-induced expression of
c-fos is part of the mechanism underlying the
trophic effect or is an epiphenomenon.
Finally, instead of a summary, the proposition
is put forward that processes involving EAA-
mediated transmission may playa role, besides
triggering plastic changes in the central nervous
system throughout life, in underpinning the sur-
vival not only of the cerebellar granule cells but
also of other types of nerve cells during critical
periods in their development. One of the charac-
teristic features ofthe developing nervous system
is an overproduction of nerve cells and their pro-
jections including synaptic contacts, followed by
a regression of the redundant elements. For in-
stance, pyramidal cells ofthe cerebral cortex make
abundant transient projections during develop-
ment, even to areas (that may be quite distant)
that they do not innervate in the adult (see, e.g.,
Cowan et aI., 1984). As it is believed that many
of the corticofugal projections are glutamatergic
(Monaghan et aI., 1989), such redundant projec-
tions may fulfill a role by providing, at the right
time, a trophic influence ensuring the survival of
their transynaptic partners.
Acknowledgments. R.B. and N.H. acknowledge
the support provided by ZWO and the Van den
Houten Foundation. O.S.l. received support for
this work from the Danish Medical Research
Council. Weare indebted to Michael Corner for
critically reading the manuscript and to Olga
Pach for typing it.
3. Granule Cells and Excitatory Amino Acids
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Wong, E.H.F., Kemp, J.A., Priestley, T., Knight, A.R.,
Woodruff, G.N., and Iversen, L.L. (1986): The anti-
convulsant MK-801 is a potent N-methyl-o-aspar-
tate antagonist. Proc. Natl. Acad. Sci. USA, 83,
7104-7108.
Wong, E.H.F., Knight, A.R., and Woodruff, G.N.
(1988): [3H]MK-801Iabels a site on the N-methyl-
o-aspartate receptor channel complex in rat brain
membranes. J. Neurochem., 50, 274-281.
4
Microtubule-Associated Proteins in
Cerebellar Morphogenesis
Andrew Matus, Richard P. Tucker, and Christopher Viereck
Ramon y Cajal's Description
of Neurogenesis in the Cerebellum
In his studies of the cerebellum, Ramon y Cajal
(1888, 1960) noted, with characteristic clarity, the
major features of morphogenesis in the various
cell types. Many of his observations are now so
familiar as to be commonplace, whereas others
are less frequently cited, perhaps because they
deal with cellular phenomena whose regulation
is still not properly understood. For example,
Ramon y Cajal was the first to report that the
growth ofaxons and dendrites involves several
distinct steps. He observed that the neuroblast
first extends short processes of rather irregular
outline, as, for example, the granule cell does
during its initial horizontal, bipolar phase. Only
later do these initial protrusions narrow and
lengthen to form a proper "axis cylinder." Den-
drites appear later still. Thus, Purkinje cells have
already produced extensive, well formed axons
long before dendrites appear. At this early stage
the Purkinje cell body is decorated with multiple
short protoplasmic extensions that are subse-
quently resorbed to be replaced by the growth of
the mature dendritic tree. Neurogenesis thus in-
volves an initial outgrowth of short, irregular
processes followed by the formation of a distinct
axon and finally by the gradual development of
the dendrites.
A century after these events were first described
there are still important unresolved questions
about them. The most fundamental of these relate
to the cellular mechanisms that regulate these
morphogenetic events. What promotes the out-
72
growth of a neuritic process? What determines
whether a process becomes a dendrite or an axon?
Ramon y Cajal invariably refers to an "axis cylin-
der," rather than an axon, to distinguish a mature
process with a smooth outline and regular caliber
from the initial protoplasmic extension, which is
highly irregular in outline and diameter. How is
this change from one form to another achieved,
and what regulates the timing ofthis event? Recent
work has implicated the cytoskeleton in these
events, especially the microtubule network. By
studying the expression and cellular localization
of microtubular proteins during nervous system
development, the molecular events that underlie
axon and dendrite growth and differentiation are
beginning to emerge.
The Putative Role of the
Cytoskeleton in Neurogenesis
The silver stain revealed to the pioneer light micro-
scopists a feature of the internal organization of
the neuron that we now know is crucially in-
volved in neurogenesis, the neurofibrillar network.
Ramon y Cajal described its appearance in the
developing Purkinje cell in detail and was aware
of its continuity from dendrites through the cell
body into the axon. He observed that when the
neurofibrils enter the axon they "are concentrated
and welded into a compact and intensely stained
fibre" (Ramon y Cajal, 1960).
The neurofibrillar network stained in Ramon
y Cajal's preparations is now known to comprise
neurofilaments, the intermediate filaments of
4. Microtubule-Associated Proteins in Cerebellar Morphogenesis
neurons. Ramon y Cajal does not seem to have
entertained any idea of this neurofibrillar network
performing a cytoskeletal function, but he made
an observation that with hindsight strikes one as
extraordinarily prescient. He described distor-
tions ofthe neurofibrillar reticulum ofthe Purkinje
cell in young mammals subjected to cold, and
showed that the same phenomenon could be in-
duced experimentally in the embryonic avian cere-
bellum by chilling eggs before fixation. Although
unable to observe them directly, Ramon y Cajal
had made the first demonstration of the role of
microtubules within neurons. Microtubules are
known to disintegrate when exposed to cold, and
it has recently been reported that the microtubules
found within the dendrites of developing Purkinje
cells are particularly susceptible to low tempera-
ture-induced depolymerization (Faivre et aI.,
1985). We can now speculate that the distortions
observed by Ramon y Cajal were the result· of
altered cellular morphology due to microtubule
disassembly, or were perhaps a reflection of the
disruption of neurofilament-microtubule cross-
bridges. Many decades after Ramon y Cajal's
observations, it was demonstrated that conditions
that induce microtubule disassembly, like cold
and specific drug treatments, lead to the inhibition
of neurite outgrowth in primary cell cultures and
the retraction of neurites that had already formed
(Black and Greene, 1982; Daniels, 1972; Seeds
et aI., 1970; Yamada et aI., 1970). These experi-
ments demonstrated the importance of micro-
tubules during the initial phases of neuronal
morphogenesis.
Microtubules also contribute to the develop-
ment of the distinct morphologies ofaxons and
dendrites. When microtubules are depolymerized
by drugs (Bray et aI., 1978; Matus et aI., 1986) or
other treatments (Anglister et aI., 1982), the neu-
ronal processes not only shrink in length, but they
also lose their cylindrical form: the outline be-
comes irregular and lateral filopodia appear. In
many ways this conversion resembles a reversal
of the transition from initial outgrowth to "axis
cylinder" that Ramon y Cajal remarked on. These
observations indicate that the micro tubules pro-
vide the basis ofthe structural support for neurite
elongation, as well as the constraint that holds in
check the tendency to lateral spreading in the
neuritic cytoplasm. The cold-sensitive elements
73
indirectly observed by Ramon y Cajal, now re-
vealed to be microtubules, thus appear to be
essential determinants of the neuritic axis.
The Putative Role of
Microtubule-Associated Proteins
in Axon and Dendrite Growth
The above considerations lead readily to the idea
that whatever determines the state of assembly of
microtubules inside developing neurons must play
an important part in determining neuronal mor-
phology. Recently, much attention has focused
on the microtubule-associated proteins (MAPs)
because several of these ha:ve been shown to lower
the critical concentration of tubulin needed for
microtubule assembly in vitro (Cleveland et aI.,
1977; Murphy and Borisy; see review by Olmsted,
1986). Molecules such as MAP2 and tau, as pro-
moters of microtubule assembly in dendrites and
axons, are probably of great importance as stabi-
lizers of both longitudinal and cross-sectional
axis. It follows that any variation in their effec-
tiveness as promoters of tubulin polymerization
could have a significant effect on neuronal mor-
phology. Several findings speak in favor of such
modulatory changes in MAP properties. First, it
is known that MAP2 and tau from developing
brain are less effective in promoting tubulin poly-
merization than those from adult brain (Francon
et aI., 1982; Mareck et aI., 1980). This correlates
with the lesser stability of micro tubules in den-
drites of Purkinje cells in developing cerebellum
compared to those of adult cerebellum (Faivre
etaI., 1985). Second, the efficacy of MAP2 and
tau in promoting tubulin polymerization is pro-
foundly affected by phosphorylation, and both
molecules are known to be highly phosphorylated
in vivo (Murthy and Flavin, 1983; Tsuyama et aI.,
1987).
There is now a considerable body of additional
evidence indicating that MAPs are involved in
neuronal morphogenesis. One of the most strik-
ing findings is the temporal correlation between
the expression of different juvenile and adult
forms of neuronal MAPs and brain maturation
(Matus, 1988). For example, in the developing
brain MAP2 is present as a high-molecular weight
form, MAP2b, and a low molecular weight form,
74 Andrew Matus, Richard P. Tucker, and Christopher Viereck
MAP2c. Each form is encoded by a separate
mRNA derived from the single MAP2 gene
(Garner and Matus, 1988). In the developing brain
both MAP2b and MAP2c, with their respective
mRNAs, are present, but in the adult brain MAP2c
and its mRNA are reduced to trace levels (Garner
and Matus, 1988; Riederer and Matus, 1985). In
rat brain the disappearance of MAP2c, as well as
various other changes in MAP abundance and
form, occur between post-natal days 10 and 20.
It is during this period that axon and dendrite
growth come to an end and mature neuronal
morphology is attained, suggesting that expres-
sion of the "early" MAPs, such as MAP2c, is
correlated with neurite growth, whereas expres-
sion of "late" MAPs, such as the high molecular
weight MAP2a, is correlated with neurite stabili-
zation (Matus, 1988).
A third feature of the neuronal MAPs that
implicates them in morphogenesis is their associa-
tion with particular aspects of neuronal structure.
The most striking case of this is the polarization
of high molecular weight MAP2 and tau in neurons
throughout the brain, with MAP2 being concen-
trated in dendrites whereas tau is localized in
axons (Binder et aI., 1985; Caceres et aI., 1984; De
Camilli et aI., 1984; Matus et aI., 1981; Tucker
et aI., 1988a).
Figure 4.1. The cerebellar cortex
of an adult Xenopus laevis
stained with monoclonal anti-
bodies against MAP2 (A) and
tau (B). MAP2 is dendrite-
specific and tau is enriched in
axons in the amphibian nervous
system, just as they are in the
mammalian brain. gl, granule
cell layer; ml, molecular layer;
P, Purkinje cell body.
4. Microtubule-Associated Proteins in Cerebellar Morphogenesis
Evolutionary Conservation
of MAP Properties
That these associations of timing and location are
no mere coincidence is shown by recent studies
of MAPs in other species. In the amphibian
Xenopus laevis the disappearance of MAP2c and
the appearance of MAP2a coincide with neuronal
maturation, that is, during metamorphosis, just
as it does in the rat (Viereck et ai., 1988). The
dendritic localization of high molecular weight
MAP2 and the axonal localization of tau have
been demonstrated in birds and amphibia in ad-
dition to mammals (Tucker et ai., 1988a; Viereck
et ai., 1988). The amphibian cerebellum has a
more primitive cytological structure than that of
birds and mammals: its granule cell dendrites do
not form postsynaptic "claws," the Purkinje cell
bodies do not form a monolayer, and their den-
drites are less well organized than the stereotype
of birds and mammals (Hillman, 1969; Sotelo,
1969). Despite the lack of these organizational
features, high molecular weight MAP2 in Xenopus
laevis is exclusively dendritic, and tau is concen-
trated within axons, exactly as they are in mam-
mals (Fig. 4.1).
We have also found that the molecular forms
of each of the known neuronal MAPs are closely
similar in mammals, birds, and amphibia (Tucker
et ai., 1988a, b; Viereck et ai., 1988). For example,
MAP2 is represented in all cases by a high mole-
cular weight doublet of around 200,000 Da and
a low molecular weight form (approximately
70,000 Da) that is found primarily in the develop-
ing nervous system. Thus, three of the fundamen-
tal properties of neuronal MAPs, their molecular
form, cellular locati~n, and developmental regu-
lation, have been conserved throughout vertebrate
evolution, indicating that these proteins play
fundamental roles in neuronal morphogenesis.
MAP Expression in the Cerebellum
The essential features of MAP expression in the
cerebellum have been described in a series of
publications from our laboratory (e.g., Bernhardt
and Matus, 1982, 1984; Bernhardt et ai., 1985).
The first indication that high molecular weight
MAP2 was selectively associated with dendrites
was found in the cerebellar cortex, where strong
75
MAP1 MAP2
Figure 4.2. Adjacent sections through the adult rat
cerebellum were stained with diluted hybridoma super-
natants reactive with MAP! (A) or MAP2 (B). MAP!
is concentrated within the dendrites of Purkinje cells
(P) and Golgi cells (G), whereas MAP2 is most abun-
dant within the cell bodies and dendrites of granule
cells (gl), as well as within varicose processes coursing
vertically through the molecular layer (arrowheads).
Note the absence of anti-MAP2 staining in the white
matter (wm).
anti-MAP2 staining of Purkinje cell dendrites
contrasted with the absence of staining of granule
cell axons (Matus et ai., 1981). Later immuno-
histochemical studies showed that other MAPs
have different patterns of expression in cerebellar
cells. Thus, MAP3, a 180,000-Da protein, is found
in astroglial cells and neurofilament-rich axons
such as basket cell endings (Huber et ai., 1985).
In contrast, MAPS, a 320,000-Da "early" MAP,
appears exclusively in neurons where it is present
in both axons and dendrites (Riederer et ai., 1986).
An interesting contrast exists between MAP1
and MAP2 expression in granule and Purkinje
cells. MAP1 is highly concentrated in Purkinje
cells, where it is present in both axons and den-
drites, but is much less abundant in granule cells
(Huber and Matus, 1984). Exactly the opposite is
the case for MAP2, which is much more abundant
in granule cells than Purkinje cells. This is most
readily seen in sections stained with diluted hy-
bridoma supernatants (Fig. 4.2).
76 Andrew Matus, Richard P. Tucker, and Christopher Viereck

antibodies do not (Fig. 4.4). High molecular weight

Figure 4.3. MAP2 is abundant in Purkinje cells in the
developing (embryonic day 12) quail cerebellum (A),
but cannot be detected in Purkinje cell bodies or their
dendrites in the mature (posthatching day 9) cerebellum
(B). The staining within the remainder of the mature
cerebellum is indistinguishable from the rat. gl, granule
cell layer; ml, molecular layer; P, Purkinje cell bodies;
wm, white matter.
MAP2 appears soon after MAP2c, at a time that
corresponds with the formation of dendrites
(Tucker et aI., 1988a, b). MAP1 levels, on the
other hand, remain low until the second week of
life and then slowly increase in step with neuronal
maturation (Riederer and Matus, 1985). The timing
of expression of MAP1 thus shows a much better
correlation with stabilization of neuronal struc-
ture than does that of MAP2.
In developing Purkinje cells of the rat, anti-
MAP2 staining is strong from birth onward, with
particularly high concentrations found in actively
growing domains of the dendritic tree (Fig. 4.5).
Staining of growing Purkinje cell dendrites by
antitubulin is far weaker. This could be because
tubulin epitopes in the developing dendrites are
masked. However, a simpler explanation is that
MAP2 concentrations rise faster than those of

In a recent study of the quail cerebellum, we

found that MAP2 is entirely absent from mature
Purkinje cell dendrites (Fig. 4.3). Three different
monoclonal antibodies as well as a polyclonal
antiserum failed to stain the quail Purkinje cell
dendrites, although neighboring interneurons
(granule, basket, Golgi, and stellate cells) were all
strongly stained, just as they are in the rat. It
seems that MAP2 is not essential for the mainte-
nance of an extensive dendritic arborization, nor
for the correct organization of microtubules within
these mature processes. MAP2 is, however, ex-
pressed in Purkinje cell dendrites-in the developing
quail cerebellum (Fig. 4.3). This suggests that the
major contribution of MAP2 to neuronal mor-
phology may be to dendritic development rather
than the maintenance of mature form.
A variety of observations support this conclu-
sion. First, there is the contrast between the early
appearance of MAP2 and the late appearance of Figure 4.4. Monoclonal antibodies that recognize the
low molecular weight form of MAP2 in addition to
MAPl. In the rat cerebellum, MAP2 appears as
high molecular weight MAP2 (monoclonal antibody
soon as neurons have finished their final mitosis C) stain the external granule layer of the postnatal day
and begin to differentiate. The form of MAP2 that 7 rat cerebellum (A). In contrast, antibodies specific for
appears first is MAP2c, since antibodies that re- the high molecular weight form of MAP2 (monoclonal
cognize this form ofMAP2 on Western blots will antibody AP14) stain only Purkinje cells and their
stain the external granular layer of the cerebellum, dendrites. P, Purkinje cells; ml, molecular layer; egl,
whereas high molecular weight MAP2-specific external granule layer.
4. Microtubule-Associated Proteins in Cerebellar Morphogenesis
Figure 4.5. The developing rat cerebellum immuno-
peroxidase stained with a polyelonal antiserum against
MAP2. Immunochemical analysis showed that this
antiserum detects only high molecular weight MAP2.
In these sections, taken from cerebella of rats 2-days-
old (B), 7-days-old (C), 12-days-old (A), and 21-days-
old (D), anti-MAP2 staining is very strong in the den-
drites of Purkinje cells bur is absent from surrounding
granule cell axons in the molecular layer (ml in A).
Within Purkinje cells the anti-MAP2 staining appears
weaker in cell bodies (P 1- P 4 in A; asterisks in B, C, and
D) than in more distal areas ofthe dendritic tree (arrow-
heads in C and D). A and E show cerebellar granular
layer in which granule cells and their processes (arrow-
heads in E) are particularly strongly stained. Bars = A,
30 J1m; D, 20 J1m; E, 15 J1m. egl, external germinal layer;
gel, granule cell layer.
tubulin in developing Purkinje cell dendrites
(Bernhardt and Matus, 1982). This observation
is supported by a study of MAP2 and tubulin in
the dendrites of hippocampal neurons developing
in culture where MAP2 is not restricted to micro-
tubules, but is present throughout the dendritic
77
cytoplasm (Matus et aI., 1986). This would imply
that the role of MAP2 in dendritic development
goes beyond merely promoting microtubule for-
mation.
A similar argument can be made for another
"early" MAP, MAP5. This protein is the earliest
major MAP to be expressed in neurons: it is
already present throughout the external granular
layer (Riederer et aI., 1986), and it appears in the
axons of both retinal ganglion cells and motor
neurons as soon as they form (Tucker and Matus,
1987,1988; Tucker et ai., 1988a). In vitro studies
with PC12 cells also support a role for MAP5 in
neurite outgrowth. When PC12 cells are induced
to grow processes by exposure to nerve growth
factor, MAP5 levels increase more than 10-fold
(Brugg and Matus, 1988). However, this MAP5
is not colocalized with microtubules until several
days after neurite growth. It may therefore initially
have some function other than or in addition to
its involvement with microtubules.
MAP2 Expression
in Cerebellum-Derived Cultures
Culture systems offer the opportunity to study
morphogenetic events with surrounding tissue
influences removed. We have examined two varie-
ties that provide different circumstances: tissue
slice cultures (Giihwiler, 1981), where the histo-
typic organization is initially present but where
neurons are substantially denervated compared
to intact brain, and dispersed cell cultures, where
individual neurons are completely removed from
histological influences and are totally denervated.
Slice cultures were made from sagittal slices of
19-day rat embryo cerebellum and cultured for
up to 3 weeks. In these cultures the Purkinje cells
are contacted by far fewer granule cell axons, both
because fat fewer granule cells complete their
migration and differentiation under these condi-
tions, and also because much of the Purkinje cell
innervation in the intact cerebellum originates
from granule cells outside their own sagittal plane.
In addition, a cerebellar slice lacks the climbing
fiber input from the inferior olive.
Under these circumstances, Purkinje cells sur-
vive in reasonable numbers and their placement
within the tissue is close to normal, with the cell
bodies roughly but not exactly in line (Fig. 4.6A).
78 Andrew Matus, Richard P. Tucker, and Christopher Viereck
As in whole cerebellum, the granule cells migrate
to form an inner granular layer but this does not
have the strictly sub ganglionic nature found in
intact brain and neither are all the granule cells
clustered into groups. Instead, the granule cells
occupy a broad area on both sides of the line of
Purkinje cell bodies (Fig. 4.6A) and the cell bodies
are scattered rather than clustered. The effect of
this loss of innervation and his to typic abnormality
has interesting consequences for neuronal mor-
phogenesis and for the organization of the cyto-
skeleton, at least as far as the distribution of
MAP2 is concerned. In slice cultures the Purkinje
cell dendrites are stunted. Primary dendrites are
formed but give rise to ~ather few secondary
branches, which are abnormally short and termi-
Figure 4.6. Slice cultures of rat cere-
bellum showing the organization of
MAP2. The same antiserum was used
as in Fig. 4.5 but with immunofluo-
rescence detection. After 10 days in
culture these cerebellar slices have
attained the major histotypic features
of the cerebellar cortex, with Purkinje
cell bodies (P) mostly aligned in a
planar array (A) and unipolar
dendrites having formed (d). Granule
neurons (g) are sparse and scattered.
Bars = A and B, 30 J.lm; C, 20 11m.
nate abruptly (Fig. 4.6A, B). The dendrites stain
strongly with anti-MAP2, indicating that the
molecule is abundantly produced despite the lack
of innervation and hypotrophy. The cell body is
also full of MAP2 in contrast to Purkinje cells in
intact brain where there is a tendency for anti-
MAP2 staining to be more concentrated in the
distal primary and secondary dendrites (Fig. 4.5).
The morphology of granule cells in slice cultures
is also abnormal. Although readily recognizable
by their typical small, spherical cell body (Fig. 4.6),
these granule cells do not form the short, claw-
shaped dendrites characteristic of these cells in
intact cerebellum, but instead form longer MAP2-
rich processes (see Figs. 4.6B, D, in particular).
Despite these abnormalities, the anti-MAP2 stain-
4. Microtubule-Associated Proteins in Cerebellar Morphogenesis
Figure 4.7. Cerebellar granule cells in dispersed cell
culture showing the distribution ofMAP2 detected by
immunofluorescence with the same polyc\onal antibody
as in Figs. 4.5 and 4.6. A, Immunofluorescence image
ing in these slice cultures resembles that of intact
brain in two important respects: it is still limited
to neurons and appears only in dendrites. This
indicates that whatever the nature of the mech-
anism that targets MAP2 to dendrites, it is not
sensitive to loss of innervation even when this has
significant consequences for the development of
neuronal morphology.
An interesting extension to these observations
can be drawn from dispersed cell cultures from the
cerebellum (Fig. 4.7). These consist overwhelm-
ingly of granule cells and do not contain Purkinje
cells (Burgoyne and Cambray-Deakin, 1988). As
in the slice cultures, the granule cells develop cell
bodies of the characteristic size and spherical
form even when denied contact with other cells
(Fig. 4.7) (see Alaimo-Beuret and Matus, 1985,
and Burgoyne and Cambray-Deakin, 1988). How-
ever, their processes are abnormal in an interesting
way. These have a varicose initial portion followed
by a long, thin unbranched process (Fig. 4.7B).
This distal domain ofthe process closely resembles
a granule cell axon even to the extent that it
frequently bifurcates at some distance from the
cell body (Fig. 4.7B) in a manner reminiscent of
a parallel fiber. The axonlike nature of this distal
process is further suggested by the observation
that it is always devoid of MAP2 (Fig. 4.7A).
Instead, MAP2 is limited to the initial varicose
portion o( the process. This partitioning of MAP2
79
with anti-MAP2. n, neuronal cell bodies. Arrowheads
mark the ends of initial MAP2-rich segment of the
processes seen by phase contrast in B. Bar = 20 /lm.
within the processes ofthese isolated granule cells
is independent of the distribution oftubulin, anti-
tubulin staining being present throughout the
length of the entire process (Alaimo-Beuret and
Matus, 1985).
These observations demonstrate that the parti-
tioning of MAP2 into separate microdomains of
the neuronal cytoplasm can occur within a single
process. Interestingly, MAP2 is at first present
throughout emerging processes of cultured granule
cells; only later does it become restricted to the
initial segment (Alaimo-Beuret and Matus, 1985;
see also Burgoyne and Cam bray-Deakin, 1988).
Since these cells develop in isolation, it appears
that the partitioning of MAP2 in the cytoplasm
is under the control of an endogenous genetically
determined program that is independent of the
actual morphology attained by the cell. Thus,
although MAP2 may well contribute to the struc-
ture of the dendrites in which it normally occurs,
it alone is not sufficient to induce a separate
dendritic process.
Tubulin Isotypes in the Cerebellum
Recently, the cerebellum has been used as a model
system for describing the distribution of beta-
tubulin isotypes. Five distinct beta-tubulin forms
are expressed in mouse brain; they can be dis-
80 Andrew Matus, Richard P. Tucker, and Christopher Viereck
MAP1
Figure 4.8. Adjacent sagittal sections through the cere-
bellum of a postnatal day 12 rat were hybridized with
cDNAs specific for beta-tubulin (A), high molecular
weight MAP2 (B), MAPI (C), and MAP5 (D) tran-
scripts. Note the differences in the relative amount of
tinguished from one another by variable amino
acid sequences at the carboxy terminus. Since
many MAPs are known to bind to tubulin near
the carboxy terminus, Burgoyne and colleagues
(1988) undertook a comparative immunohisto-
chemical study of the distribution of MAPs and
beta-tubulin forms in the cerebellum. Although
they demonstrated that these forms display cellu-
lar specificity (e.g., isotype 5 is concentrated in
astrocytes, and isotype 6 is found exclusively in
neurons), there was little evidence of compart-
mentalization ofisotypes within any given cell or
colocalization of any isotype with the major brain
MAPs. This observation is in agreement with the
work of Lewis et al. (1987) and Lopota and
Cleveland (1987), who used tissue culture cells to
demonstrate that beta-tubulin isotypes do not
sort into distinct microtubules. One must con-
clude from these studies that the compartmentali-
zation of MAPs within neurons is not regulated
by the distribution of beta-tubulin isotypes.
MAP2
MAP5
hybridization within the external granular layer (egl)
and granular layer, the white matter (wm) and gray
matter, and the cerebellum and inferior colliculus (ic).
n, deep cerebellar nucleus.
These observations also emphasize the impor-
tance of MAPs in generating functional hetero-
geneity within the neuronal microtubule network.
Although tubulin may contribute to microtubule
function in a way that is not yet understood, the
major role of this protein appears to act as the
backbone of a filament system that is regulated
and given diverse functions by MAPs.
MAP mRNA in the
Developing Cerebellum
We have recently completed an in situ hybridiza-
tion study of the location of MAP mRNAs in the
developing rat brain. The results clearly show
that the developmental regulation of MAP ex-
pression is taking place at the transcriptional
level. For example, comparing the density of the
cDNA hybridization signal between the external
and internal granular layers shows that the mRNA
4. Microtubule-Associated Proteins in Cerebellar Morphogenesis
for the "early" MAPS is more abundant in the
zone of granule cell proliferation and differentia-
tion, whereas the mRNAs for both high molecular
weight MAP2 and MAP 1 are more abundant in
the internal granular layer, where more mature
granule cells are found (Fig. 4.8). Another obser-
vation made within the cerebellum concerns the
relative abundance of hybridization within gray
and white matter. The MAP2 eDNA probe does
not hybridize significantly within white matter,
whereas the MAPI eDNA probe does. This indi-
cates that MAP 1 is expressed in both neurons
and oligo den droglial cells, whereas MAP2 is
neuron-specific.
The most striking feature to emerge from the
in situ hybridization studies is the clear indication
that the mRNA for MAP2 is found in dendrites
(Garner et aI., 1988). This has been most clearly
demonstrated in the developing cerebral cortex,
where dendrites are bundled and have a distinctive
radial orientation. The autoradiographic signal
with the MAP2 eDNA probe is unequivocally
associated along the length of these processes,
and not in the pyramidal cell bodies, where the
hybridization with a beta-tubulin eDNA probe
produces a punctate pattern. MAP2 mRNA is
apparently exported so that protein synthesis can
occur in the dendrite, whereas for tubulin the
protein is synthesized in the cell body and then
transported. The presence of MAP2 mRNA in
dendrites has interesting implications for the po-
tential -regulation of MAP2 synthesis: it seems
possible that the rate of synthesis may be modu-
lated immediately and locally within the process,
perhaps via cues provided from the afferent path-
ways.
Implication of MAP
Chemical Structure
The primary sequences of tau and MAP2 have
now been determined (Goedert et aI., 1988; Lee
et aI., 1988; Lewis et aI., 1988), and it transpires
that the tubulin binding sites of MAP2 and tau
are essentially identical, consisting of 18 amino
acid repeats near the carboxy termini ofthe mole-
cules (Lewis et aI., 1988). Indeed, these data show
that the entire carboxy terminal domains of MAP2
and tau are highly homologous, indicating that
these molecules are probably derived from a com-
mon ancestral gene and also suggesting a related
81
primitive function, possibly associated with tubulin
binding and the stimulation of polymerization.
However, the amino termini of tau and MAP2
differ greatly. This bipartite structure, a common
carboxy terminus and disparate amino termini,
lends itself to speculation concerning functional
significance. Certainly the close homology of the
tubulin-binding domains rules out the possibility
that binding to different classes of microtubules
is responsible for their partitioning into axons
and dendrites. Presumably, the specific functions
of tau in axons and MAP2 in dendrites must be
connected with the primary structure ofthe amino
terminal domains.
Conclusions
Our growing knowledge of the microtubule-
associated proteins, their specific localization in
axons or dendrites, and the temporal regulation
of their expression being so closely linked to pro-
cess outgrowth or stabilization in many species
all indicate a fundamental role for these proteins
in neuronal morphogenesis. The recent introduc-
tion of recombinant DNA techniques for study-
ing brain MAPs has provided insights into the
mechanisms that regulate their expression and
has also begun to reveal the sites at which this
regulation can occur. The power of these techni-
ques promises greater insights, as the recent elu-
cidation of the MAP2 and tau tubulin-binding
domains show. For the same reasons that Ramon
y CajaI's attention was turned to the cerebellum
when he sought to unravel the complexities of
brain interconnectivity, that is, its orderly organi-
zation and development and distinct fields of
axons and dendrites, future studies designed to
manipulate MAP gene expression experimentally
will again turn to this system to determine the
precise function of the microtubule network in
neuronal morphogenesis.
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5
Cerebellar Grafting as a Tool to
Analyze New Aspects of
Cerebellar Development and Plasticity
Constantino Sotelo and Rosa-Magda Alvarado-Mallart
The cerebellum, especially the cerebellar cortex,
is one of the central nervous system regions in
which ordered organizational patterns are obvi-
ous. Its apparent simplicity has attracted many in-
vestigators interested in the mechanisms involved
in the development of the nervous system. A
hundred years ago, the study of the cerebellum
of nonmammalian species, and particularly the
study of its development, allowed Ramon y Cajal
(1888a-c) to obtain the first direct proof of the
independence of nerve cells, leading to the form4-
lation of the "Neuron Theory." In the cerebellum,
Ramon y Cajal (1890, 1911) also accurately des-
cribed for the first time the processes of neuronal
migration and neuronal differentiation as well as
the role of regressive processes during the forma-
tion of specific neuronal networks. From these
and other studies, it has been concluded that the
precise spatial positioning of the neurons- percei-
ved as a static arrangement in the adult animal-
is the result of strict temporal organization during
development. Neuronal proliferation and migra-
tion, together with dendritic differentiation and
axonal outgrowth, proceed according to strictly
defined timing and kinetics, resulting in the estab-
lishment of complex neuronal networks with a
high degree of specificity. Furthermore, the pro-
gression of most ifnot all of these sequential steps
takes place through cell-to-cell interactions, ap-
parently programmed to occur at precise matu-
rati'onal stages of both neural partners. Indeed,
absence or even delay in maturation of one of
these partners results in structural changes of the
cerebellar circuits. For example, proper inter-
actions between developing parallel fibers and
84
growing Purkinje cell dendrites are needed for
the normal acquisition of the tridimensional ar-
rangement of the dendrites (Legrand 1982, 1983;
Sotelo 1978) and for the stabilization of the pre-
synaptic parallel fibers (Sotelo and Changeux,
1974).
Neural grafting is a valuable approach to the
study of cerebellar development, since it allows
one to bring together immature and adult neural
components. The study of the developmental steps
of the grafted embryonic neurons will determine
whether or not the age mismatch between inter-
acting partners prevents the immature neurons
from proceeding with their developmental pro-
gram.
Neurons are highly differentiated cells that lose
their proliferative ability at a distinct step of their
development; loss of neurons cannot be compen-
sated for by proliferation ofthe surviving neurons.
One experimental way to palliate the loss of neu-
ronal populations in the adult cerebellum is to
replace the missing neurons by grafting homotypic,
young postmitotic neurons, taken from isogeneic
embryos. In systems organized in a "point-to-
point" manner like the cerebellum (Sotelo and
Alvarado-Mallart, 1986), neuronal replacement
is effective only when the grafted neurons succeed
in reestablishing the anatomical and functional
integrity of the deficient networks by constituting
an equivalent synaptic circuit. Such a recon-
stitution is only possible when the embryonic
neurons grafted into the adult deficient cere-
bellum are able to pursue their developmental
program in such a way that the cell-to-cell inter-
actions between immature and adult neurons
5. Cerebellar Grafting
recapitulate those taking place during normal
ontogeny.
In this chapter, we first report results obtained
with long-term survivals after cerebellar grafting
into the cerebellum of adult mutant mice devoid
of Purkinje cells, to provide evidence that neuro-
nal replacement and subsequent synaptic inte-
gration does occur. Thereafter, we describe results
obtained with short-term survivals to analyze the
developmental steps, and their temporal organi-
zation, that are followed by the grafted immature
Purkinje cells to achieve the neuronal replace-
ment. Finally, we compare the cellular mechanisms
underlying this replacement with those governing
the normal ontogeny ofthe cerebellum. This com-
parison permits the conclusion that embryonic
and adult neurons do interact, according to a
tempo imposed by the immature grafted Purkinje
cells, which follow a predetermined pattern of
maturation, apparently regulated by an internal
clock.
Long-Term Survivals
The biological system we have used to determine
the degree of neuronal replacement, and therefore
the degree of structural restoration of the cere-
bellar circuits resulting from neural grafting, has
been the cerebellum of adult mice affected by the
Purkinje cell degeneration (ped) mutation. In the
ped mouse (Landis and Mullen, 1978; Mullen et
aI., 1976), the cerebellum develops normally until
the end of the second postnatal week. Between
P14 and P45 virtually all Purkinje cells degenerate
(less than 120 of these neurons remain in 3-month-
old mutants, most ofthem located in the nodulus)
(Wassef et aI., 1986). Although all Purkinje cells
degenerate in a relatively short period of time,
their susceptibility to the pathologic process is
positionally determined (Wassef et aI., 1987). The
cerebellar cortex of mutants aged from 20 to 27
days can be subdivided into compartments con-
taining an almost normal number of Purkinje
cells and apposed compartments where most of
them hav.e disappeared. Thus, at these ages, the
surviving Purkinje cells are arranged in a repro-
ducible pattern of longitudinal zones, which is
symmetric relative to the midline.
In most of the experiments reported here, donor
tissue was taken from cerebellar primordia of
85
t2-day-old (E12) mouse embryos (C57BL), and
the hosts were 3- to 4-month-old homozygous
ped mice with a C57BL genetic background. In
a few experiments, 22- to 27-day-old ped mice
were used as hosts. Two grafting procedures were
performed (Fig. 5.1): either the cerebellar primor-
dia were mechanically dissociated in tissue culture
medium and the cell suspensions used for grafting,
or the primordia were sliced into small pieces,
and individual pieces were used in solid trans-
plantation. In both instances, the embryonic
material was injected with small glass pipettes
at variable depths within the parenchyma of the
host cerebellum. The obtained results were almost
identical with the two procedures and, therefore,
they have been pooled together.
In order to repair the cerebellar circuits of the
ped mouse, the Purkinje cell substitution needs
to achieve three essential prerequisites: a) from
all the postmitotic neurons and progenitors pre-
sent in the grafts, only neurons of the Purkinje
cell category must leave the implanted cellular
mass and move to the correct position previously
occupied by the missing Purkinje cells. Indeed,
the restoration of the cortical circuits implies that
the embryonic Purkinje cells are able to integrate
themselves, as the missing link, into the host defi-
cient network, b) once they reach the proper loca-
tions, the grafted neurons must not only follow
their differentiation program and build up their
dendritic trees, but must participate in synapto-
genesis with specific host afferents. These develop-
mental events would lead to the synaptic integra-
tion of the grafted Purkinje cells into the deficient
cortical circuitry, and c) finally, no functional
improvement can be accomplished ifthe disrupted
corticonuclear projection is not reestablished. For
that, the grafted Purkinje cells need to grow axons
that, navigating throughout the adult host cere-
belllum, reach their appropriate targets in the
deep nuclei, and synaptically contact the proper
host nuclear neurons.
Selective Invasion of Grafted Purkinje Cells:
The Role of Competition with
Host Purkinje Cells
By staining Purkinje cells with immunohisto-
chemistry [using selective markers: either an anti-
body against calbindin (Jande et aI., 1981) or
86
Figure 5.1. Schematic representation of the grafting
procedures, indicating the age of the donor cerebellar
primordium (E12 mouse) and the two methods of graft-
ing. In the upper part, the arrows point to the various
steps in obtaining cellular suspensions, and their
implantation into the adult host cerebellum. The
large arrow in the lower part indicates the direct implan-
tation of solid pieces of cerebellar primordium.
Constantino Sotelo and Rosa-Magda Alvarado-Mallart
2
Figure 5.2. Midsagittal section of the cerebellum of
an adult pcd mouse 3 months after grafting. The material
has been immunostained with an antibody against cal-
bindin. This light micrograph illustrates the areas of
host molecular layer occupied by the grafted Purkinje
cells. Bar = 600 flm .
5. Cerebellar Grafting
against cyclic guanosine monophosphate-depen-
dent protein kinase (De Camilli et ai., 1984)], we
have been able to identify grafted Purkinje cells
in the host cerebella 2 to 5 months after implanta-
tion. Portions of the grafts evolve into small
remnants still containing some Purkinje cells.
However, the vast majority ofthese neurons have
moved out and occupy the host molecular layer
(Fig. 5.2), with ectopic cell bodies dispersed
throughout the superficial four fifths of this layer,
in several folia in direct contact with the graft
remnant. In the largest transplants, the diameter
of the spread is about 1.4 mm, suggesting that
embryonic Purkinje cells can migrate through the
adult cerebellum for distances as long as 0.7 mm.
One of the most important observations in this
study has been to establish that the invasion of
grafted neurons is not random. Only neurons of
the category of the missing ones penetrate the
host parenchyma, whereas the other grafted neu'-
ronal populations remain in the implant remnant.
Two lines of evidence have allowed us to reach
this conclusion. The first derives from the cytolo-
gical examination of the cortical areas adjacent
to the transplants. In these areas, the only visible
change in the mutant cortex is the presence, in
the molecular layer, of scattered perikarya of large
neurons exhibiting the ultrastructural features of
Purkinje cells, particularly the hypolemmal cistern
(Palay and Chan-Palay, 1974). With the exception
of these ectopic Purkinje cells, the appearance of
the remaining cell classes-basket and stellate
cells-and their density are similar to those en-
countered in the mutant molecular layer, at a
distance from the graft.
The second line of evidence arises from experi-
ments designed to visualize specifically all neuro-
nal categories originated from the transplants
wherever their position in the host. Most neurons
are provided with a cell surface glycoprotein,
Thy-I, that exists in mice in two allelic forms
called Thy-l.l and Thy-1.2 (see refs. in Morris,
1985), and can be distinguished immunologically.
Although the expression of Thy-1 by granule cell
bodies has not been clearly demonstrated in vivo,
it is present at high levels in regions containing
granule cell processes such as glomeruli and the
molecular layer (Fig. 5.3). Thus, immunohisto-
chemistry allows the distinction between graft
and host neurons when the cerebellar primordium
87
taken from a mouse bearing one allele is trans-
planted into the cerebellum of an adult host mouse
that is of the other allelic form (as has been illus-
trated in the hippocampus by Zhou et ai., 1985).
Since the C57BL strain bearing the pcd muta-
tion is homozygous for Thy-1.2, we have used as
donors 12-day-old embryos of the AKR strain,
homozygous for Thy-l.l (Fig. 5.3). Although
C57BL and AKR strains differ in their major histo-
compatibility antigen haplotypes (Green, 1981),
the transplants survived for 1 to 2 months in the
cerebellum of the adult pcd mice. The Thy-l.1
alloantigenic determinant was visualized immu-
nohistochemically by using the OX7 monoclonal
antibody (Mason and Williams, 1980). The graft
remnants comprise Thy-1.1 immunoreactive neu-
rons disposed in a disorganized manner, giving
them the appearance of dense immunopositive
masses, surrounded by immunonegative host
tissue. In the host cortex, within 1.4 mm around
the remnants, Thy-1.1 immunoreactivity is visible
only in the molecular layer (Fig. 5.4), correspond-
ing to zones that, in cresyl violet-stained sections,
can be seen to contain Purkinje cell bodies. In the
most distant regions from the remnants, at the
end of the host territory invaded by grafted cells
and where their density is low, it is possible to
determine the shape of single Thy-l.1 positive
neurons, and all of them are easily identified as
Purkinje cells because of their peculiar dendritic
arbors (Fig. 5.5). Immunoreactive cells are absent
from either the granular layer or the deep nuclei.
The immunohistochemical results indicate the
specificity of the mechanisms involved in the pro-
cess of invasion of the mutant host cerebellum by
grafted cells: only Purkinje cells, the cell category
missing in the host cortex, are able to leave the
grafts and to enter the cerebellar parenchyma of
the host.
The question still to be resolved concerns the
nature of the "mechanisms" involved in the selec-
tive migration of immature Purkinje cells from
the grafts to the molecular layer of the adult host
cerebellum. In some previous experiments (Sotelo
and Alvarado-Mallart, 1985) we have shown that
when pieces of cerebellar primordia are implanted
in the neocortex or the hippocampus, virtually
all grafted Purkinje cells do not leave the implants,
and instead form a we\! organized minicerebellum
with a small trilaminated cortex and, occasion-
88 Constantino Sotelo and Rosa-Magda Alvarado-Mallart

Figure 5.3. Cerebellar cortex of the vermis of an adult
AKR mouse, immunostained with an anti- Thy-l.l
antibody. The visualization of this cell surface antigen
permits the recognition of all cerebellar cell classes. In
the molecular layer (ML), Purkinje cell dendrites and
molecular layer interneurons appear as negative struc-
tures; the cell membranes of these cellular elements as
well as those of the processes in the neuropil, particu-
larly parallel fibers, provide the immunoreactivity of
the molecular layer. The plasmalemma of Purkinje cell
bodies (arrows) are also immunoreactive. In the granu-
lar layer (GL), the glomeruli are densely stained, and
the granule cells appear as negative rounded cells
surrounded by a very thin circular staining (stars).
Bar = 80flm.
5. Cerebellar Grafting
ally, a central nuclear zone. Recently, we have
performed new experiments aimed at disclosing
whether or not the presence of host Purkinje cells
interferes with the invasion of grafted Purkinje
cells into the host molecular layer. Three lines
of evidence, indicating that the Purkinje cell-
deficient molecular layer exerts a "positive neuro-
tropic" effect (Ramon y Cajal, 1910), have been
obtained.
Cerebellar Grafting in Normal Cerebellum
Young adult C57BL mice were implanted with
cerebellar primordia taken from 12-day-old em-
bryos, and the transplanted cerebella were analyzed
1 to 3 months later (Figs. 5.6, 5.7). As is often the
case in ped transplanted mice, the graft remnants
remain squeezed between two folia, lying against
their pial surfaces. Small numbers of Purkinje
cells do migrate out ofthe remnant, but the grafted
and host Purkinje cells are easily distinguished.
Host cells have their cell bodies arranged in a
monolayer between molecular and granular layers
(Fig. 5.6), whereas the grafted cells have their cell
bodies ectopically located in the host molecular
layer (Figs. 5.6, 5.7). Despite important individual
variations between mice, the general pattern of
disposition of grafted Purkinje cells can be sum-
marized as follows: In cortical areas lesioned by
the cannula used for the implantation, there are
narrow zones devoid of host Purkinje cells. These
areas contain abundant Purkinje cells with ectopic
cell bodies spreading thrc ·;hout the superficial
four fifths of the host molecular layer (Fig. 5.6).
In cortical zones juxtaposed to the cannula track,
the molecular layer contains overlapping host
and grafted Purkinje cells; the former always
at the interface between molecular and granular
layer, the latter with ectopic cell bodies occupying
~----------------------------------------------------------------------
Figure 5.4. Low magnification of an AKR graft in the
cerebellum of an adult pcd mouse 2 months after im-
plantation, visualized by Thy-l.1 immunoreactivity.
The arrow in this sagittal section points to the grafted
cells that have penetrated the host parenchyma. Note
that, with exception of this area of host molecular
layer, the host cerebellum is immunonegative. Bar =
600/lm.
Figure 5.5. High magnification of an area similar to
89
the superficial four fifths of the molecular layer.
However, this zone of direct invasion extends
only 100 J1.m from the cannula track. Beyond this
distance, the only ectopic cell bodies of grafted
Purkinje cells have a subpial position with short,
inverted dendritic trees that overlap with those
of host Purkinje cells (Fig. 5.7). As we show below,
these subpial Purkinje cells are derived not from
direct invasion of the molecular layer but rather
by interfolial spread with subsequent penetration
into the outer molecular layer. This is far less than
the extensive integration of grafted Purkinje cells
seen in ped mutant cerebella; in such cerebella,
grafted Purkinje cells may spread 700 J1.m from
the cannula track (see above).
Cerebellar Grafting in 20- to
27-Day-Old ped Mice
As stated above, the cerebellum of these young
ped mice still contain abundant Purkinje cells
arranged in precise longitudinal zones (Wassef
et aI., 1987). Therefore, cerebellar grafting in these
mice offers an optimal control experiment to deter-
mine the presumptive role of host Purkinje cells
in regulating the extension of host invasion by
grafted neurons in animals of an identical genetic
background to those used in our long-term sur-
vival experiments. Moreover, these experiments
could disclose whether or not the Purkinje cell
death results from toxic environmental factors,
which theoretically may be present in the ped
cerebellum during this phase of maximal neuronal
degeneration. In all these mice, the large majority
of the implanted tissue remains as an organized
grafted minicerebellum with an interfolialloca-
tion (Fig. 5.8). Grafted Purkinje cells do invade
the host molecular layer but to a much lesser
extent than in older ped mutants (Figs. 5.8, 5.9).
that illustrated in Fig. 5.4. Thy-l.1 immunoreactivity
is confined to the host molecular layer. At the deep
part of the fissure, where only a few scattered grafted
Purkinje cell bodies were observed in the consecutive
paraffin section stained with cresyl violet, the immuno-
staining permits the identification of the dendrites
(arrow heads) of these grafted Purkinje cells. No other
cellular element is immunoreactive. GL, granular layer.
Bar= 30/lm.
90
Figure 5.6. Cerebellar grafting in normal mouse cere-
bellum I month after transplantation. Immunostaining
with anticalbindin. Sagittal section of the vermis, illus-
trating an area in which host Purkinje cells have been
destroyed by the implantation cannula (arrows) . Host
Purkinje cells have cell bodies at the interface between
molecular (ML) and granular layer (GL) (white squares).
Grafted Purkinje cells (white triangles), with ectopic
cell bodie~ dispersed through the molecular layer, fill
the gap created by the lesion. Note that only one
Purkinje cell (double arrow) has invaded the territory
occupied by the host Purkinje cells. Bar = 30.um.
Constantino Sotelo and Rosa-Magda Alvarado-Mallart
Figure 5.7. Same material as for Fig. 5.6 but far from
the implantation cannula track. The graft remnant
(GR) containing grafted Purkinje cells (white squares)
lies on the surface of a folium that contains a normal
contingent of host Purkinje cells (identified by their
dendritic trees). Note that some of the grafted Purkinje
cells (arrowheads) have penetrated the host cerebellum
but their cell bodies keep a subpial location. Bar =
30.um.
5. Cerebellar Grafting
GL
Figure 5.8. Calbindin immunostaining of the cerebel-
lum of a pcd mouse grafted at 24 days of age and fixed
3 months after the implantation. A large graft remnant
(GR) organized as a normal trilaminated cerebellar
cortex is visible between two folia of the host cerebel-
lum. The arrows indicate areas of host molecular layer
containing grafted Purkinje cells. Bar = 500 !tm.
91
9
Figure 5.9. Higher magnification of the same material
as that in Fig. 5.8. The grafted Purkinje cells that have
invaded the host cerebellum have cell bodies located
in the superficial half of the host molecular layer and
inverted dendritic trees. Overlying this folium, Purkinje
cells remaining in the graft remnant (GR) are visible.
GL, granular layer. Bar = 50 !tm.
92 Constantino Sotelo and Rosa-Magda Alvarado-Mallart
Furthermore, the cell bodies of the invading
Purkinje cells remain in the superficial quarter
of the host molecular layer (Fig. 5.9). Similar
differences in the extent of grafted Purkinje cell
invasion between young and old pcd hosts have
been reported (Chang et aI., 1988). The survival of
large numbers ofPurkinje cells argues against the
existence of toxic factors responsible for the degene-
ration of mutant Purkinje cells.
Cerebellar Grafting in Nervous Mutant Mice
Nervous (nr) is a neurological mutation also
affecting Purkinje cells (Landis, 1973; Sidman
and Green, 1970). In this mutant cerebellum, the
degeneration of Purkinje cells is slower and less
complete than in the pcd mouse, and about 15%
of them survive. The survival is also positionally
related: all Purkinje cells degenerate in the hemi-
spheres, some survive in the flocculus and in the
intermediate cortex, and half in the vermis, where
they are grouped into distinct longitudinal stripes
that are symmetric with relation to the midline
(Fig. 5.10) (Wassef et aI., 1987). In our experiments,
E 12 isogenic cerebellar primordia were implanted
into the intermediate cortex, and the cerebella
were studied 2 to 4 months later. By analyzing
the molecular layer ofthe host at its interface with
the graft remnant, we have determined that, in
general, the invasion of grafted Purkinje cells is
oriented toward the region devoid of host Purkinje
cells. Furthermore, when the grafted neurons reach
a zone of the nr molecular layer containing the
dendritic trees of the host Purkinje cells, they
normally stop their migration. The zones of over-
lap between grafted and nr Purkinje cells are
small; in them the cell bodies belonging to the
grafted neurons are almost abruptly excluded
from the deeper half of the host molecular layer
(Fig. 5.11).
The results obtained with these three groups
of experimental animals point in the same direc-
tion: the extent of the host molecular layer inva-
sion by grafted Purkinje cells in these mice is
much smaller than that obtained when the host
is completely devoid of Purkinje cells at the
time of transplantation. Thus, the Purkinje cell-
deficient molecular layer seems to exert a "positive
neurotropic" effect, of still unknown nature, selec-
tive for neurons of the same category as those
missing.
Synaptic Integration of Grafted
Purkinje Cells into the Circuits of the
Host Cerebellar Cortex
Immunohistochemical, ultrastructural, and elec-
trophysiological studies ofthe grafts demonstrate
that, with some limitations, the prerequisites dis-
cussed above for the repair of deficient circuits
can be fulfilled.
Location and Dendritic Arrangement
of Grafted Purkinje Cells
In order to facilitate their synaptic integration,
the grafted Purkinje cells must move out of the
implant to the location previously occupied by
the missing neurons. In our material, the grafted
neurons do not completely succeed in this task,
because their cell bodies remain ectopic (Figs.
5.12,5.13). However, since more than 95% of the
synaptic inputs to these neurons are dendritic, the
distribution and organization of the dendritic
trees belonging to grafted Purkinje cells become
much more important than the location of their
cell bodies. These dendritic trees are able to spread
throughout the host molecular layer (Figs. 5.12,
5.13), stopping abruptly at the interface with the
granular layer. In this respect, host and grafted
Purkinje cells have identically located dendrites,
spanning the whole depth of the mutant molecular
layer.
The dendrites of grafted Purkinje cells do not
fully acquire the characteristic espalier shape seen
in control animals, and their shapes are related
to the position of their cell bodies (Figs. 5.12,
5.13). Those with cell bodies located under the
pial membrane commonly have inverted dendrites,
whereas those at the center ofthe layer are bipolar
and even multipolar. Despite this variety of shapes,
all these dendrites have acquired the two principal
features that characterize normal Purkinje cell
dendrites: a) they comprise a proximal compart-
ment of thick, almost smooth branches, and a
distal compartment of spiny branchlets (Fig. 5.12)
and b) they have achieved a flattened arrangement
with a maximal extension in the sagittal plane
(Fig. 5.12), and a minimal one in the transverse
plane (Fig. 5.13). They are therefore confined to
the plane perpendicular to the bundles of parallel
fibers of the mutant cerebellum (Fig. 5.14). Consi-
dering the features of these dendrites, we can
5. Cerebellar Grafting
Figure 5.10. Low magnification of an adult nr cerebel-
lum cut in the coronal plane and immunostained with
an anticalbindin antibody. Note that the remaining
Purkinje cells are not randomly located but are loca-
lized into precise longitudinal compartments, providing
mirror images in relation to the midline. Bar = 600 11m.
Figure 5.11. Cerebellum of an adult nr mouse 3
months after implantation of a cerebellar graft into the
intermediary cortex. The arrows indicate the interface
93
'10
11
in the host molecular layer between a zone containing
host Purkinje cells (arrowheads) and another devoid of
these neurons. Note that the area lacking host Purkinje
cells contains numerous grafted Purkinje cells, with
somata dispersed throughout the host molecular layer.
Only a few grafted neurons (white triangles) overlap
with the zone containing host Purkinje cells. Immuno-
staining with anticalbindin. Bar = 45 p.m.
94
Figure 5.12. pcd grafted cerebellum 2 months after the
implantation. Distal area of the host molecular layer
containing grafted Purkinje cells. The arrow points to
one of these neurons with a dendritic tree resembling
that of normal Purkinje cells. Note the presence of
thick dendritic branches, forming the proximal com-
partment, as well as distal thin branches of the distal
compartment. The dendritic tree does not have the
Constantino Sotelo and Rosa-Magda Alvarado-Mallart
13
normal outward orientation, but exhibits a maximal
extension in this parasagittal plane of sectioning.
Immunostaining with an anticyc\ic GMP-dependent
protein kinase. Bar = 90 flm.
Figure 5.13. Same material as in Fig. 5.12 but sec-
tioned in the coronal plane. Note the flattening of the
grafted Purkinje cell dendrites in this plane of section-
ing. Bar = 40 flm.
5. Cerebellar Grafting 95

Figure 5.14. Diagrammatic

representation of Purkinje
cell dendritic trees in control
mice and in transplimted
pcd cerebella. Note that the
flattening of the trees in
the coronal plane is more
pronounced in the control
(mono planar) than in the
grafted Purkinje cells.

conclude that grafted Purkinje cells have indeed
succeeded in occupying the host territory where
the lost mutant Purkinje cells had received their
whole contingent of synaptic inputs.
Grafted Purkinje Cells and Host Cortical
Cerebellar Circuits
The ultrastructural study of grafted Purkinje cells
within the pcd molecular layer discloses a synaptic
14
investment qualitatively similar to that of the
normal cerebellum. Each thick dendritic segment
bears clusters of stubby spines synaptically con-
tacted by climbing fiber varicosities, whereas the
distal branches, from which emerge numerous
long-necked spines, are the postsynaptic targets
for parallel fibers. Despite their ectopic location,
the perikarya receive synaptic inputs from either
stellate cell axons or ascending collaterals of the
96 Constantino Sotelo and Rosa-Magda Alvarado-Mallart

bllsket fibers. These two classes ofaxons, belong- possible restoration of the mutant cerebellar
ing to inhibitory interneurons of the host mole- circuits.
cular layer, also establish synaptic contacts with
the smooth surface of the stem dendrites. These Corticonuclear Reconnection: The Problem
ultrastructural observations clearly indicate that oj Growth and Navigation oj Axons
the synaptic investment of grafted Purkinje cells
Originated Jrom the Grafted
is almost normal, although some few qualitative
Purkinje Cells
(Sotelo and Alvarado-Mallart, 1987a) and quanti-
tative (Sotelo and Alvarado-Mallart, 1987b) dif- To obtain functional recovery in pcd mice, it is
ferences have been reported. imperative that the information processed by the
The function of the newly formed synapses repaired cerebellar cortex reach its target neurons,
between the adult host neurons and the grafted located mostly in the deep cerebellar nuclei. Since
Purkinje cells has been investigated, in collabora- the only known cortical output is made by axons
tion with Francis Crepel and colleagues (Gardette of Purkinje cells, it is obvious that the recovery
et aI., 1988), with electro physiologic techniques is dependent on the formation of a new corti-
on pcd cerebellar slices in vitro. The grafted neu- conuclear projection originated from the implan-
rons were impaled with intracellular microelec- ted Purkinje cells. This formation implies a pro-
trodes and their synaptic interactions were analy- cess of oriented axonal growth that, owing to the
zed by electrical stimulation of the white matter nature of the neurons involved and to their cellular
at the base of the folium to activate anterogradely milieu, will share some of the mechanisms under-
climbing and mossy fibers. All grafted Purkinje lying axonal pathfinding in the developing central
cells tested responded to this white matter stimu- nervous system (CNS) as well as axonal regenera-
lation by a typical all-or-none climbing fiber exci- tion in the adult CNS. The similarities and dif-
tatory postsynaptic potential (EPSP) (complex ferences between these processes can be summari-
spike), indicating that not only have all grafted zed as follows: a) the distance to be covered by
Purkinje cells formed synapses with axons of the the differentiating axons of grafted Purkinje cells
host inferior olivary neurons, but also that they and the cellular milieu faced by their growth cones
are, as in control animals, monoinnervated by during navigation are those offered by the adult
climbing fibers. Purkinje cell responses, due to host cerebellum. Thus, the obstacles that these
the activation of the mossy fiber-granule cell axons must overcome are those encountered by
pathway, were also elicited in the grafted neurons regenerating axons in the adult CNS, b) the target
when the stimulus intensity was low enough not neurons in cerebellar nuclei are presumably adult
to give rise to any climbing fiber response. The and may not exert the same effects on Purkinje
latencies of the disynaptic responses (ranging axons that occur during development, c) Purkinje
between 2.4 and 5.2 ms) were longer than those cells are grafted either as precursor cells, devoid
resulting from the monosynaptic activation of of neuritic expansions, or as young postmitotic
the climbing fibers. These disynaptic EPSPs were neurons, with neurites remaining within the solid
markedly graded with the stimulus intensity as pieces of implanted cerebellar primordium. Since
in normal cerebellum. Finally, well developed these neurons are not axotomized, the molecular
inhibitory postsynaptic potentials (IPSPs) were signals induced by axotomy are not expressed in
also evoked in grafted neurons, although of the grafting experiments, and d) the immaturity
shorter duration than in control Purkinje cells. of the grafted neurons leads us to suppose that
These electrophysiQlogic experiments confirm that they are capable of greater plasticity than adult
all the excitatory and inhibitory inputs forming neurons, so that they could adapt better to the
the synaptic investment of grafted Purkinje cells existing conditions for their axonal outgrowth.
are functional and have characteristics compar- These considerations suggest that the formation
able to those in control mice. Thus, the grafted of a corticonuclear projection in the mutant cere-
Purkinje cells have been synaptically integrated bellum will need to overcome most ofthedifficul-
into the cortical circuitry of the deficient host ties encountered by central mammalian neurons
cerebellum, fulfilling the second requirement for in their abortive axonal regeneration. It is there-
5. Cerebellar Grafting
fore expected that the fulfillment of this pre-
requisite will be the most difficult to attain. The
results obtained in our experiments, using Purkinje
cell immunomarkers to trace their axons, confirm
this expectation. Only in a low percentage of
grafted cerebella is a corticonuclear projection
formed, and the latter only involves a few of the
axons emerging from those Purkinje cells that
have succeeded in invading the mutant molecular
layer.
The growth of grafted axons may be limited by
two complementary mechanisms. First, the target
neurons may not present the same influences as
occur during development. Second, grafted deep
cerebellar neurons in the implant remnants may
impair contact with host deep cerebellar nuclear
neurons because they are close to the grafted
Purkinje cells, and may preempt their growing
axons when the distance between integrated graf-
ted Purkinje cells and host deep nuclei exceeds
600 11m. Thus, 600ilm seem to be the longest
distance over which the grafted axons can navigate
and form a new corticonuclear projection.
Normal development depends on interactions
between axonal growth cones and local cellular
elements, provided with specific cues, that stimu-
late (permissive) or inhibit (nonpermissive) the
oriented axonal outgrowth. In the developing
nervous system, such guidance mechanisms are
based on the recognition of cell-surface and extra-
cellular matrix molecules present in those cells
signaling the pathway (Dodd and Jessell, 1988).
Whether these clues persist in the mature nervous
system remains unknown; our observations sug-
gest a loss of such clues in the cerebellum of the
adult pcd mouse. Indeed, the majority of the axons
emerging from grafted Purkinje cells remain within
the molecular layer (Fig. 5.15), where they may
contact other Purkinje cells or form aberrant
plexuses at the interface between the molecular
and the granular layers, as if they are lost in their
search for appropriate target neurons. A small
minority of Purkinje cell axons do leave the
molecular layer for unknown reasons and cross
perpendicularly the granular layer (as normal
Purkinje cells do), reaching the white matter axis
of the folium. The fate of these axons depends, as
already discussed, on their position with respect
to the deep cerebellar nuclei. If they are close to
the graft remnants, they will end by synapsing on
97
grafted deep nuclear neurons. If, conversely, they
are far from the graft remnants and within 600 11m
from the host deep nuclei, they will direct their
outgrowth toward the latter and, once they enter
this territory, differentiate into branched terminal
arborizations, bearing numerous varicosities. Our
ultrastructural analysis has proven that the vari-
cosities establish specific synaptic connections on
the somata and on the dendrites of host deep
nuclear neurons. Therefore, the third needed pre-
requisite is, by far, the most difficult to fulfill. A
corticonuclear reconnection is obtained only in
a few transplanted cerebella, and the extent of the
reconnection is always very limited.
In some of the transplants with solid pieces of
cerebellar primordia and in all of those using cell
suspensions (Sotelo and Alvarado-Mallart, 1986),
some of the grafted Purkinje cells remain dispersed
throughout the cannula track and a few within
the host white matter. These cells have stellate
shapes with several main dendritic stems emerging
from the cell bodies. The dendrites are short,
studded with spines, and poorly ramified, suggest-
ing that they are deprived of most of their normal
synaptic inputs (Sotelo, 1978). These grafted
Purkinje cells have been extremely useful in indi-
cating that one of the main limiting factors for
the corticonuclear reconnection lies in the irritial
disorientation of their developing axons within
the host molecular layer. Indeed, these Purkinje
cells outside the gray matter are able to grow axons
for long distances that follow the longitudinal axis
of the folium, parallel to the bundles of myelina-
ted fibers present in the white matter (Fig. 5.16).
These observations raise an interesting issue.
In a recent study by Caroni and Schwab (1988),
membranes from CNS myelin have been proven
to be nonpermissive substrata for axonal out-
growth, and the inhibitory oligodendroglial-
surface molecules have been identified. In theory,
the immunoblockade of these proteins could per-
mit the regeneration of central neurons in adult
mammalian CNS. Our in vivo observations with
the transplants suggest that oriented axonal growth
can occur within the white matter. Although the
distance reached from this growth is rather small,
it is enough to allow in the mouse an important
rate of regeneration of the corticonuclear projec-
tion. However, even though Purkinje cells have
a great resistance to axotomy (Ramon y Cajal,
98 Constantino Sotelo and Rosa-Magda Alvarado-Mallart

GL

Figure 5.15. Calbindin-immunofluorescent grafted
Purkinje cells in the molecular layer of the pcd cere-
bellum, cut in the coronal plane 1 month after implan-
tation. At the distal border of the host molecular layer
containing the grafted neurons, the axons ofthese cells
16
do not cross the granular layer but remain within tQe
molecular layer, forming a loose bundle of parallel
axons. GL, granular layer; WM, white matter axis of
the folium. Bar = 60/lm.
Figure 5.16. Coronal section through the pcd cerebel-
 5. Cerebellar Grafting
1959; Sotelo, 1978), they are unable to reestab-
lish a corticonuclear projection after cutting their
axons. The results obtained by Ramon y Cajal
(1959) in young mammalian cerebellum after
Purkinje cell axotomy, the so-called Purkinje cells
with arciform axons, suggest the existence of some
axonal regeneration but the inability of these
axons to find their pathway to the white matter.
Thus, as Ramon y Cajal wrote: "A result of the
disappearance of the extracollateral portion of
the axon and of the compensatory hypertrophy
of the collaterals and initial portion of the axon
is the transformation of the cell of Purkinje into
a neurone with a short axon." All these observa-
tions point to an essential question: What prevents
regenerating axons and axons of grafted embryo-
nic neurons from finding their ways to their specific
target neurons? Without this answer, it is difficult
to imagine how neuronal grafting could succeed
in replacing neurons with the complete synaptic
integration needed for the restoration of deficient
systems wired in a "point-to-point" manner.
General Conclusions
The results obtained in this series of grafting
experiments, studied after long-term survival,
demonstrate that specific synaptic integration of
grafted neurons into the deficient cerebellar cir-
cuitry can take place, although with important
limitations. The replacement of missing Purkinje
cells by grafted homologous neurons, that can
involve 5% to 15% of the volume of the mutant
molecular layer (Sotelo and Alvarado-Mallart,
1987b), occurs in all the implanted cerebella of
the adult pcd mutant mice. It results in the re-
storation of corti co cerebellar circuitry, owing to
the complete synaptic integration of the grafted
Purkinje cells into the cortex of the mutant cere-
bellum. However, the corticonuclear projection
is reestablished in only a few percent of grafted
cerebella and involves a low number of grafted
Purkinje cells.
~-----------------------------------------------------------------
lum grafted 2 months before fixation and immunostain-
ed with anticalbindin antisera. The upper part of the
micrograph corresponds to the host molecular layer
containing grafted Purkinje cells with dendrites delinea-
ting the interface (arrowheads) between the molecular
99
Short-Term Survivals
The results obtained with long-term survivals
imply that, in the adult mutant cerebellum, the
replacement of missing Purkinje cells occurs in a
precise manner, most probably recapitulating the
processes followed by these neurons during their
normal ontogeny. The principal events in cerebel-
lar morphogenesis take place in sequential critical
steps from the cellular proliferation of stem cells
in the primitive cerebellar neuroepithelium to the
functional validation and selective elimination of
synaptic connections (Changeux and Danchin,
1976), resulting in the formation of the cerebellar
cortex and its specific circuitry. These orderly
steps (see refs. in Cowan, 1981) concern the follow-
ing processes: a) a phase of cellular proliferation,
followed by b) an outward migration of young
postmitotic Purkinje cells from the subventricular
zone to the cortical plate (Goffinet, 1983), c) a
phase of cytodifferentiation with the formation of
neuritic expansions, which will result in the acqui-
sition of tridimensionally arranged dendritic trees
(see refs. in Sotelo, 1978) and in the establishment
of highly organized efferent projections, d) simul-
taneously with the formation of dendritic arbori-
zations, Purkinje cells begin their synaptogenetic
period, with the formation of redundant connec-
tions (Crepel et aI., 1976), that will be followed by
a secondary period of numerical adjustment, in-
volving regressive processes resulting in e) the
selective elimination of some synaptic connections
and the f) synaptic stabilization of the remaining
ones. According to the hypothesis of Changeux
and Danchin (1976), these last two processes are
regulated by the function ofthe forming cerebellar
circuits.
In order to determine whether or not grafted
Purkinje cells are able to recapitulate their de-
velopmental history within the adult host cere-
bellum, and to disclose the cellular mechanisms
underlying the successful neuronal replacement
discussed in the preceding section, new grafting
and the granular layer (GL). The left side corresponds
to the grafted Purkinje cells distributed all along the
cannula track. Note that the axons of these neurons
have succeeded to grow into the white matter axis
(WM) of the folium. Bar = 40 11m.
100 Constantino Sotelo and Rosa-Magda Alvarado-Mallart

GL
Figure 5.17. Autoradiogram of a pcd cerebellum graft-
ed I month before fixation. The grafted mouse was
intraperitoneally injected with tritiated thymidine 6
and 24 hours after the implantation. The cerebellum
was cut in the sagittal plane and counterstained with
cresyl violet. In this double exposure (bright and dark
fields), the auto radiographic silver grains appear white.
19
The arrows point to grafted Purkinje cell bodies con-
taining labeled nuclei. Bar = 40 /lm.
Figure 5.18. Calbindin immunostaining of grafted
Purkinje cells during their tangential migration at the
surface of the host molecular layer (ML). Note the
elongated shapes of the migrating neurons, 5 days after
grafting. Bar = 40 /lm.
 5. Cerebellar Grafting
experiments in pcd cerebellum have been carried
out. Most of them have been analyzed in a timed
sequence from 3 up to 21 days after grafting (3-
21 DAG) in order to compare at a given age the
phase of maturation of grafted Purkinje cells with
that normally occurring in cerebellar ontogenesis
(Sotelo et al., 1990). In the following description
we report the results obtained for each of these
developmental steps.
Proliferation Period
of Grafted Purkinje Cells
During the morphogenesis of the mouse cerebel-
lum, Purkinje cell proliferation starts the embryo-
nic day 11 and ends 72 hours later (from Ell to
E13) (Fujita, 1967; Miale and Sidman, 1961). Since
in our experiments donor tissue is taken from
12-day-old embryos, less than 25% of Purkinje
cells have already finished proliferation. Thus, the
majority ofthis neuronal population is grafted as
neuroblasts, with potential mitotic activity for at
least 48 hours. The trauma associated with the
implantation and the abnormality of the cellular
milieu in which the grafted primordium will under-
go its development could influence the prolifera-
tion period of grafted Purkinje cell progenitors.
They could either arrest their proliferative period
or prolong it beyond the expected 48 hours. To
study this question we have used the following
approach: Adult pcd mice were grafted with solid
pieces of E 12 cerebellar primordium. One group
of recipients was injected intraperitoneally with
tritiated thymidine (5p.Ci/g) 6 and 24 hours after
the operation. In another group, the isotope was
injected 60 and 70 hours after grafting. Mice of
both groups were fixed between 45 and 60 days
after grafting, their cerebella were embedded in
paraffin, and serial sections were used for the
autoradiographic detection of the tritiated thy-
midine. The sections Were counterstained with
cresyl violet.
In the cresyl violet-stained preparations, graf-
ted Purkinje cells can be identified by their cyto-
logical features. They appear as large, ovoid cell
bodies of about 22p.m in diameter, containing a
~----------------------------------------------------------------------
Figure 5.19. Seven days after grafting, the migrating
calbindin immunoreactive neurons have changed
polarity, crossing the host molecl,llar layer in a radial
101
large nucleus provided with a single spherical
nucleolus. The wide cytoplasm around the nucleus
is filled with numerous small and irregular Nissl
bodies. Neurons of this sort can be encountered
in two locations. Some ofthem are present within
the graft remnant, but the majority is dispersed
within the upper three quarters ofthe host mole-
cular layer (Fig. 5.17), in folia adjacent to the graft
remnant. In the group of mutant mice treated
with tritiated thymidine 6 and 24 hours after
implantation, grafted Purkinje cells with auto-
radiographic labeled nuclei are present in both
locations. About 25% of those Purkinje cells
integrated in the host molecular layer exhibit
labeled nuclei (Fig. 5.17). Conversely, in those
implanted mice injected with the isotope 60 and
70 hours after grafting, no labeled Purkinje cells
have been observed.
These results show that the Purkinje cell pro-
genitors are able to proliferate when implanted
in an adult cerebellum. More importantly, this
proliferation seems to take place over an interval
similar to that occurring during normal cerebellar
ontogeny. The incorporation of tritiated thymi-
dine does not take place 60 hours after grafting,
a time at which the implanted Purkinje cells have
reached an age equivalent to 15 embryonic days,
the age at which Purkinje cells have ceased their
proliferative activity in normally developing fetal
mice.
Migratory Routes of Grafted
Purkinje Cells
During the first days after transplantation, the
grafts, even when implanted as solid pieces of
donor cerebellar primordium, modify their shapes
according to physical constraints. The pressure
exerted by the parenchyma ofthe host cerebellum
forces the grafts to adapt themselves to regions
of less resistance either created by the cannula
track or already present in the host cerebellum
like the cerebellar fissures. Thus, 3 to 4 days after
grafting (3-4 DAG) they have acquired irregular
shapes consisting of a main elongated mass that
crosses several folia of the host cerebellum, and
or oblique direction. Note that the descending proces-
ses of these grafted neurons (arrows) do not enter into
the host granular layer (GL). Bar = 40 pm.
102
Figure 5.20. One-Ilm thick section stained with tolui-
dine blue, taken from a pcd grafted cerebellum 6 days
after grafting. The upper part of the micrograph cor-
responds to a lateral swelling of the graft (LSG) lying
on the surface of a folium of the host cerebellum. The
graft contains large neurons ofthe Purkinje cell category
Constantino Sotelo and Rosa-Magda Alvarado-Mallart
(arrowheads). The arrows point to the edges of a basal
lamina hole. Note the large processes of grafted
Purkinje cells penetrating the host molecular layer
(star). The asterisks mark grafted Purkinje cells either
in a subpial position or intermingled with the neuropil
of the host molecular layer (ML). Bar = 20 tim.
 5. Cerebellar Grafting
one or several lateral swellings expanding between
the surfaces of adjacent folia, at both sides of the
main elongated mass. Hence, grafted neural
elements are apposed to the host cerebellar
parenchyma in two distinct fashions: a) there is a
direct apposition (graft-host interface) between
the trilayered cortex ofthe host folia and the main
elongated mass, and b) the lateral swellings ofthe
graft lie on the pial surface of the host folia,
separated from the host parenchyma by the pial
cells and their basal lamina.
Owing to this special disposition ofthe implants,
grafted Purkinje cells can invade the host paren-
chyma following two different migratory pathways,
one originating from the graft-host interface and
the other through the pial surface and its basal
membrane. Indeed, 4-5 DAG a migratory stream
has been formed at the periphery of the host folia
directly attached to the graft. This stream emerges
from the graft and moves off, between the subpial
surface and the glial-limiting membrane, in a fun-
neling manner (at its emergence it consists of a
five- to seven-cell-deep layer and at its end, about
700 Jlm away, it is formed of a single row of
elongated neurons), without disrupting the com-
pact neuropil of the host cerebellar cortex.
Six DAG, the tangential migratory stream at-
tains its maximal extension, about 1.5 mm in
maximum diameter around the graft remnant.
The cellular composition within this superficial
stream appears homogeneous. With the excep-
tion of some degenerating cells and macro phages
close to the host- graft interface, the stream com-
prises young undifferentiated bipolar neurons of
about 20 Jlm in diameter, similar to migrating
Purkinje cells in E18 normal mouse embryos.
The confirmation of the nature of the immature
neurons has been obtained with immunohisto-
chemical markers since the migrating neurons
express calbindin (Fig. 5.18), as do migrating
Purkinje cells in control fetuses (Wassef et aI.,
1985).
~----------------------------------------------------------------------
Figure 5.21. Calbindin-immunoreactive cells are
tightly packed in a lateral swelling of the graft (LSG)
lying on the host cerebellar surface. Note the direct
penetration of immunoreactive Purkinje cells (arrows)
into the upper region ofthe host molecular layer (ML)
6 days after grafting. Bar = 30 tlm.
Figure 5.22. Same material as that in Fig. 5.21, but
103
The second early migratory pathway is formed
by calbindin-immunoreactive, postmitotic Pur-
kinje cells that, from the lateral swellings of the
graft lying on the surface ofthe host folia, are able
to pass directly into the host molecular layer
through discontinuities in the subpial basal lamina
(Figs. 5.20, 5.21). Glial markers (anti-glial fibrillary
acidic protein and antivimentin antibodies), used
on consecutive sections to those immunostained
with anticalbindin, provide a clear picture of the
size and disposition of the glia limitans below the
basal lamina. The endfeet of the host Bergmann
fibers facing the discontinuities in the subpial
basal lamina seem to grow out and invade the
graft, forming an unusual tuft of astrocytic proces-
ses among the grafted neurons (Fig. 5.22). Thus,
the holes in the subpial basal lamina provide a
direct communication between the graft and the
host molecular layer, with passage of glial fibers
(from host to graft) and of Purkinje cells as well
as glial processes (from graft to host) (Figs. 5.20,
5.22). We have no precise information about the
mechanisms underlying the breakage ofthe basal
lamina since in our material glial fiber sprouts
and migrating Purkinje cells are simultaneously
seen at the broken patches. However, the known
physiology of these two categories of neural cells
lead us to predict that the migrating Purkinje cells
could be responsible for this breakage. Indeed,
migrating neurons are provided with a leading
process and its terminal growth cone, and it has
been proven that neuronal growth cones are able
to release proteolytic enzymes. One of these re-
leased serine proteases is the urokinase-type
plasminogen activator that, by activation of
plasminogen to plasmin, can degradate matrix
components such as laminin and break the basal
lamina. Similarly, metalloproteinases, also com-
prised between the cell-associated proteases
present in neuritic growth cones, can be involved
in the breakage of the basal lamina (see refs. in
Monard, 1988).
immunostained with an antivimentin antiserum. The
arrowheads mark the external surface of the host mole-
cular layer. Note the fine immunopositive sprouts emerg-
ing from the host Bergmann fibers penetrating the
lateral swelling ofthe graft (LSG), and delineating the
discontinuities of the host subpial basal lamina. Bar =
30tlm.
104
P5
f20 PO
Figure 5.23. Calbindin-immunoreactive grafted Pur-
kinje cells in the molecular layer of a pcd mouse 11 days
after grafting. Note that the neurons have lost their
long smooth processes (compare Figs. 5.19 and 5.23)
and that they exhibit the morphology of Purkinje cells
in the second phase of dendritic development, with
short processes emerging in all directions from the cell
Constantino Sotelo and Rosa-Magda Alvarado-Mallart
PO
25
bodies. GL, granular layer; ML, molecular layer.
Bar = 40 11m.
Figure 5.24. Calbindin-immunoreactive grafted Pur-
kinje cells in the molecular layer of a pcd mouse 14
days after grafting. The arrow points to a distal Purkinje
cell in its third phase of dendritic development. ML,
molecular layer. Bar = 40 11m.
 5. Cerebellar Grafting
It is important to recall that both types of early
Purkinje cell migrations commonly coexist, and
that both of them are at the origin of the inward
migration of the grafted neurons. Indeed, 6-7
DAG, grafted Purkinje cells of both origins mas-
sively penetrate the host molecular layer. Those
arising from the tangential migratory stream do
so by changing polarity and adopting a bipolar
radial or somewhat oblique position. Those
directly passing from the lateral swellings of the
graft do so traversing the basal lamina and invad-
ing the host molecular layer. The inward-oriented
processes of both groups penetrate the whole
depth ofthe molecular layer, but do not enter the
granular layer, as if their permissive microen-
vironment abruptly stops at the molecular layer-
granular layer interface (Fig. 5.19). Occasionally,
the inward migrating Purkinje cells run parallel
to the host Bergmann fibers; we have even ob-
served direct appositions between these categories
of cells (Sotelo and Alvarado-Mallart, 1987c;
Sotelo et aI., 1990). More frequently, in single thin
sections there are no apparent cellular interactions
between the migrating Purkinje cells and the glial
axes (Fig. 5.26). In any case, this later phase of
radial migration resembles that occurring during
cerebellar ontogeny with one essential difference:
it takes place inward instead of outward. Normally,
Purkinje cells originate from the ventricular neu-
roepithelium and migrate outward to the cerebel-
lar plate, toward the surface of the nascent cere-
bellum.
The radially or obliquely oriented Purkinje
cells have, as migrating neurons do, a bipolar
shape with asymmetrically long processes emerg-
ing from the opposite poles of their elongated
cell bodies (Fig. 5.19). The latter are dispersed
throughout the superficial three quarters of the
host molecular layer. Ten DAG, the grafted Pur-
kinje cells have completely changed the shape and
disposition oftheir processes (see below) but main-
tain unchanged the position of their cell bodies.
This observation suggests that the migratory
~-----------------------------------------------------------------
Figure 5.25. Diagrammatic representation of the deve-
lopment of Purkinje cell dendrites in a control rat. The
drawings at E20 and PO illustrate Purkinje cells in
their first phase or "phase of the fusiform cells." The
drawing at P5 illustrates a Purkinje cell in its second
105
phase of the grafted neurons and the consecutive
perikaryal translocation are frozen when the
leading processes of the migrating neurons reach
the lowest limits of the host molecular layer. This
likely explains why the grafted Purkinje cells do
not attain their normal location at the interface
between the molecular and the granular layers,
and remain ectopic in the long-term survivals
(Sotelo and Alvarado-Mallart, 1986, 1987a).
Dendritic Differentiation
of Grafted Purkinje Cells
The anticalbindin immunostaining, which pro-
vides Golgi-like pictures of developing Purkinje
cells, has allowed a complete analysis of the den-
dritogenesis of the grafted Purkinje cells as well as
the evaluation ofthe kind of cellular interactions
taking place between immature and adult neurons
in the process of building up the dendritic trees
of the former.
Normal Purkinje cells develop their dendritic
trees following three sequential phases (Fig. 5.25),
called by Ramon y Cajal (1926): a) the phase of
the fusiform cell, b) the phase of the stellate cell
with disoriented dendrons, and c) the phase of
orientation and flattening of the dendrites. Re-
cently, Armengol and Sotelo (1991) have studied
the variety offorms present in the first phase, and
the time sequence as well as the cellular interac-
tions taking place during the passage from the
first to the second phase. Postmigratory Purkinje
cells, in 19- and 20-day-old rat embryos, preserve
their bipolar shape, although the long apical den-
dritic process branches into two asymmetrical
segments. These processes are smooth and long
and can receive synaptic contacts. During the first
two postnatal days, the shape of some of these
neurons evolves into more complicated forms
with basilar dendrites. An important change
occurs during the second through the fourth post-
natal day when the long dendrites disappear (as
well as their transient synaptic contacts), and
phase or "phase of the stellate cell with disoriented
dendrons." The drawing at P8 illustrates one of these
neurons in its third phase or the "phase of orientation
and flattening ofthe dendrites." Finally, the last draw-
ing to the right illustrates an adult Purkinje cell.
106 Constantino Sotelo and Rosa-Magda Alvarado-MaIIart
instead the neurons proceed to the formation of
a great number of perikaryal long filopodia, giv-
ing them the appearance of stellate cells (second
phase). By 3 days postnatally, when the synapto-
genesis between climbing fibers and Purkinje cells
starts (Crepel, 1971), most Purkinje cells have
finished the resorption or retraction of their pri-
mitive long apical and basilar dendrites and have
entered the "phase of stellate cells with disoriented
dendrons." The passage from the first to the second
phase seems to be intrinsic to Purkinje cells since
it occurs even in the absence of climbing fibers
(Armengol and Sotelo, 1991). By the end of the
first postnatal week, the growth cone at the apical
end of the stellate cell body has grown out a
flattened dendritic tree, with enrichment of termi-
nal spiny branch lets , and \-'1ith the disappearance
of most of the long somatic filopodia. The cell
has entered into the third and final phase of its
dendritic development. The passage from the
second to the third phase requires specific cell-
to-cell interactions, dealing mostly with the
synaptogenesis between the developing parallel
fibers and the growing dendrites of Purkinje cells
(see refs. in Sotelo, 1978).
The dendritic differentiation of grafted Purkinje
cells follows a process remarkably similar to that
just described for normal developing Purkinje
cells (Sotelo et aI., 1990). During their migratory
stage, these neurons exhibit a bipolar shape closely
resembling those commonly observed in the "phase
of the fusiform cell" (Fig. 5.19). The essential dif-
ference is that the long and asymmetrically branch-
ed dendritic process has not an apical position,
but instead it is basal and perpendicularly or
obliquely oriented toward the molecular layer-
granular layer interface. Grafted Purkinje cells
with bipolar shapes constitute the majority of
these neurons observed between 5 and 9 DAG,
corresponding to the biological ages of 17 em-
bryonic days and 1 postnatal day (the gestation
in the mouse lasts for 19 days). The main regres-
sive process that characterizes the formation of
Purkinje cell dendrites, the retraction of the long
and smooth dendrites, and the appearance of
long somatic filopodia (the passage from the first
to the second developmental phase) also occurs
in grafted Purkinje cells (compare Figs. 5.19 and
5.23). Thus, 10-11 DAG, the shape of these neu-
rons has completely changed. Despite their ectopic
location, the cells have acquired the typical ap-
pearance of cells in the "phase of the stellate cell
with disoriented dendrons" (Fig. 5.23). This change
in shape, which occurs when the grafted neurons
attain the biological age of 2 to 3 postnatal days,
coincides with the beginning of the synaptogenesis
between host climbing fibers and grafted Purkinje
cells (see next section), as is the case during cere-
bellar ontogeny. It is of interest to note that at
this age an important proportion of the grafted
neurons has also attained the third develop-
mental phase: these cells exhibit multiple, profusely
branched primary dendrites, like those commonly
observed 14-15 DAG.
In the cerebellum of mutants studied 14-15 DAG
(biological age for Purkinje cells: 6-7 postnatal
days), the perikarya of the ectopic neurons have
developed multiple primary dendritic segments
(two to six), profusely branching into secondary
and distal branches (Fig. 5.24). The latter are
studded with spines and correspond to incipient
spiny branchlets. Furthermore, the perikarya have
lost most of their long filopodia, but shorter and
thinner processes still remain. A main feature
characterizes the dendritic trees of these grafted
Purkinje cells: they span most of the host mole-
cular layer with arbors flattened in the sagittal
plane. This important feature is most evident in
those Purkinje cells at the most distal border of
the host molecular layer containing a low density
of grafted neurons: the dendrites of these cells
have a tangential disposition, with their axis
parallel to the cerebellar surface and perpendi-
cular to the bundles of host parallel fibers. Hence,
despite the anomalies in number and orientation
of the primary branches (due to the ectopic loca-
tion of their perikarya), grafted Purkinje cells at
this stage of differentiation resemble those reach-
ing the "phase of orientation and flattening of the
dendrites." This stage coincides with a phase of
active synaptogenesis between host parallel fibers
and grafted Purkinje cells (see next section), syn-
aptogenesis that will proceed for at least the whole
next week with an expanding rate. Thus, by 21
DAG, the grafted Purkinje cells are provided with
dendritic trees qualitatively similar to those re-
ported before in long-term survivals, indicating
the end of their maturation.
5. Cerebellar Grafting
Figure 5.26. Electron micrograph of a grafted Purkinje
cell with a subpial location (arrowheads point to the
basal lamina) penetrating the host molecular layer's
parenchyma. Note the immature appearance of the
cytoplasm and the absence of axon terminals synapsing
on the cell body. pcd cerebellum, 7 days after grafting.
Bar=2/lm.
107
Figure 5.27. Electron micrograph taken from aped
cerebellum 7 days after grafting. This micrograph illus-
trates a large descending process belonging to a grafted
Purkinje cell (PCD). The arrow points to a synaptic
contact between a host axon terminal and the grafted
Purkinje cell process. Bar = 1 /lm.
108 Constantino Sotelo and Rosa-Magda Alvarado-Mallart
Synapse Formation Resulting
in the Synaptic Integration
of Grafted Purkinje Cells
In the transplanted cerebella, grafted Purkinje
cells during their tangential and radial migration
(5-9 DAG) exhibit ultrastructural features of im-
mature neurons: absence of Nissl bodies and
presence of free polyribosomes suspended in the
cytoplasmic matrix (Fig. 5.26). In this respect, the
grafted neurons resemble Purkinje cells of similar
ages (E17 to PI) in normal perinatal mice. Hence,
the large diameter of the cell bodies and processes
as well as their immature neuronal appearance
provide good markers for the ultrastructural
identification of the grafted Purkinje cells in the
adult neuropil of the host molecular layer.
A careful analysis of the synaptic investment
of the migrating Purkinje cells (Sotelo et at, 1990)
shows that, despite their proximity to the adult
cerebellar neuropil containing abundant axon
terminals, they practically lack synaptic inputs
(Fig. 5.26). Only occasionally, small axon termi-
nals establish a few synaptic junctions on the long
and smooth dendritic processes of the migrating
neurons (Fig. 5.27). These rare synapses resemble
those observed in the cerebellum of perinatal rats,
from E 19 to P2 (West and Del Cerro, 1976). They
most probably represent, as in the normal cere-
bellum (Armengol and Sotelo, 1991), transient
synapses, since they are established on tran-
sient dendritic branches that regress when the
Purkinje cells reach the second phase of their den-
dritic development (see preceding section). The
functional meaning of these transient synaptic
contacts is not yet elucidated.
Ultimate synaptogenesis between host axons
and grafted Purkinje cells starts 10-11 DAG,
when the neurons reach the biological age of 2 to
3 postnatal days. At this developmental stage, the
neurons have lost their long and smooth dendrites
and have begun to grow their final dendritic trees
("phase of the stellate cell with disoriented den-
drons"). One ofthe main features of this phase is
the production of perikaryal filopodia (Fig. 5.28),
which in normal ontogeny of the cerebellum cor-
respond to the postsynaptic elements of the early
synapses between climbing fibers and Purkinje
cells (stage of pericellular nest of Ramon y Cajal,
1911). Some of the somatic processes emerging
from the grafted Purkinje cells are directly appos-
ed to host-beaded axons, some oftheir varicosities
establishing asymmetric synaptic contacts on the
somatic filopodia (Fig. 5.28). These beaded axons,
in addition to the typical organelles of climbing
fibers (densely packed rounded agranular vesicles,
a few large granulated vesicles, abundant micro-
tubules, etc.) can also contain numerous tubular
profiles of the smooth endoplasmic reticulum that
resemble those in growth cones, and therefore
have been considered to be axonal sprouts of the
host climbing fibers.
The number of climbing fibers synapsing on
somatic filopodia of the grafted Purkinje cells is
relatively low, and the vast majority of the filo-
podial and smooth perikaryal membrane is free
of synaptic inputs. Despite this low incidence of
synapses, in some rare instances it is possible to
observe an axon terminal with pleiomorphic vesi-
cles establishing a symmetric synaptic contact on
the soma of a grafted neuron (Fig. 5.29). The axon
terminals engaged in these rare synapses belong
to axons of host molecular layer interneurons,
stellate, and/or basket cells. Furthermore, the
growing dendrites of the grafted Purkinje cells
begin to produce a few spines that occasionally
are synaptically contacted by varicosities of the
host parallel fibers. Hence, although the majority
of the observed synapses on Purkinje cells at
10-11 DAG involve host climbing fibers, some
rare synapses belonging to axons of host stellate
or basket cells as well as to host parallel fibers
have also been established at this early stage of
the grafted Purkinje cells' development.
Between 12 and 14 DAG, synaptogenesis is
very active. Host climbing fibers succeed in form-
ing complete pericellular nests around the somata
of the grafted Purkinje cells, and their en passant
axon terminals establish numerous asymmetric
synaptic contacts on the thin somatic filopodia.
Simultaneously, the frequency of symmetric syn-
aptic complexes between host axon terminals,
arising from stellate or basket cell axons as well
as from axon collaterals of the grafted Purkinje
cells, and the smooth surface of the bodies of the
latter rapidly increases. Hence, during this short
phase of development there is a coexistence of
climbing fibers and stellate or basket cell axons
on the surface ofthe grafted Purkinje cell somata.
The dendritic trees of the latter proceed with their
5. Cerebellar Grafting
Figure 5.28. This electron micrograph illustrates the
early formation of host climbing fiber-grafted Purkinje
cell synapses. Two varicosities of a host climbing fiber
(CF) are partially covered by long somatic filopodia
(asterisks). The arrow points the synaptic contact
between one of these varicosities and a somatic filo-
podium. pcd grafted cerebellum 11 days after grafting.
GPC, grafted Purkinje cell body. Bar = 1 flm .
109
Figure 5.29. Same material as that in Fig. 5.28. A
host axon terminal (AT), most probably belonging to
an ascending collateral of a basket cell axon, containing
tubular profiles of the smooth endoplasmic reticulum
and pleiomorphic synaptic vesicles, establishes a sym-
metric synaptic contact (arrow) on the smooth surface
of the grafted Purkinje cell body (GPC). Bar = 1 flm.
110 Constantino Sotelo and Rosa-Magda Alvarado-Mallart

Figure 5.30. Electron micrograph from a pcd grafted
cerebellum, 14 days after grafting. This micrograph
illustrates the translocation of a host climbing fiber.
One varicosity of this fiber (ATi) has a perisomatic
location around the cell body of a grafted Purkinje cell
(GPC) and synapses on somatic filopodia (asterisks).
Another varicosity (AT2) has a peridendritic location
(GPD, grafted Purkinje cell dendrite) and synapses on
primary dendritic spines (arrowheads). Bar = 1 pm.
Figure 5.31. Same material as that in Fig. 5.30, but
treated with a postern bedding immunogold method to
detect GABA. An axon terminal containing pleiomor-
5. Cerebellar Grafting
ultimate. growth, and axonal varicosities of the
host parallel fibers establish abundant asymmetric
synaptic contacts on newly formed dendritic
spines.
By 14 DAG, the grafted Purkinje cells have
reached their third phase of dendritic develop-
ment. At this stage, the process of climbing fiber
translocation, from their somatic to their final
dendritic location, has already started. Hence,
climbing fiber varicosities, although still numer-
ous at the somatic level, are also present parallel
to the primary dendritic branches of the grafted
neurons, establishing asymmetric synapses on
clusters of stubby spines that emerge from those
dendritic branches (Fig. 5.30). Similarly, axon ter-
minals belonging to the host stellate or basket
cell axons either keep their somatic location or
begin to synapse on the smooth surface of the
proximal dendrites. The use of postembedding
gold immunostaining, with an anti-gamma-
aminobutyric acid (GABA) antibody (as previous-
ly described by Angaut and Sotelo, 1987), has
revealed the GABA-ergic nature of the axon ter-
minals with pleomorphic vesicles and symmetric
synaptic contacts. Both those synapsing on the
smooth perikaryal surface (Fig. 5.31) and on the
smooth dendritic surface of the grafted Purkinje
cell exhibit a positive immunoreaction.
As stated above, by 14 DAG the grafted Purkinje
cells are provided with sagittally oriented, flatten-
ed dendrites comprising a proximal and a distal
compartment. The latter consists of rapidly form-
ing spiny branchlets. Synaptogenesis between
host parallel fibers and branchlet spines proceeds
at a high rate. This situation is favorable for the
analysis of the sequential phases in the develop-
ment of host parallel fiber-grafted Purkinje cell
synapses, since newly formed and more mature
synapses coexist. The distal dendritic spines, as
in control mouse cerebellum by the end of the
first postnatal week (Larramendi, 1969), appear
as long and slender processes, generally devoid
of postsynaptic differentiation, growing within
the adult neuropil of the host molecular layer.
~------~--------------------------------------------------------------
phic synaptic vesicles and immunogold particles is in
synaptic contact with the smooth surface of a grafted
Purkinje cell body (GPC). The arrow points to the
symmetric synaptic complex. Bar = 0.5 11m.
Figure 5.32. Same material as that in Fig. 5.30. This
111
The most immature synaptic contacts are estab-
lished on the distal region of the head of these
slender spines. They are characterized by their
long synaptic complexes that can reach a length
more than twice that of adult parallel fiber-
Purkinje cell synapses. More mature synaptic
contacts are more numerous and can be identified
easily because they bear synaptic complexes of
decreased size (Fig. 5.32), similar to those encoun-
tered in control adult molecular layer. Hence,
the formation of host parallel fiber- Purkinje cell
synapses follows a sequence of events similar to
those operating during cerebellar ontogeny of
control mouse, with the same regressive processes
("synaptic adhesion waning" of Larramendi, 1969).
By 21 DAG, the synaptic investment of the
grafted Purkinje cells is qualitatively similar to
that reported in the first part of this chapter in
long-term survivals. Thus, after this period of
development, only quantitative changes result
from the continuing synaptogenesis.
General Conclusions
The results obtained in the series of short-term
survivals indicate that grafted Purkinje cells are
able to pursue their developmental program in
the cerebellum of the adult host mutant mouse.
Indeed, the sequential critical steps followed by
Purkinje cells during normal cerebellar morpho-
genesis are recapitulated by the grafted cells.
Cellular proliferation, migration, dendritic devel-
opment, and synaptogenesis proceed according
to the same time sequence as in the control cere-
bellum.
As far as our results allow us to determine,
Purkinje cell proliferation, which proceeds with
a time window similar to that characterizing
Purkinje cell precursors in control mouse embryos,
occurs only in the grafted primary neuroepithelium,
within the transplanted cerebellar mass. What-
ever the nature of the attracting forces of the host
Purkinje cell-deficient molecular layer, these are
only exerted on young postmitotic Purkinje cells.
electron micrograph illustrates synaptic contacts (arrow-
heads) between host parallel fiber varicosities and
spines emerging from a distal spiny branchlet of a
grafted Purkinje cell (GPD). Bar = 1 Jim.
112
In this respect, the environmental changes implied
in the transplantation do not alter the normal
behavior of the Purkinje cell precursors.
Purkinje cell migration seems to be the result
of the specific attraction of the deficient molecular
layer of the host cerebellum. This attraction can-
not be exerted before the physical integration of
the implants into the adult host cerebella, integra-
tion that is completed only 3 DAG. The early
pathways followed by the young postmitotic
Purkinje cells exhibit abnormalities with either
tangential migration over the surface of the neuro-
pil of the host molecular layer or direct penetra-
tion from the lateral swelling of the graft, after
breaking the subpial basal lamina. Despite these
abnormalities, these neurons penetrate the host
molecular layer through a radial and/or oblique
migration, resembling that followed by postmito-
tic Purkinje cells during normal development,
although they follow an inward instead of an
outward direction. Our study also shows that the
migratory phase lasts for 4 days and, therefore,
that the biological age of grafted Purkinje cells
at the end of their migratory period is E19, the
age at which the migration of these neurons is
also completed in mouse fetuses. The cellular
mechanisms involved in normal Purkinje cell
migration are not well established. It has been
suggested, in view of the radial and/or oblique
trajectory of their migration from the subventri-
cular zone to the cortical plate, that they may use
a radial glial axis, as other cortical neurons do
(Rakic, 1984). The grafted Purkinje cells also
exhibit radial and/or oblique trajectories during
the last phase of their migration, and some of
them appear directly apposed to Bergmann fibers
(Sotelo and Alvarado-Mallart, 1987c). We thus
can conclude that their migration may follow
cellular mechanisms similar to those used during
the normal cerebellar morphogenesis. In any case,
the time window of the migratory period is the
same for Purkinje cells in normal cerebellum as
that in implants grafted into adult pcd cerebellum.
At the end of their migration, the grafted
Purkinje cells have typical bipolar shapes and
begin to build up their ultimate dendritic trees.
Our morphological analysis has revealed that the
three developmental phases described by Ramon
y Cajal (1926) for Purkinje cell dendritogenesis
are recapitulated by the grafted neurons. These
Constantino Sotelo and Rosa-Magda Alvarado-Mallart
observations indicate that the cell-to-cell interac-
tions regulating the molding of these extraordinary
dendritic arbors in cerebellar ontogeny (see refs.
in Sotelo, 1978) are also operative in the trans-
planted cerebella. The acquisition of the two es-
sential features of a Purkinje cell dendritic tree
(the flattening of the dendrites as well as their
compartmentalization with issuing input segre-
gation) also occurs in the grafts simultaneously
with the phase of highest synapse formation,
particularly between host adult parallel fibers
and newly emerging spines from grafted Purkinje
cell dendrites. Hence, as already suggested by
Ramon y Cajal (1960) for normal cerebellar mor-
phogenesis, the parallel fibers are the main
organizing element for the flattened disposition
of the grafted Purkinje cell dendrites and for the
induction of distal spiny branchlets. Therefore,
the adult parallel fibers are able to proceed accord-
ing to the same developmental rules that apply
during their normal morphogenesis, when they
are experimentally confronted with specific im-
mature postsynaptic partners.
Despite the apparent similarities in the cellular
mechanisms involved in Purkinje cell dendrito-
genesis, the timing in the maturation of dendritic
trees from grafted Purkinje cells is slightly advanc-
ed when compared with that observed during
normal dendritogenesis. This precocity is effec-
tive only from the passage from the second to the
third phase of dendritic maturation. Indeed, the
acquisition of the bipolar shape needed for migra-
tion (first phase) and the regression of the long
and smooth dendrites (second phase) seem to be
the consequence of intrinsic mechanisms regulat-
ed by the Purkinje cells themselves (Armengol
and Sotelo, 1991) and independent from synapto-
genesis. Conversely, as stated above, the forma-
tion of the ultimate dendritic tree depends on
synaptogenesis, and the latter is somewhat more
precocious in the grafts than in normal cerebellar
ontogeny (see below).
It is also remarkable to note that synapse for-
mation between grafted Purkinje cells and adult
host presynaptic axons proceeds according to a
precise program, recapitulating all of the different
steps described during normal cerebellar synap-
togenesis (Larramendi, 1969). These steps can
be summarized as follows: a) early appearance of
transient synapses on the long and smooth dend-
5. Cerebellar Grafting
rites of grafted Purkinje cells during their migra-
tion, b) beginning of earliest synaptic contacts
between host climbing fiber sprouts and filopodia
from grafted Purkinje cell perikarya, to establish
pericellular nests (Ramon y Cajal, 1890), c) synap-
togenesis between slender, distal dendritic spines
and host parallel fibers, together with that of ax on
terminals from host molecular layer interneurons
and the smooth surface of grafted Purkinje cell
perikarya, and d) the translocation of host climb-
ing fibers from their perisomatic to their final
peridendritic location.
Despite the accelerated rhythm ofthe synapto-
genesis (the above-mentioned sequential steps
taking place somewhat faster in the grafts than
in normal development), the timing for its begin-
ning is the same in both situations. In both in-
stances, the starting point seems to be marked by
the maturity of the Purkinje cells, since the synap-
togenic process begins only when these neurons
attain the second phase of their dendritic devel-
opment. These results indicate that the presence
of immature Purkinje cells in the molecular layer
of the deficient mutant cerebellum provokes the
sprouting of specific adult host axons with sub-
sequent synaptogenesis. The observed differences
in timing may be explained by the fact that, dur-
ing reactive synaptogenesis, all presynaptic ele-
ments are available for synaptic contact in the
vicinity of grafted Purkinje cells from the beginning
oftheir invasion into the host molecular layer. In
contrast, during normal development most of the
neurons providing presynaptic axons to the matur-
ing Purkinje cells (granule, stellate, and basket
cells) do not end their proliferative period before
the first postnatal week (Fujita, 1967; Miale and
Sidman, 1961).
It has been established (Mariani and Changeux,
1981;Crepelet aI., 1976, 1981)thatoneimportant
feature of normal cerebellar synaptogenesis is the
presence of a phase, lasting about 12 days, of re-
dundancy of the connections between climbing
fibers and Purkinje cells. This phase is followed
by a regressive process of numerical adjustment,
allowing the adult one-to-one relationship. Re-
cently, Gardette et al. (1990) have performed an
electrophysiological study of the reactive synap-
togenesis in grafting experiments and have shown
that during the establishment of synaptic connec-
tions between adult host climbing fibers and grafted
113
Purkinje cells there is also a transient stage of
multiple innervation. This stage lasts only between
10 and 13 DAG and can be detected by the step-
wise variation in amplitude of the climbing fiber
EPSPs recorded during this period. The electro-
physiological results strongly suggest that the
cell-to-cell interactions regulating the synapse
formation between adult climbing fibers and im-
mature Purkinje cells are of the same nature as
those taking place during normal development.
Moreover, since the transient period of multiple
innervation is shorter and, as discussed above,
synaptogenesis between host parallel fibers and
grafted Purkinje cells also occurs in a shorter time
window than in normal cerebellar morphogene-
sis, we suggest that in the grafts, the regression of
the multiple innervation could be in part due
to competition with parallel fiber-Purkinje cell
synapses.
The most important conclusion from all these
studies on short-term survivals is the striking
similarity in the cellular mechanisms and chrono-
logy ofthe sequential steps leading to the matura-
tion and synaptic investment of Purkinje cells
during their normal ontogeny and when grafted
to the adult pcd cerebellum. This remarkable cor-
respondence allows us to postulate that matura-
tion of grafted Purkinje cells follows an internal
clock that regulates all their developmental pro-
grams, independently of environmental signals.
Thus, the presence of immature Purkinje cells in
the deficient molecular layer of the host would
allow adult neurons and glial cells to behave
transiently as if they were young postmitotic cells,
and to interact with the grafted Purkinje cells
according to a tempo imposed by the latter. The
most likely, but not necessarily unique, interpre-
tation ofthis adaptive behavior is that the grafted
Purkinje cells themselves regulate gene expression
of adult neural cells by generating a transient
permissive microenvironment. The end result of
this plastic process will be the quasinormal devel-
opment of the grafted Purkinje cells and, above
all, their synaptic integration into the deficient
cortical circuitry. The interplay between hosts
and grafts, allowing the specific migration of the
missing Purkinje cells, and the regulatory role
played by the latter in the plastic behavior of adult
neurons of the host lead to the restoration of the
impaired circuitry of the mutant cerebellar cortex.
114 Constantino Sotelo and Rosa-Magda Alvarado-Mallart

Acknowledgments. We thank D·rs. D.M.D. Landis Dodd, J., and Jessell, T.M. (1988): Axon guidance and
and R.V. Rouse for helpful criticism of the manu- the patterning of neuronal projections in vertebrates.
script; Drs. P. Greengard, D.E.M. Lawson, Science, 242, 692-699.
S. Sharma, A.M. Hill, and D. Dahl for gifts, res- Fujita, S. (1967): Quantitative analysis of cell prolifera-
pectively, of anti-GMP cyclic dependent protein tion and differentiation in the cortex ofthe postnatal
mouse cerebellum. J. Cell Bioi., 32, 277-288.
kinase, calbindin, Thy-l.1, vimentin, and GFAP
Gardette, R., Alvarado-Mallart, R.M., Crepel, F., and
antibodies; Dr. J.L. Guenet for a continuous sup- Sotelo, e. (1988): Electrophysiological demonstration
ply of ped mutant mice; and Dr. S. Martinez for of a synaptic integration of transplanted Purkinje
the schematic drawings. cells into the cerebellum of the adult "Purkinje cell
degeneration" mutant mouse. Neuroscience, 24,
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6
Light and Electron Microscopic
Immunocytochemistry of Putative
Neurotransmitter Amino Acids in the
Cerebellum with Some Observations on the
Distribution of Glutamine
Ole P. Ottersen and Jon H. Laake

Ramon y Cajal (1888, 1889) was the first to des- Mathisen et aI., 1983), glycine (Dale et aI., 1986;
cribe accurately the different cell types in the Ottersen et aI., 1986, 1987), taurine (Madsen et aI.,
cerebellum and their interconnections. With the 1985; Ottersen et aI., 1985), glutamate (Ottersen
development ofthe electron microscope, the ultra-and Storm-Mathisen, 1984b; Storm-Mathisen
et aI., 1983), aspartate (Ottersen and Storm-
structural features of the different types of cells
and synapses were soon characterized in great Mathisen, 1985), and glutamine (Laake et aI.,
detail, so that today the synaptology of the cere-
1986). All sera were raised against protein-glutaral-
dehyde conjugates of the respective amino acids
bellar cortex must be regarded as well established
(Mugnaini, 1972; Palay and Chan-Palay, 1974). (Storm-Mathisen et aI., 1983) and were subjected
In contrast, our understanding of the chemical to one or several purification steps in solid or
nature of the cerebellar circuitries is still incom-
liquid phase before satisfactory selectivity was
obtained (for review see Ottersen and Storm-
plete. Early biochemical studies and investigations
based on immunocytochemistry of the gamma- Mathisen, 1987). The observations presented here
amino butyric acid (GABA) synthesizing enzyme, were made in postembedding-labeled sections
glutamic acid decarboxylase (GAD), strongly of Durcupan-embedded rat cerebella. For light
suggested that amino acids played major roles as microscopy, semithin (0.5 /lm) sections were etched
transmitters in the cerebellum, as in other partswith sodium ethanolate and subsequently process-
of the central nervous system (CNS) (for reviews ed according to the peroxidase-antiperoxidase
see Mugnaini and Oertel, 1985; Ottersen and procedure as previously described (Ottersen, 1988;
Storm-Mathisen, 1984a). However, it was not Somogyi et aI., 1984). For electron microscopy,
until recently that the neuroactive amino acids ultrathin sections from the same blocks were col-
themselves could be visualized by immunocyto- lected on mesh grids and then treated sequential-
chemistry (Storm-Mathisen et aI., 1983), thus ly with HI0 4 , NaI0 4 (to remove osmium), the
becoming amenable to precise anatomical analysis.primary amino acid antiserum, and a secondary
In this chapter we show how amino acid im- antibody coupled to colloidal gold particles (for
munocytochemistry has provided new insight in details, see Ottersen et aI., 1988a; Somogyi and
the organization of the amino acid transmitter Hodgson, 1985). The density of gold particles in
systems in the cerebellum. the different neuronal profiles could be assessed
by a computer program (Morforel) designed for
this purpose (Blackstad et aI., 1990).
Methods Most of the material presented is from rats that
Work in our laboratory has led to the production were perfusion fixed through the heart with a
and characterization of antisera against GAB A mixture of 1%paraformaldehyde and 25% gluta-
(Ottersen and Storm-Mathisen, 1984b; Storm- raldehyde. Alternatively, in order to study the

116
6. Immunocytochemistry of Putative Neurotransmitter Amino Acids
synaptiG: handling of the amino acids, some cere-
bella were rapidly removed from anesthetized
rats, cut into sagittal slices, and kept in artificial
cerebrospinal fluid in vitro before immersion in
the same fixative as above. Some ofthe slices were
exposed to 5 mmoljI K + (= physiological concen-
tration) throughout, whereas others were exposed
to 55 mmoljl K + (after an initial period of stabi-
lization in physiological K +) to evoke synaptic
release. A similar strategy was previously used
by Storm-Mathisen et ai. (1986a, b) to study the
synaptic handling of amino acid transmitters in
the hippocampus. The calcium dependency of
the release process was examined in the presence
of 0.1 mmoljI Ca2+ and 1Ommoljl Mg2+.
Specificity of the antisera was tested by screen-
ing them against more than 40 different small
molecular compounds that had been coupled to
rat brain protein by means of glutaraldehyde and
subsequently spotted on Millipore filters for in-
cubation together with Vibratome or frozen tissue
sections (Ottersen and Storm-Mathisen, 1984b),
or embedded in resin for incubation together
with plastic sections (Ottersen, 1987). A series of
such test conjugates was routinely included in the
immunocytochemical experiments for continuous
monitoring of specificity (see Figs. 6.3, 6.8, 6.10,
6.11). The latter test conjugates were prepared
from the six most abundant amino acids in the
brain and were incorporated in a sandwich that
could be processed in the same drops of sera as
the tissue sections. This strategy insured identical
conditions for testing and immunocytochemistry
on the light microscopical as well as on the elec-
tron microscopical levels. With the electron micro-
scope it was possible to quantify the immuno-
labeling of the different amino acid conjugates by
assessment of the density of gold particles over
them (Ottersen, 1987). All antisera produced a
highly selective labeling of the conjugate pre-
pared from the amino acid used for immuniza-
tion (see Figs. 6.3,6.8,6.10,6.11). No significant
affinity could be detected toward glutaraldehyde-
treated brain macromolecules, indicating that
amino acids incorporated in proteins were not
recognized by the specific antibodies.
The antisera were also tested against the small
molecular fraction of a crude rat brain homo-
genate. This was done after separation of the frac-
tion by thin layer chromatography. The chro-
117
matograms were sprayed with glutaraldehyde
and poly-L-Iysine and then processed exactly as
for immunocytochemistry (Ottersen, 1988). The
taurine antiserum, for instance, only labeled one
band in this system, the position of which corres-
ponded to that of authentic taurine (see Fig. 6.3D).
The glutamate antiserum labeled a different band
that had comigrated with authentic glutamate.
Similar results were obtained with the other anti-
sera tested. This test system should in principle
pick up any unknown cross-reacting small mole-
cule, provided it occurs in a sufficiently high con-
centration.
The closest one can come to proving specificity
is to show that the immunoreactivity disappears
when the proposed antigen is selectively removed
from the tissue. This can be achieved with taurine,
but only in the species that are totally dependent
on dietary taurine due to lack of a synthetic
machinery. Cats fall into the latter category
(Sturman et aI., 1985). Cerebellar sections ob-
tained from 8-week-old kittens deprived of dietary
taurine both prenatally and postnatally displayed
an almost total lack of taurinelike immunoreac-
tivity (taurine-LI), supporting the specific nature
of our antiserum (Madsen et aI., 1990).
Results and Discussion
GABA and Glycine
Of all neuroactive amino acids in the cerebellum,
GABA is the one that has been studied in greatest
detail. Early biochemical and immunocytoche-
mical studies revealed the presence of GAD in
basket, stellate, and Golgi cells (Fonnum et aI.,
1970; McLaughlin et al., 1974; Oertel et aI., 1981;
Ribak et aI., 1978; Saito et aI., 1974), and the
same cell types have also been found to contain
GABA-LI (Gabbott et aI., 1986; Ottersen et aI.,
1988a; Ottersen and Storm-Mathisen, 1984b;
Seguela et aI., 1985; Somogyi et aI., 1985). Both
markers also occur in the axons and axon ter-
minals of the Purkinje cells (cf. Fonnum and
Walberg, 1973), whereas results differ somewhat
concerning the level of each of these markers in
the Purkinje cell bodies (see, e.g., Gottlieb et aI.,
1986; Mugnaini and Oertel, 1985; Wu et aI., 1986).
Our rat material consistently shows a low level
of GABA-LI in the Purkinje cell bodies, at the
118
Figure 6.1. Adjacent semi thin sections through perfu-
sion-fixed rat cerebellar cortex, showing the distribution
of GABA-LI (A) and glycine-LI (B). The majority of
the Golgi cells (long arrows) contain both immunore-
activities, whereas others are single-labeled for
glycine (crossed arrow) or GABA (not shown). Most of
the glomeruli (single arrowheads) also appear to colo-
light as well as at the electron microscopic levels
(Figs. 6.1, 6.2).a The Purkinje cells differ from the
other types of putative GABA-ergic neurons in
the cerebellum by the length of their axons, and
the low level of GABA in the Purkinje cell bodies
may simply reflect a rapid transfer of newly formed
GAD into the axons, allowing little time for GABA
synthesis in the cell bodies.
Glycine-LI occurs in a sizeable subpopulation
of the Golgi neurons (Fig. 6.1). Analysis of conse-
cutive semi thin sections revealed that about 70%
of the Golgi cells contained immunoreactivities
a Purkinje cells of chicken (Matute and Streit, 1986)
and pigeon (Domenici et ai., 1988) have been reported
to contain a higher level of GABA-LI than Purkinje
cells of rats. In the pigeon the Purkinje cells were
particularly strongly labeled in folium X.
Ole P. Ottersen and Jon H. Laake
'GLY
'-
calize GABA and glycine. The vast majority of the cell
bodies in the molecular layer are labeled for GABA,
but unlabeled for glycine (double arrowhead). Asterisks,
Purkinje cells; short arrows, glycine immunoreactive
fibers in the molecular layer. MO, molecular layer; GC,
granule cell layer; WM, white matter. Bar = 50 pm.
(Modified with permission from Ottersen et ai., 1988a.)
for GABA as well as glycine (Fig. 6.1); (Ottersen
et aI., 1988a). The remaining Golgi neurons were
labeled for either GABA-LI or glycine-LI. Colo-
calization of the two amino acids was also de-
monstrated at the level of the Golgi cell terminals
by means of the postembedding immunogold
technique (Ottersen et aI., 1988a). In contrast,
the stellate and basket cells appeared to contain
GABA, but, with few exceptions, little or no glycine
(Fig. 6.1). The modest number of glycine immu-
nopositive terminals that were encountered in
the molecular layer displayed the ultrastructural
characteristics of stellate cell terminals and were
found to be double-labeled for GABA whenever
this was tested (Ottersen et aI., 1988a).
The biological significance of a colocalization
of GAB A and glycine in the same neurons remains
to be explored. The Golgi cell terminals are
6. Immunocytochemistry of Putative Neurotransmitter Amino Acids
Figure 6.2. Electron micrographs showing the distri-
bution of GABA-LI at the transition between the
Purkinje cell and granule cell layers (main picture), and
in the deep part of the granule cell layer (inset). From
same specimen as Fig. 6.1. Note strong immunolabeling
of basket cell terminals (B) and of Golgi cell bodies
(Go). High densities of gold particles are also found
over basket cell axons (asterisks). Purkinje cell bodies
depleted of both immunoreactivities on stimulation
by high K+(Ottersen et al., 1990a}, showing that
both amino acids belong to a releasable pool.
This is in agreement with data on biochemically
recorded eH] GABA and eH] glycine release
119
(P) are weakly immunolabeled. Gr, granule cell. Inset
shows part of a glomerulus. The Golgi cell boutons
(b) display a high concentration of gold particles,
whereas the granule cell dendrites (d) and the mossy
fiber boutons (Mf) display gold particle concentrations
similar to that over empty resin. Bars: main picture =
0.7 flm; inset = 0·4 flm.
from purified glomerulus fractions (Morales and
Tapia, 1987). Both amino acids were reported to
show a Ca 2 +-dependent release, although the
glycine release appeared to be more strictly Ca 2 +
dependent than the GABA release (Morales and
120
Figure 6.3. Photomicrographs showing distribution of
taurine-LI in a sagittal section of perfusion-fixed rat
cerebellar cortex (posterior vermis). A: Taurine-LI
occurs in Purkinje cell bodies (large asterisk), Purkinje
cell dendrites (small arrowheads), and in Purkinje cell
axons (crossed arrow), but is not detectable in stellate
and basket cell bodies (large arrowheads), Bergmann
glial processes (small arrows), and in the granule cell
layer (GC). Small asterisks: Pial surface. M 0, molecular
layer. B: Enlarged detail from A, showing that taurine-
LI extends into the Purkinje cell dendritic spines (arrow-
heads). Inset in B: Test section containing brain protein-
glutaraldehyde-amino acid conjugates prepared from
the six most abundant amino acids in the brain. Code:
1. GABA, 2. glutamate, 3. taurine, 4. glycine, 5. none
(i.e., glutaraldehyde-treated brain protein), 6. aspartate,
and 7. glutamine. Note selective labeling of the taurine
conjugates. The test section was incubated together
with the tissue section shown in A and B. C: Taurine-
Ole P. Ottersen and Jon H. Laake
LI in the nucleus interpositus anterior. Note strong
immunolabeling ofaxons (arrows) and nerve terminal-
like puncta, some of which (arrowheads) appear to
contact unlabeled cell bodies (asterisks). D: Specificity
testing. The soluble fraction of a total brain extract (E)
was applied to a cellulose gel together with free gluta-
mate (G) or free taurine (T). After separation in n-
butanol: acetic acid: H2 0 (4: 1: 1), the chromatograms
were sprayed with glutaraldehyde and poly-L-lysine,
and then processed exactly as for immunocytochemis-
try. The taurine antiserum labeled a single spot that
had comigrated with authentic taurine (right half of
figure), whereas the glutamate antiserum labeled a
separate band that had comigrated with authentic glu-
tamate (left half of figure). E: Tissue section and test
section (inset) treated with a taurine antiserum that
had been preadsorbed with glutaraldehyde-taurine com-
plexes (200 liM with respect to the amino acid). Bar =
25 lim. (From Ottersen, 1988).
6. Immunocytochemistry of Putative Neurotransmitter Amino Acids 121

Tapia, 1987). However, the issue is complicated

by the fact that Triller et al. (1987), using im-
munocytochemistry, found glycine receptors to
be localized postsynaptic to GAD-negative Golgi
cell terminals but not to GAD-positive ones. b
Thus, the possibility should be considered that
the released glycine partly acts on strychnine-
i~sensitive glycine receptors that are not recog-
nIzed by the receptor antibodies used.
Our finding of glycine-LI in Golgi cells receives
s~pport from an immunocytochemical investiga-
tIon based on a glycine antiserum different from
that used here (Campistron et aI., 1986a), and
from autoradiographic data showing that Golgi
cell boutons are endowed with high affinity uptake
mechanisms for [3H] glycine (Wilkin et aI., 1981).

Taurine
Taurine has attracted a great deal of interest as
a possible transmitter in the cerebellum following Figure 6.4. Electron micrograph of ultrathin section
through the molecular layer, treated with a taurine
the work of Yarbrough et ai. (1981) and Okamoto
antiserum. From the same specimen as in Fig. 6.3. The
et al. (1983a-d). These groups showed in the rat
Purkinje cell dendritic spines (S) are greatly enriched
and guinea pig, respectively, that the synaptically in taurine-LI, whereas the parallel fiber terminals (PI)
evoked inhibition of the Purkinje cells produced are moderately labeled (see Fig. 6.6). Bar = O.4jlm.
by electrical stimulation of the superficial part of
the molecular layer was blocked or substantially
reduced by 6-aminomethyl-3-methyl-4H, 1,2,4-
hibit the morphological features of stellate cell
benzothiadiazine-l,l-dioxide (TAG). The same
terminals and that can be found to contain GABA
compound also blocked the action of microin-
in adjacent sections (Fig. 6.6) (Ottersen et aI.,
jected taurine, but not that of GABA, on the
1988b). The electrophysiological studies described
Purkinje cells. These and additional results sug-
above and the immunocytochemical data from
gested that the actual transmitter in the stellate
this and other laboratories (Campistron et aI.,
cell synapses with the Purkinje cells was taurine.
1?86b; Ida et aI., 1987; Yoshida et al., 1986) are
It was therefore surprising to find, in our initial
dIfficult to reconcile; possible explanations have
study oftaurine in the cerebellum, that only a very
been discussed previously (Ottersen, 1988; Ottersen
small proportion ( < 2%) of the stellate neurons
et aI., 1988b). Importantly, the stellate cell termi-
were labeled with our taurine antiserum (Madsen
nals are depleted ofGABA-LI in slices exposed to
et aI., 1985). In contrast, taurine-LI occurred in
high K + in vitro (Ottersen et aI., 1990a), showing
high concentrations in the Purkinje cells. These
that the GABA that they contain belongs to a
results have since been confirmed and extended by
releasable pool. This effect is largely Ca 2 + de-
postembedding immunocytochemistry (Figs. 6.3,
pendent.
6.4, 6.5) (also see Ottersen, 1988). Quantitative
The enzyme cysteine sulphinic acid decarboxy-
electron microscopic analysis of ultrathin sections
lase (CSAD) has been proposed to be of major
treated with the taurine antiserum shows a modest
importance in the synthesis oftaurine in the CNS
density of gold particles over terminals that ex-
(for review see Wright et aI., 1986). Immunocyto-
~hemical studies have suggested that this enzyme

b Araki et al. (1988) and van den Pol and Gorcs (1988) IS concentrated in a subpopulation of Purkinje
~ound no or very low glycine receptor immunoreactivity cells that form sagittally oriented bands through-
III the granule cell layer of the cerebellum. out the cerebellar cortex (Chan-Palay et aI., 1982).
122
Within these bands, CSAD-LI also occurred in
other cell types, including stellate and granule
cells. Similar bands could not be detected by
visual inspection of transverse cerebellar sections
treated with our taurine antiserum. Specifically,
a careful analysis of consecutive semi thin sections
through the vermis and intermediate zone (where
the CSAD-positive bands are reported to be very
conspicuous) showed that all cells that could be
identified as Purkinje cells were taurine immuno-
reactive (Ottersen, 1988). Moreover, optical den-
Ole P. Ottersen and Jon H. Laake
Figure 6.5. Electron micrographs
showing colocalization of taurine-LI
(A) and GABA-LI (B) in a Purkinje cell
terminal (PT), which establishes a sym-
metric contact (arrowheads) with a
small dendrite (D). From nucleus inter-
positus anterior (same specimen as in
Fig. 6.3C). Strong immunolabeling is
also found in a large proportion of the
myelinated axons (AX). E, endothelial
cell; V, vessel lumen. Bar = 0.4 11m.
sitometry carried out on the same sections revealed
modest variations in the level oftaurine-LI among
the Purkinje cells (Fig. 6.7). The small variations
observed did not conform to any zonal or banding
pattern.
That taurine and CSAD are differentially dis-
tributed does not necessarily mean that the
taurine synthesis is independent of CSAD. Thus,
the very low turnover rate of taurine (5.5 days;
Huxtable, 1981) may allow for a considerable
redistribution of this amino acid after its forma-
6. Immunocytochemistry of Putative Neurotransmitter Amino Acids 123

TAURINE - LI IN CEREBELLUM
180

160
N
E 140
::1.
.....
UI 120
Q)

- 100
u
.Cl..
a. 80
"0
0 60
Cl

40

20~
UI ..:

.....
E E
..
"0
...
E Q)
E I: I:
..: ...
E
...
E .;
...
--
0
a.
.c
0 Q)
E Q)

..
Q)

- E
..
"0 "0 Q) Q) UI

- -
UI UI
I: ci.
·c .I:
Q) Q)
~ .!!.
I:
Q)

I:
Q)
Q)
"0 .c 0
UI
Q)

Cl Q)
u Q)

Cl :>.

... ... ... ...:::J C C

..
Cl
~ UI
..:
...Cl
~ ~

...Cl
~ ~
UI Cl UI
Q) Q)
:::J :::J :::J 0 Cl Cl 0
a.. a.. a.. a.. (!) (!) a.. (!) (J) m (!) (J) ::E

Figure 6.6. Quantitative assessment of the distribution
oftaurine-LI in perfusion-fixed rat cerebellum (immu-
nogold technique). The densities of gold particles over
the different profiles were calculated by means of a
computer program (Morforei). Bars indicate standard
errors ofthe mean. Note that the Purkinje cell terminals
are enriched in taurine-LI compared to the other parts
of the Purkinje cells. Hatched zone indicates back-
ground labeling (gold particle density over empty resin).
From dorsal part of dentate nucleus with overlying
cortex. Based on data from Table 1 in Ottersen, 1988
which should be consulted for statistical analysis.

100
r---------------------------------------------------------
6 90
(/)
(/)

~
(/)
z
: 60

~ l~t~--------------------------------------------------------
- TOWARDS MIDLINE

Figure 6.7. Level of taurine-LI in 110 consecutive
Purkinje cell bodies, as assessed by optical densitometry
in a transverse 0.5-Jlm section through posterior vermis
(same staining procedure as in Fig. 6.3). Each dot re-
presents one Purkinje cell body. Note absence of varia-
tions that could be interpreted as zonal labeling. Straight
line indicates the mean labeling intensity of the stellate/
basket cell bodies (n = 30). 100% transmission is defined
as value obtained by measurement through the slide
outside tissue section.
124
tion, due to active or passive release and uptake
processes. It should be pointed out that taurine
has been suggested to participate in osmoregula-
tion in the CNS (Solis et aI., 1988; Wade et aI.,
1988), as previously established for other tissues
(Fugelli and Thoroed, 1986), in which case one
must envisage that taurine can pass between dif-
ferent cellular compartments. It should also be
recalled that alternative routes for taurine syn-
thesis have been described (Ca vallini et aI., 1976),
and that a substantial amount of taurine may be
derived from the diet (Sturman et aI., 1985). Ir-
respective of the mechanisms involved, the dif-
ferential distributions of taurine and CSAD
Figure 6.8. Distribution of glutamate-LI in cerebellar
slices incubated in artificial cerebrospinal fluid contain-
ing 5 mmolil K + (A) or 55 mmoljl K + (B). Increasing
the K + concentration leads to a loss of glutamate-LI
from structures resembling mossy fiber terminals
(arrows) and from the interstices between the Purkinje
cell dendrites in the molecular layer (corresponding to
the situation of the parallel fiber terminals). These
changes are associated with an enhanced labeling of
Ole P. Ottersen and Jon H. Laake
clearly indicate that CSAD is unsuitable as a
marker of taurine-containing neurons.
By virtue of the immunogold technique, it was
possible to study the intracellular distribution of
taurine in the Purkinje cells in a quantitative
manner (Fig. 6.6). Whereas there were no signifi-
cant differences in the levels of immunoreactivity
between perikaryon, dendrites, and dendritic
spines, the Purkinje cell terminals in the deep
cerebellar nuclei seemed to be enriched in taurine-
LI relative to other parts of the neuron. Coloca-
lization of taurine-LI and GABA-LI could be
demonstrated by analysis of adjacent ultrathin
sections through Purkinje cell terminals (Fig. 6.5).
Bergmann glial cell bodies (crossed arrows) and pro-
cesses (small arrows). The diffuse labeling in the granule
cell layer in B probably also resides in glial elements.
Arrowheads, stellate/basket cell bodies; asterisks,
Purkinje cell bodies. MO, molecular layer; GC, granule
cell layer. Inset in B: Test section incubated together
with the tissue sections shows selective labeling of
glutamate conjugates (code, see Fig. 6.3). Bar = 50 11m.
6. Immunocytochemistry of Putative Neurotransmitter Amino Acids
At the organelle level, taurine-LI was enriched in
mitochondria, but also over clusters of synaptic
vesicles (Fig. 6.5) (also see Otters en, 1988). A pos-
sible role of taurine, if released from the Purkinje
cell terminals, could be to interact with the post-
synaptic GABA receptors to modulate the res-
ponse to GAB A (Horikoshi et ai., 1988).
Glutamate and Aspartate
Glutamate has long been considered to be the
neurotransmitter released by the majority of the
excitatory synapses in the CNS (for review see
Fonnum, 1984; Ottersen and Storm-Mathisen,
1984a). Immunocytochemical studies of gluta-
mate are complicated by the fact that a large
fraction of the glutamate in the brain participates
in various metabolic pathways rather than in
synaptic mechanisms (Fonnum, 1984). In agree-
ment, in perfusion-fixed brains, glutamate-LI is
found in all neuronal compartments, although
quantitative electron microscopic studies have
pointed to a differential distribution with highest
levels in terminals of proposed glutamatergic
pathways (Ji et ai., 1991; Ottersen, 1987, 1989;
Otters en and Bramham, 1988; Somogyi et ai.,
1986; also see Rinvik and Ottersen, 1988). The
differences between putative glutamatergic ter-
minals and other cell compartments are accen-
tuated in brain tissue that is immersion fixed after
incubation in vitro, rather than fixed by perfusion
(Fig. 6.8) (also see Storm-Mathisen et ai., 1983,
1986a, b). This accentuation may reflect a high
synthetic capacity in the terminals, which provides
a significant build-up of the transmitter during
the in vitro incubation (for a discussion of other
mechanisms, see Storm-Mathisen and Ottersen,
1988a, b). Another advantage with the slice pre-
paration is that it permits us to decide whether
the glutamate can be subject to synaptic release.
In slices that have been incubated under physi-
ological K + concentrations, glutamate-LI is con-
centrated in the interstices between the Purkinje
cell dendrites in the molecular layer (Fig. 6.8A).
The labeling has a granular appearance, suggest-
ing a localization in nerve terminals. Electron
C partate from the cerebellar cortex (Cuenod et ai.,
C A similar pattern of glutamate-LI has been obtained
in perfusion-fixed material by means of a sensitive
postembedding staining technique (Liu et aI., 1989;
Matute et aI., 1987a; Streit et aI., 1988):
125
microscopic analyses ofthe same material confirm
that the labeling resides in parallel fiber terminals
(not illustrated). In the granular layer, glutamate-
LI is concentrated in profiles with the light and
electron microscopic characteristics of mossy fiber
terminals (Figs. 6.8, 6.9). Glial cells are weakly
labeled. This pattern changes dramatically after
exposing the slice to 55 mmol/l K + to induce
synaptic release (Ottersen et ai., 1990b). In such
preparations, the labeling of the nerve terminals
is strongly depressed, whereas that of the glial
cells is increased (Figs. 6.8, 6.9). These changes
are largely Ca2+ dependent and similar to those
found in hippocampal slices (Storm-Mathisen
et ai., 1986a, b). The findings are compatible with
biochemical studies, suggesting that a substantial
proportion of the released glutamate is taken up
into glial cells (for review see Fonnum, 1984). In
conclusion, the slice experiments show that the
mossy and parallel fiber terminals not only are
enriched in glutamate, but are also endowed with
the ability to release glutamate on depolarization.
Taken together with electron microscopic analyses
of the distribution of glutamate-LI in perfusion
fixed material (Ottersen, 1987, 1989; Ottersen and
Bramham, 1988; Somogyi et ai., 1986; Hamori
et ai., 1990), the present results from slices favor
the idea that glutamate is a transmitter in both
of these fiber systems. However, particularly in
the case of the mossy fiber system, it is likely that
some fibers release other compounds, in addition
to, or instead of, glutamate (see, e.g., Morales and
Tapia, 1987; discussion in Beitz et ai., 1986).
The third main type of excitatory fibers in the
cerebellum is the climbing fiber system. Several
lines of evidence suggest that these fibers (or a
subpopulation) may use L-aspartate as a trans-
mitter. Thus, D-[3H] aspartate is taken up by
climbing fibers and subsequently transported re-
trogradely to the parent cell bodies in the inferior
olivary complex (Matute et ai., 1987b; Wiklund
et ai., 1982, 1984). Further, destruction of the
inferior olivary complex by 3-acetylpyridine leads
to a decreased calcium-dependent release of as-
1989; Streit et ai., 1988; Toggenburger et ai.,
1983).
Despite extensive search with the light and
electron microscope in all parts of the cerebellum
in several different species, we have never suc-
126
Figure 6.9. Electron micrographs from the same speci-
men as in Fig. 6.8. From granule cell layer (A, 5 mM K +;
C, 55 mM K +) and molecular layer (B, 55 mM K + ).
Confirming the light microscopical observations, the
immunogold technique shows that depolarization with
high K + is followed by a depletion of glutamate-LI
from mossy fiber terminals (Mf) and an accumulation
Figure 6.10. Photomicrographs showing the distribu-
tion of aspartate-LI in the cerebellar cortex of a slice
that had been immersion-fixed after incubation in arti-
ficial cerebrospinal fluid containing 5 mM K + (A) or
55 mM K + (B). (The same slice is represented in Figs. 6.8,
6.9). Note strong labeling of beaded fibers interpreted
as climbing fibers (arrows). The Purkinje cell dendrites
(small arrows) and stellate/basket cell bodies (crossed
Ole P. Ottersen and Jon H. Laake
of glutamate-LI in glial cell processes (G 1 in B). Gr,
granule cell body; b, unlabeled bouton, probably belong-
ing to a Golgi cell. Asterisks in A, swollen profiles of
uncertain identity; asterisks in B, parallel fibers; asterisk
in C, granule cell dendrite. Arrow in C points to a gap
junction between glial lamellae. Bar = 0.4 j1m.
,,
arrows) are unlabeled. Depolarization with high K +
abolishes labeling of climbing fibers, but not that of
the Bergmann cell bodies (arrowheads). Mo, molecular
layer; GC granule cell layer. Inset in B: Test section
incubated together with the tissue section. Only the
aspartate conjugates are labeled (code, see Fig. 6.3).
Bar = 50j1m.
6. Immunocytochemistry of Putative Neurotransmitter Amino Acids
ceeded ,in displaying a significant concentration
ofL-aspartate-LI in climbing fibers after perfusion
fixation (however, see Campistron et aI., 1986c).
Even quantitative studies with the immunogold
technique have failed to associate climbing fibers
with endogenous aspartate (Zhang et aI., 1990).
In the slice preparation, however, aspartate-LI is
sometimes selectively concentrated in structures
that are reminiscent of climbing fibers (Fig. 6.10).
Analogous to the situation for glutamate discus-
sed above, one interpretation ofthis finding is that
the climbing fibers synthesize aspartate rapidly
enough to cause a net accumulation during the
Figure 6.11. Electron micrographs of test section simi-
lar to those represented in Figs. 6.3, 6.8, and 6.10, incu-
bated with an antiserum against glutamine. Represent-
ative conjugate clumps are enlarged in right part of
127
in vitro incubation. (Our failure to find a signifi-
cant concentration of aspartate-LI in these fibers
in perfusion-fixed material may simply indicate
that the transmitter pool of the climbing fibers is
very small under physiological conditions in vivo.)
The aspartate-LI in the climbing fibers can be
abolished by in vitro depolarization with high
K + (Fig. 6.10).
It should be emphasized that the in vitro find-
ings are open to an alternative explanation. Thus,
it is clear from our electron microscopical studies
of perfusion-fixed cerebellum (Zhang et aI., 1990)
that aspartate occurs rather diffusely in many cell
the figure. Note selective labeling of the glutamine
conjugate. Spacer sections are from rat hippocampus.
Bar = 2/lm.
128
<;ompartments, probably reflecting a diversity of
metabolic roles (also see Madl et aI., 1987;
Ottersen and Storm-Mathisen, 1985). During the
in vitro incubation, part of this metabolic aspar-
tate could leak into the medium and be subject to
uptake by the climbing fibers. Thus, it remains to
Figure 6.12. Electron micrographs showing the distri-
bution of glutamine-LI in the Purkinje cell layer (A)
and granule cell layer (B) of perfusion-fixed rat cerebel-
lar cortex. Gold particles occur over all cell profiles,
including basket cell and mossy fiber terminals (B and
Mf), but are particularly concentrated over glial cells
Ole P. Ottersen and Jon H. Laake
be determined whether aspartate uptake could
explain or at least contribute to the enrichment
of aspartate-LI in the climbing fibers in vitro.
Even when taking into account our in vitro data,
we feel that the sum of immunocytochemical
evidence speaks against a transmitter role of
(G in upper panel). The test results (see Fig. 6.11) suggest
that the gold particles represent the presence of gluta-
mine (and not unspecific labeling), even at sites with
very low gold particle concentrations. P, Purkinje cell
body; G, in lower panel, granule cell body; d, granule
cell dendritic digits. Bar = 0.4 Jim.
6. Immunocytochemistry of Putative Neurotransmitter Amino Acids
aspartate in the climbing fibers (see discussion in
Zhang et aI., 1990).
As is the case for the mossy fiber system, climb-
ing fibers appear to contain several different neuro-
active substances. It has been claimed that the
climbing fibers that are capable of releasing aspar-
tate are preferentially distributed to the cerebellar
hemispheres (Cuenod et aI., 1989; Streit et aI.,
1988). Other substances that may be released
from climbing fibers include homocysteate and
adenosine (Cuenod et aI., 1989; Do et aI., 1986).
Recently, corticotropin-releasing factor has attract-
ed interest as an additional neuromodulator in
this fiber system (Cummings et aI., 1988; Palkovits
et aI., 1987; Powers et aI., 1987). The nature of
the interaction between the different neuroactive
compounds present in the climbing fibers remains
to be explored.
Glutamine
Glutamine is regarded as an important precursor
of transmitter glutamate and has also been as-
signed a role in GABA synthesis (Bradford et aI.,
1978; Hamberger et aI., 1979; Kvamme, 1983).
Biochemical studies have suggested that gluta-
mine is synthesized in the glial cells before being
transferred to the neurons for subsequent con-
version to glutamate by means of phosphate-
activated glutaminase (Benjamin and Quastel,
1972; Van den Bergh et aI., 1975). In agreement
with this theory, immunocytochemical studies
indicate that the major glutamine synthesizing
enzyme, glutamine synthetase, is preferentially
localized in glial cells (Norenberg and Martinez-
Hernandez, 1979).
The results obtained with our glutamine anti-
serum (Laake et aI., 1986), which shows no de-
tectable cross-reactivity with glutamate or other
amino acids tested (Fig. 6.11), are consistent with
the above data. The highest level of glutamine-LI
is found in glial cells (both astrocytes and oligo-
dendrocytes), but modest labeling also occurs
in putative glutamatergic as well as in putative
GABA-ergic terminals (Fig. 6.12) (also see Ji et aI.,
1991; Zhang et aI., 1991) compatible with the
proposed precursor role of glutamine. The glial
labeling is strongly enhanced in slices exposed to
high K + (not shown). This probably reflects an
increased glial uptake of glutamate (and GABA)
129
and subsequent conversion of these amino acids
to glutamine by glutamine synthetase.
Conclusion
All amino acids with proposed transmitter roles
in the cerebellum can now be studied by direct
immunocytochemical methods. The postembed-
ding immunogold technique permits a quanti-
tative assessment ofthe levels of immunoreactivity
and offers a resolution that is high enough to
allow for analysis of the amino acid distribution
at the organelle level. Having established the
pattern of amino acid distribution in perfusion
fixed tissue (reflecting the in vivo distribution),
the stage is set for exploiting immunocytochem-
istry for more dynamic studies. In this chapter
we have shown that the technique can be used to
demonstrate synaptic release and glial uptake of
amino acids. We have also used our antisera to
analyze the redistribution of amino acids that
occurs in different models of experimental epilepsy
(Meldrum et aI., 1987). Future applications of
this technique will include its use as a tool to
study the synthesis and metabolism of the amino
acids in the different cell compartments, in normal
as well as in pathological tissue.
Acknowledgments. The expert technical assistance
of A.T. Bore (preparation of sera), K. H~genhaven,
J. Knutsen, and B. Riber (immunocytochemistry)
is gratefully acknowledged. We also wish to thank
C. Ingebrigtsen and G. Lothe for photographi-
cal assistance, T. Eliassen for typing and editing
the manuscript, and Drs. J. Storm-Mathisen and
F. Walberg for constructive criticism ofthe manus-
cript. Supported by the Norwegian Council for
Science and the Humanities, the Norwegian
Council on Cardiovascular Disease, the Nor-
wegian Society for Fighting Cancer, and Odd
Fellows Research Fund.
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7
The Expanding Role of the Basilar Pontine Nuclei
as a Source of Cerebellar Afferents
G.A. Mihailoff, R.J. Kosinski, S.A. Azizi, H.S. Lee, and B.G. Border
Among those studying the structural and func-
tional organization of the cerebellum, one might
expect that there would be almost universal agree-
ment that information transmitted to the cere-
bellum from the cerebral cortex plays an impor-
tant role in cerebellar function. In fact, the lateral
cerebellar hemispheres are often referred to as the
neocerebellum since this portion of the cerebellar
cortex is thought to derive the majority of its
input from neocortex. Furthermore, although this
has not been analyzed or quantified in a rigorous
manner, it is obvious that as the volume of the
neocortical mantle increases across phylogeny,
there is a parallel increase in the volume of the
cerebellar hemispheres that presumably reflects
an increase in cerebral cortical input. It is perpaps
unexpected then that there are no direct projec-
tions extending from the cerebral cortex to the
cerebellum except for rather sparse transient con-
nections present during a relatively brief period
early in the postnatal development of the central
nervous system (Distel and Hollander, 1980;
Tolbert and Panneton, 1983, 1984). Thus, corti-
cal signals to the cerebellum are transmitted
indirectly via cortical projections to various pre-
cerebellar nuclei, which then provide access to the
cerebellar nuclei and cortex. Based on numbers
of fibers (Allen and Tsukahara, 1974), it appears
that the principal precerebellar cell group involved
in the linkage of the cerebral and cerebellar cor-
tices is the basilar pontine nuclei (BPN).
In line with these considerations, the BPN are
often viewed from a narrow functional perspective
as faithful transmitters of cerebrocerebellar infor-
mation. Again, because of the vast number of cel-
lular elements involved in these circuits, one might
assume that such activities represent the domi-
nant functional operation of the BPN. However,
recent observations regarding the connectivity
and intrinsic cytologic and synaptic organiza-
tion of the BPN suggest that this brain stem cell
group is involved in a more diverse array of pre-
cerebellar activity than has previously been recog-
nized. First, it has been shown that the dorsal
column nuclei (and spinal cord neurons) project
to the BPN (Swenson et aI., 1984) and ultra-
structural studies suggest further that two types
of dorsal column nuclei (DorCN) cells might be
involved in this projection (Kosinski et aI., 1986).
This finding, in conjunction with the observations
of Shambes et ai. (1978), which describe multi-
synaptic, peripheral cutaneous inputs to granular
cells ofthe cerebellar cortex suggest that the BPN
might possibly represent one cell group that
conveys these signals to the cerebellar cortex.
Second, the presence of gamma-aminobutyric acid
(GABA)-ergic neurons was reported in the BPN
of the rat (Border and MihailofT, 1985) and more
recently in the cat and monkey (Brodal et aI.,
1988; Thier and Koehler, 1987). These findings
correlate with the observations of Ramon y Cajal
(1911), who illustrated a heavily spine-laden cell
within the BPN that gives rise to an axon that
ramifies extensively within the dendritic domain
of the parent neuron and presumably represents
a local-circuit type neuron. In addition, previous
Golgi and electron microscopic studies have
demonstrated cellular and synaptic features that
also suggest the possible existence oflocal-circuit
type neurons in the BPN (Cooper and Beal, 1978;
135
136
(ooper and Fox, 1976; Mihailoff and McArdle,
1981; Mihailoff and King, 1975; Mihailoff et aI.,
1981a). The implication then is that the BPN
might function in more of an integrative capacity
than has previously been considered.
In this chapter we review a series of observ-
ations that can be divided into four major cate-
gories: a) a survey of noncortical BPN afferent
systems, b) a demonstration that some BPN af-
ferent projections contain GABA-ergic compo-
nents, c) an illustration of certain BPN cytological
and synaptic features, and d) a review of recently
obtained findings regarding the functional pro-
perties of BPN neurons. These observations pro-
vide additional support for the concept that the
BPN, in addition to being involved in the trans-
mission of cerebrocerebellar information, also
receive a substantial variety of other afferent in-
formation that is processed and integrated in
some manner before eventually being forwarded
to the cerebellar cortex. Consequently, the BPN
appear to playa broader role as a source of
cerebellar afferent information than has common-
ly been recognized in the past.
Results
Survey of Noncortical BPN Afferent Systems
The retrograde transport of wheat germ agglutinin
conjugated to horseradish peroxidase (WGA-
HRP) was used in rats to identify the cell bodies
of origin for all afferent projections to the BPN
with the exception of the cerebral cortex (Mihailoff
et aI., 1988b). With a parapharyngeal surgical
approach, the injection micropipette was placed
into the basilar pons under visual control. Although
technically difficult, this approach was particu-
larly advantageous because it avoided the comp-
lication of false-positive labeling that might have
resulted from a dorsal approach to the BPN and
the possible transection and injury-filling ofaxo-
nal systems coursing longitudinally through the
brain stem immediately dorsal to the BPN. Cases
were discarded if histological sections through
the injection site illustrated any tissue disruption
by the micropipette outside the BPN or if there
appeared to be an extension ofthe injected tracer
outside the BPN. For orientation, a Nissl-stained
transverse section through the midportion of the
G.A. Mihailoff et al.
basilar pons is shown in Figure 7.1 along with
illustrations of two representative WGA-HRP
injection sites in the BPN.
Much to our surprise, more than 50 cell groups
were identified in these experiments as having a
projection to the BPN. Their distribution is plotted
in Figures 7.3 and 7.4. Among these afferent sys-
tems are a number of newly identified inputs such
as the central amygdaloid nucleus, the nucleus
basalis of Meynert, and the horizontal nucleus of
the diagonal band of Broca. Many BPN afferent
cells were also newly identified in the midbrain;
these included somata adjacent to the red nucleus
(perirubral cells), the medial and lateral terminal
nuclei, nuclei of the posterior commissure,
Edinger-Westphal nucleus, nucleus of Darksche-
witsch, periaqueductal gray, substantia nigra pars
lateralis and reticulata, peri peduncular nucleus,
and portions of the mesencephalic reticular for-
mation. Among the newly identified inputs from
medullary and pontine levels of the brain stem
were the trigeminal chief sensory nucleus, nucleus
X of the vestibular complex, the pedunculopon-
tine tegmental and dorsal tegmental nuclei, numer-
ous components of the medullary reticular form-
ation, and certain raphe nuclei (dorsalis, obscurus,
magnus, pallidus). Also newly identified as BPN
afferents were several cell groups known to have
direct projections to the cerebellum such as the
lateral reticular nucleus, nucleus prepositus, the nu-
cleus of Roller, the parabrachial nuclei, and the locus
coeruleus and subcoeruleus. In addition to each of
these newly identified inputs, all cell groups reported
in previous studies to give rise to BPN afferents were
confirmed. Included in this category are neurons in
several hypothalamic nuclei, the zona incerta, various
pretectal nuclei and the nucleus of the optic tract,
superior colliculus, ventral lateral geniculate nu-
cleus, cerebellar nuclei, spinal trigeminal nucleus,
dorsal column nuclei, external cuneate nucleus,
and nucleus dorsalis at lumbar levels of the spinal
cord.
Because a number of the labeled cell groups
in these studies were known to have direct projec-
tions to the cerebellum, there was a concern that
some or all of such neurons might have been
labeled by virtue of the fact that their axon passed
through the BPN to enter the cerebellum via the
middle cerebellar peduncle (brachium pontis).
Consequently, such axons might have been
7. The Basilar Pontine Nuclei
Figure 7.1. A: Nissl-stained transverse brain stem sec-
tion illustrates the location of the basilar pontine nuclei
(BPN), cerebral peduncle (CP), medial lemniscus (ML),
and nucleus reticularis tegmenti pontis (NRTP). B:
Same section as in A but at higher magnification to
transected and injury-filled at the BPN injection
site. To assess this possibility, two series of rats
were prepared. In both series, 12 to 16 placements
(0.08-0.12 pI per site) of WGA-HRP were distri-
buted throughout the cerebellar hemispheres and
vermis (Fig. 7.2C). In addition, in one of these
series, the WGA-HRP injections were initiated
30 to 60 minutes after bilateral transection of the
brachium pontis lateral and dorsal to the BPN
in a region where this fiber bundle was about to
enter the cerebellum (Fig. 7.2A, B). In the series
in which the WGA-HRP injections were not
137
illustrate medial (Med), ventral (Vent), and lateral (Lat)
subdivisions of BPN. C, D: Representative lateral (C)
and medial (D) WGA-HRP injection sites in the BPN
performed using a ventral, parapharyngeal surgical
approach.
preceded by a brachium pontis lesion, each ofthe
cerebellar-projecting cell groups in question ex-
hibited labeled somata as anticipated. In the series
in which the WGA-HRP injections followed the
bilateral brachium pontis lesions, the nuclei in
question continued to exhibit labeled somata in
approximately the same number as the nonlesion
cases. These findings were interpreted to suggest
that the cell groups in question project to both
the BPN and the cerebellum, but their axons
do not enter the cerebellum via the brachium
pontis.
138
Figure 7.2. A: Nissl-stained transverse brain stem sec-
tion illustrating the position of the brachium pontis
(BP). B: Transverse section taken at approximately the
same rostrocaudallevel as A. Note the bilateral electro-
lytic lesions that involve the BP. C: Transverse brain
stem demonstrating multiple injections ofWGA-HRP
that essentially involve nearly the entire cerebellar cor-
tex. (A and B reprinted with permission from Mihailoff
et aI., 1988b.)
Some BP N AfJerents Contain
GABA-Ergic Components
In the course of light microscopic studies (Border
and Mihailoff, 1985) designed to demonstrate the
presence of GABA-ergic neurons in the rat BPN
G.A. Mihailoff et al.
using immunoperoxidase methods to visualize
glutamic acid decarboxylase (GAD), it seemed
unlikely that the many labeled axons and axon
terminals observed might originate solely from
the relatively sparse number of immunoreactive
somata in the rat BPN. To determine if GABA
neurons extrinsic to the pontine gray provided
afferent projections to the BPN, a double-labelling
protocol involving the retrograde transport of
WGA-HRP was employed in combination with
GAD immunocytochemistry. Tissue sections col-
lected throughout the brain of animals that
received ventral BPN injections of WGA-HRP
as part of the preceding study of BPN afferents
were reacted according to an HRP stabilization
procedure (Rye et aI., 1984) and subsequently in-
cubated with GAD antiserum (Oertel et aI., 1981)
and processed according to routine immunoper-
oxidase procedures (Border et aI., 1986).
As indicated in Figures 7.3 and 7.4, double-
labeled somata (asterisks) were observed in the
zona incerta, perirubral area, anterior pretectal
nucleus, lateral cerebellar nucleus, and in various
portions of the medullary reticular formation.
Collectively, the largest accumulation of double-
labeled somata was exhibited by the medullary
reticular formation, followed in descending suc-
cession by the perirubral area, zona incerta, and
anterior pretectal nucleus. The fewest number of
double-labeled cells were observed in the lateral
cerebellar nucleus.
The double-labeled somata exhibited a wide
range in size and morphology; a few examples are
shown in Figure 7.5. The largest of the double-
labeled cells exhibited a spheroidal-shaped or
multipolar-shaped soma and were most frequently
located in the reticular formation. However, other
double-labeled reticular formation somata were
relatively small and spindle-shaped. In the peri-
rubral area and zona incerta, most double-labeled
somata were small and multipolar or spindle-
shaped whereas in the lateral cerebellar and
anterior pretectal nuclei, a mixture of small and
medium somata was observed.
In several cases, the injection site in the BPN
extended dorsal to the medial lemniscus and cere-
bral peduncle and in such cases double-labeled
cells were observed only in the lateral cerebellar
nucleus. Although not specifically llsed in this
study, such cases in essence served as control
7. The Basilar Pontine Nuclei
Figure 7.3. The composite distribution of neuronal
somata labeled by the retrograde transport of WGA-
HRP (-), as well as those somata double-labeled with
WGA-HRP and immunoperoxidase staining for GAD
(*). This is a composite diagram depicting all retro-
139
• HAP
*HAP& GAO
gradely labeled somata observed in cases in which the
HRP injection site was restricted to the BPN as indi-
cated in section 9. (The illustrated sections are taken
from the atlas of Paxinos and Watson, 1986.)
140
Figure 7.4. The composite distribution of neuronal
somata labeled by the retrograde transport of WGA-
HRP (e), as well as those somata double-labeled with
WGA-HRP and immunoperoxidase staining for GAD
(*). This is a composite diagram depicting all retro-
experiments, which ruled out the possibility that
the double-labeled cells were HRP-positive as a
result of the inadvertent spread of the injected
tracer into the tegmentum dorsal to the BPN.
The double-labeling of cells in the lateral cerebel-
G.A. Mihailoff et al.
• HRP
*HRPS. GAO
grade\y labeled somata observed in cases in which the
HRP injection site was restricted to the BPN, as indi-
cated in section 9 of Fig. 7.3. (The illustrated sections
are taken from the atlas of Paxinos and Watson, 1986.)
lar nucleus in the latter experiments is apparently
due either to the interruption and injury-filling of
GABA-ergic cerebello-olivary axons that are
known to course through the ventral portion
of the tegmentum, or to retrograde transport in
7. The Basilar Pontine Nuclei
Figure 7.5. Two examples of somata double-labeled
by the retrograde transport of WGA-HRP (from a
BPN injection) and immunoperoxidase staining for
GAD. The cell in A is from the perirubral are a adjacent
axons of cerebellar nuclear neurons that project
to nucleus reticularis tegmenti pontis.
Synaptic Features in the Monkey BPN
Suggest the Presence of
Local-Circuit Neurons
Recently, five cynomolgous monkeys became
available for ultrastructural and immunocyto-
chemical (ICC) studies. Each animal was first
immobilized with ketamine, intubated, and then
deeply anesthetized with Nembutal. While an-
esthesia was being achieved, 2.5 ml of the vasodi-
lating agent Diamox (acetazolomide, 1 mljkg) was
infused intravenously and 15 minutes later a tho-
racotomy was initiated while the animal was arti-
ficially ventilated. The heart was immediately
freed from the pericardial sac and a blunt 10-
gauge needle introduced into the left ventricle.
This needle was connected via polyethylene tubing
and a peristaltic pump to three flasks, one con-
taining physiological saline, the other a mixture
of 1% paraformaldehyde- l % glutaraldehyde in
0.1 M sodium phosphate buffer, and the third
contained 2% paraformaldehyde-2% glutaral-
dehyde in 0·1 M sodium phosphate buffer.
After incising the right atrium, 300 ml of warm
saline was rapidly flushed through the vascular
141
B
to the substantia nigra (SN) and red nucleus (Rn). The
midline (ml) is to the reader's right. The cell body
indicated in B is located within the ventromedial portion
of nucleus gigantocellularis.
system and immediately followed first by 1 liter
of chilled 1%- 1% fixative and then 1.5 liters of
chilled 2%-2% fixative. At the conclusion of
perfusion, the animal was left undisturbed for 30
minutes, whereupon the entire brain was removed
from the cranial cavity and immersed in 300 ml
of the 2%- 2% fixative. The brain was refrigerated
overnight and on the following morning, the entire
basilar pons was isolated from the brain stem and
subdivided under a dissecting microscope into
small tissue blocks containing pontine regions
located medial, ventral, or lateral to the cerebral
peduncle. The tissue was then osmicated, de-
hydrated, and embedded in plastic according to
previously described (Mihailoff and McArdle,
1981) routine methods for studies of the rat BPN.
Before osmication, tissue to be processed for
immunoperoxidase studies (70-l1m vibratome sec-
tions) was imlJlersed in 2% normal goat serum in
Tris buffer and then incubated for 24 hours at
4°C in rabbit anti-GABA (Maley and Newton,
1985) diluted 1: 2000 in 2% normal goat serum in
Tris buffer. After immersion in goat anti-rabbit
immunoglobulin G (IgG) (1 : 200) for 1 to 2 hours,
the free-floating sections were incubated for
1 hour with rabbit peroxidase antiperoxidase
(PAP) diluted 1:400. The tissue was then reacted
with 0.05% diaminobenzidene (DAB) and 0.01,!~
142
Figure 7.6. Two representative examples oflarge bou-
tons are shown in A and B. A: One variety exhibits a
fairly regular oval shape, contacts several dendritic
appendages on its perimeter, and is partially enclosed
by glial lamellae. B: The second variety exhibits an
irregular shape and is rarely surrounded by glial pro-
cesses. Both types primarily contain round clear vesicles
G.A. MihailofT et al.
with some dense core vesicles and both form only
asymmetrical synaptic junctions. C: Transversely sec-
tioned dendritic shaft contains three clusters of pleo-
morphic vesicles located near symmetrical synaptic
active sites. (Fig. 7.6c reprinted with permission from
MihailofT and Border, 1990.)
7. The Basilar Pontine Nuclei
Figure 7.7. A: An example ofthe type of bouton at the
small end of the size spectrum. It contains round vesicles
with an asymmetric active site. B: Dendritic appendage
postsynaptic to a bouton containing small round vesi-
cles as well as several dense core vesicles. C: An example
143
ofthe most commonly observed type of bouton, in this
instance contacting the shaft of an intermediate-sized
dendrite. D: Bouton forming a symmetrical active site
and containing a mixture of flattened, discoid, and
spheroidal vesicles as well as a dense core vesicle.
144
hydrogen peroxide in Tris buffer (pH 7.4) for 3 to
6 minutes. Subsequently, the tissue was lightly
osmicated and prepared for electron microscopy
in a routine manner as previously described.
In addition to the above procedure, the post-
embedding immunogold method was used to
identify GABA-immunoreactive profiles within
the BPN. Briefly after collecting ultrathin sections
on nickel grids, the tissue was exposed to 1% per-
iodic acid (4 minutes), 1% sodium metaperiodate
(4 minutes), and 2% normal goat serum (NGS;
45 minutes) with gentle washing in distilled water
between each step. The tissue was then incubated
in rabbit anti-GABA diluted 1: 1000 with 1% NGS
containing 0.3% Triton X-100 for 1.5 hours. After
3 washes (5 minutes each) in a solution comprising
10 ml 0.05 M Tris containing 5 mg polyethylene
glycol, the tissue was incubated in goat anti-
rabbit IgG conjugated with 15 nm colloidal gold
particles (Janssen; 1:20 dilution) for 1.5 hours.
The sections were then gently rinsed with four
changes (5 minutes each) of distilled water and
stained with uranyl acetate (20 minutes) and lead
citrate (20 minutes). Although there was no at-
tempt to quantify rigorously the number of gold
particles overlying a given profile, we routinely
assessed the extent of potentially nonspecific label-
ing by noting the number of gold particles present
over the lumen of blood vessels and the compact
portion of axonal myelin. Such nonspecific or
background labeling was extremely sparse.
Careful examination of the neuropil in tissue
sections from control monkey brains revealed an
abundance of presynaptic profiles exhibiting a
wide variety of sizes and synaptic vesicle and
active site morphology. Included were several
varieties of round vesicle boutons (Figs. 7.6 and
7.7 A-C) that formed asymmetric active sites with
dendritic shafts and spines. Boutons containing
a population of pleomorphic (various sizes and
shapes) vesicles (Fig. 7.7D) formed symmetrical
active sites whereas boutons populated only with
large, dense-core vesicles (not illustrated) were
also observed. In some instances, profiles identi-
fied as dendrites by their content of ribosomes
and microtubules (Fig. 7.6C) contained synaptic
vesicles that varied in size and in shape from
spheroidal to discoidal (pleomorphic). These
vesicles were often aggregated near membrane
specializations that consisted of symmetrically
G.A. MihailofT et al.
disposed electron-dense material. In addition, a
curious group of dendritic profiles was distin-
guished by their electron-dense cytoplasmic matrix
(Fig. 7.8A-C). Occasionally a similar dense matrix
was clearly present within synaptic boutons
(Fig. 7.8D). In this chapter, however, we focus on
two other specific categories of synaptic profiles,
each of which involves apparent synaptic interac-
tion between two processes, both of which contain
synaptic vesicles.
In the first example (Fig. 7.9), a glial-invested
glomerular synaptic complex consisting of a
central axon terminal surrounded by several small
dendritic profiles also includes on its periphery a
vesicle-containing profile. In all examples of this
type observed to date, the peripheral vesicle-
containing profile was postsynaptic to the central
axon terminal. Only in rare instances, however,
did the peripheral bouton form a distinct synaptic
active site in the same thin section (Fig. 7.11A),
and in this circumstance the postsynaptic ele-
ment was a neighboring small dendritic profile.
Whereas the central axon terminal contained
round clear synaptic vesicles and formed asym-
metric active sites, the peripheral boutons con-
tained pleomorphic (mostly spherical) vesicles
embedded in a pale or lucent matrix and formed
symmetrical active sites.
The second category of synaptic arrangement
typically consisted of two vesicle-containing pro-
files and a dendritic element that were distributed
throughout the neuropil without glial encapsula-
tion and for that reason are referred to as non-
glomerular synaptic complexes (Fig. 7.10). As in
the first category, the pale bouton with pleomor-
phic vesicles was always postsynaptic to a bouton
that contained uniformly round vesicles and was
itself only rarely presynaptic to a dendritic profile
in the same plane of section (Fig. 7.10B, C). Those
profiles that were presynaptic to the pale boutons
always contained round clear vesicles and formed
asymmetric active sites. However, the round
vesicle boutons were quite variable in size (always
smaller than glomerular central boutons) and in
several instances, the round vesicle bouton exhi-
bited a dense background matrix (Fig. 7.10D)
that might perhaps be characteristic of a parti-
cular class of neuron in the B PN whose somatic,
dendritic, and axonal profiles exhibit an elevated
cytoplasmic matrix density. On the other hand,
7. The Basilar Pontine Nuclei 145

Figure 7.8. A: Certain dendrites exhibited an unusually such a dense dendrite is shown in B, whereas a similar
dense cytoplasmic matrix (solid block arrow) that easily dense dendrite and a spine1ike appendage are illustrated
distinguished them from other dendritic elements (open in C. Occasionally, synaptic boutons as indicated in D
block arrow) in the vicinity. An oblique section through exhibited a similar dense matrix.
146
Figure 7.9. Examples of glomerular synaptic com-
plexes are shown in A-D. A: Central axon terminal
surrounded by dendritic appendages and synaptic
boutons that are always postsynaptic to the central
axon. B-D: Other examples of glomeruli in which the
G.A. Mihailoff et al.
peripheral vesicle-containing profile (*) characteristi-
cally exhibits a pale or lucent background. Arrows
indicate polarity of synapse. (Fig. 7.9A reprinted with
permission from Mihailoff and Border, 1990.)
7. The Basilar Pontine Nuclei
Figure 7.10. Examples of nonglomerular arrangements
that include synaptic contact between two vesicle-
containing profiles. A: A simple variety shown here
involves only two profiles with the pale or lucent bouton
(*) in a postsynaptic position. B, C: When three profiles
147
are involved, the pale profile (*) is postsynaptic to
another bouton and presynaptic to a dendritic element.
D: Dense matrix bouton is presynaptic to a pale
bouton (*).
148
Figure 7.11. Examples ofGABA-immunogold labeled
pale boutons. A: In glomeruli, only the small peripheral
boutons are GABA-Ir. Here a triadic arrangement is
evident as the GABA-Ir bouton (*) is postsynaptic to
the central axon and presynaptic to an adjacent den-
dritic appendage, which i~ itself postsynaptic to the
central axon terminal. B: Nonglomerular arrangement
G.A. MihailofT et al.
in which GABA-Ir pale bouton (*) is postsynaptic to
a small axon terminal and presynaptic to a nearby
dendritic appendage. C, 0: In some instances, the GABA-
Ir pale boutons (*) are linked by thin strands. Note
also in 0 the transversely sectioned GABA-Ir dendrite
(Dd). (Reprinted with permission from MihailofT and
Border, 1990.)
7. The Basilar Pontine Nuclei
those dendrites that were postsynaptic to pale
profiles in these serial synaptic arrangements
were not distinctive in any way other than their
relatively small size, which suggests that they
probably represent more distal portions of the
dendritic tree or a type of dendritic spine or pro-
trusion. No dense matrix dendrites have yet been
observed to participate as postsynaptic elements
in a serial synapse.
In the primate material processed for immuno-
cytochemistry, the frequency with which GABA-
immunoreactive (GABA-Ir) somata and dendrites
(Fig. 7.11A) have been observed throughout the
neuropil was noticeably greater than in the rat
BPN. This is to be expected, based on light micro-
scopic studies (Brodal et aI., 1988), which revealed
a much greater density ofGABA-Ir cell bodies in
the monkey BPN compared to the rat. In addi-
tion, as previously observed in the rat (Border
and Mihailoff, 1990; Mihailoff etal., 1988a), pre-
synaptic profiles in the monkey BPN were clearly
GABA-positive (Mihailoff and Border, 1990).
Although both immunoperoxidase and immu-
nogold material provided qualitatively the same
observations, in this chapter we focus primarily
on the gold-labeled material since it was possible
to distinguish readily the intracellular contents of
the labeled profiles. GABA-immunoreactivity was
observed within BPN somata, dendrites, and a
variety of profiles that contained synaptic vesicles.
In no instance was gold labeling observed in
association with those profiles that exhibited the
dense cytoplasmic matrix, although, such struc-
tures were clearly preserved in the immunogold
material.
Synaptic boutons immunopositive for GABA
could generally be divided into two categories:
those that exhibited a distinctly pale or lucent
matrix with a sparse complement of synaptic
vesicles and those that contained a relatively large
accumulation of synaptic vesicles that gave them
a darker but more typical appearance compared
to surrounding elements in the neuropil. The pale
GABA-Ir boutons participated in both glom-
erular and nonglomerular arrangements. In the
glomerular complexes (Fig. 7.11A), the GABA-Ir
boutons were involved in a characteristic triadic
serial synapse in which they were postsynaptic to
the c~ntral bouton and presynaptic to an adjacent
dendritic profile that was itself postsynaptic to
149
the central bouton. In their nonglomerular loca-
tions, the pale GABA-Ir boutons were involved
in serial (but not triadic) synapses in which again
they were always postsynaptic to another vesicle-
containing profile and presynaptic to a dendri-
tic element (Figs.7.11B and 7.12B). Also, the
pale GABA-Ir boutons participated in relatively
simple, nonserial synapses with dendritic profiles
(Fig. 7.12A, C). Often these pale GABA-Ir bou-
tons appeared to be joined to one another by thin
strands (Figs. 7.11 C, D and 7. 12C). Occasionally,
a GABA-Ir pale bouton formed synaptic contact
with a GABA-Ir dendrite (Fig. 7.13C). In addi-
tion, transversely (Fig. 7.12D) or longitudinally
(Fig. 7.13A) sectioned dendrites containing small
aggregates of clear synaptic vesicles and an oc-
casional dense-core vesicle (Fig. 7.13B) also ex-
hibited immunogold labeling.
The second general category of GABA-Ir
bouton characteristically contained a large accu-
mulation of pleomorphic synaptic vesicles (most
being spheroidal of varying size) and formed
symmetrical active sites (Fig. 7.14A-C). Most of
these profiles were relatively small ( < 1.0 Jim dia-
meter), rounded boutons (Fig. 7.14B), although
others were larger and more irregularly shaped
(Fig. 7.14A). In addition, this type of GABA-Ir
bouton participated in serial synapses in which
they were presynaptic to the pale type GABA-Ir
bouton (Fig. 7.14C), which in turn was presyn-
aptic to an adjacent dendritic spine.
Evidence that Inhibition and Afferent
Convergence are Part of the Functional
Operations in the Rat BPN
In an effort to correlate the morphological fea-
tures of cytology and synaptic organization in
the BPN with an electrophysiologically derived
view of certain functional properties of BPN
neurons, single-unit recording experiments in the
BPN were initiated. The basic protocol for these
studies is illustrated schematically in Figure 7.1SA.
The animal was initially anesthetized through
the inhalation of 3% to 4% halothane and then
maintained on a lower concentration (2-3%) with
the use of an endotracheal tube. For orthodromic
activation of corticopontine neurons, an indi-
vidually moveable concentric bipolar electrode
(Rhodes SNE-100; outer diameter, 0.2 mm; center
150 G.A. Mihailoff et al.

Figure 7.12. Not all pale

GABA-Ir boutons are
involved in serial synapses.
A: Pale GABA-Ir bouton
(arrow) receives an en passage
synaptic contact. B: Pale
GABA-Ir bouton forms
synaptic contact with two
adjacent dendritic profiles.
C: Pale GABA-Ir bouton
forms a synapse with an
adjacent dendrite (thin
arrow) and is continuous
with a slender stalk (paired
arrows). D: Commonly
observed type of gold-labeled
profile that does not exhibit
any synaptic vesicles or
membrane specializations.
May represent a plane of
section through a pale bouton
such as that shown in C or
might be a transversely
sectioned dendritic shaft
from a GABA-Ir neuron.

exposure 0.25 mm) was held in a plexiglass holder
and inserted perpendicular to the cortical surface.
Electrodes were initially positioned on the basis
of stimulation or recording in the sensorimotor
cortex but later were located using the stereo-
taxic (somatotopic) map developed by Hall and
Lindholm (1974). Additional stimulating elec-
trodes were positioned simultaneously within a
variety of other cortical regions, and in some cases,
the deep cerebellar nuclei. For antidromic activ-
ation ofBPN neurons, an array ofthree monopolar
stimulating electrodes (approximately 500-750.um
apart) was inserted either into a contralateral
cerebellar lobule at a depth of 350.um or was
stereotaxically positioned within the brachium
pontis contralateral to the BPN recording site.
After securing each electrode or electrode array
in position, the animal was inverted carefully to
7. The Basilar Pontine Nuclei
Figure 7.13. A: Longitudinally sectioned GABA-Ir
dendrite (thick arrows) within the BPN. B: Enlargement
of portion of dendrite in A (thin arrows) to demonstrate
the presence of clear and dense core vesicles. C: GABA-
the supine position and maintained in the stereo-
taxic instrument while the BPN was exposed
through a ventral, parapharyngeal surgical ap-
proach. Finally, small bipolar-stimulating elec-
trodes were placed bilaterally under the skin of
the forepaws, hind paws, and vibrissae pad areas.
Anesthesia was continued with halothane but the
level was reduced to 0.75%-\.0% (of total oxygen)
for the remainder of the recording session.
Single BrN neuron activity was recorded with
glass microelectrodes filled with 2 M NaCI and
fast green (impedance of 3- 12 Mil) and conven-
tional apparatus was used for neuron pulse de-
tection and histogram computation. Brain elec-
trical stimulation consisted of single or double
151
Ir dendrite receiving synaptic contact from a pale GABA-
Ir bouton. Note the extensive subsynaptic specializa-
tion. (arrow). (Fig. 7.130 reprinted with permission
from Mihailoff and Border, 1990.)
cathodal pulses of 0.2 ms duration at constant
current intensities ranging from 40 to 200 jJ.A at
frequencies ranging from 1 to 10 Hz. A weak
anodal pulse (20 f..lA, 2 ms) followed the cathodal
pulse in an attempt to minimize the potential
deleterious effects of excessive current delivered
through the stimulating electrode. Peripheral
cutaneous sites were electrically stimulated with
up to double the intensity of cortical sites. For
each BPN single-unit (neuron) recorded, the
threshold response was determined by identify-
ing the lowest stimulation current from a given
cortical site that was sufficient to evoke a response
in that cell. In the case of peripheral stimulation,
when threshold values were determined, natural
152
Figure 7.14. A: Large variety ofGABA-Ir bouton that
is densely populated with synaptic vesicles giving it
a dark appearance compared to the pale GABA-Ir
boutons. B: Small dark GABA-Ir boutons are also
present throughout the neuropil. C: Serial synapse
stimulation was applied (gentle brushing of glab-
rous and hairy skin, light squeezing and tapping,
joint manipulation) to determine the sensory
modality responsible for elicitation of that re-
sponse. Finally, all BPN neurons that were tested
exhibited the appropriate response to antidromic
stimulation (followed high frequency stimulation
G .A. Mihailoff et al.
involving a dark GABA-Ir bouton that is presynaptic
to a gold-labeled pale bouton, which is in turn pre-
synaptic to a small dendritic profile. (Reprinted with
permission from Mihailoff and Border, 1990.)
of 300 Hz with constant latency) delivered through
the brachium pontis.
At the conclusion of the recording session, each
animal was administered an overdose of Nem-
butal and perfused transcardially with 10% buf-
fered formalin. The brain was removed, sectioned
transversely at 50 pm on a freezing microtome,
7. The Basilar Pontine Nuclei
pg3847
FlPer BIN " 2MS
0.0 0.2
TIME (SEC)
Figure 7.15. A: Protocol employed in the initial single-
unit recording experiments. Three stimulation sites
included cerebral cortex (Ctx), paramedian lobule
(PML) of the cerebellar cortex, and the forepaw contra-
lateral to the Ctx stimulation site. B, C: Poststimulus
time histograms (100 sweeps) illustrating the response
and the sections mounted, stained with cresyl
violet, coverslipped, and examined to determine
the location of stimulating and recording elect-
rodes. Because of the relatively large size of the
BPN and the fact that it was approached ven-
trally under visual control, positioning of the re-
cording electrode within the BPN was not a
problem.
Cerebral Cortical and Peripheral Stimulation
Shown in Figure 7.16 are several representative
poststimulus histograms that illustrate the spike
activity in single BPN units in response to cere-
bral cortical stimulation. Excitatory responses
to forelimb motor and forelimb sensory cortex
153
pg3848
FLSCx BIN = 2 MS
0.0 0.2
TIME (SEC)
of two different BPN single units to stimulation of
the forepaw (FLPer, B) and the contralateral forelimb
somatosensory cortex (FLSCx, C). Small thin arrows
indicate excitatory responses whereas small thick arrows
indicate period of suppressed electrical activity (in-
hibitory trough). Asterisk indicates stimulus artifact.
occurred at similar latencies of 4 to 6 ms after
stimulation at threshold intensity. Threshold sti-
mulation in motor and sensory areas of the face
representation produced slightly more rapid re-
sponses at 2 to 5 ms latencies. Quantitatively,
threshold stimulation produced a broad range of
spike amplitudes in responsive neurons, whereas
qualitatively the responses were consistent in that
spikes were followed by a decrease in cellular
electrical activity for periods ranging from 50 to
80 ms (Fig. 7.16A, B). This quiet period, which
is referred to as the inhibitory trough, was also
apparent, albeit less prominently, after stimula-
tion at an intensity below (subthreshold) that
which was required to elicit an excitatory response
in the BPN (Fig. 7.16C). The inhibitory trough
154 G.A. Mihailoff et al.

A B
250 / 250 I
pg42112 I pg42111
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200 200

150 150
FLMCx FLSCx BIN = lMS
BIN = 1 MS
100 100

50 50

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TIME (SEC) TIME (SEC)

C / * 0 E
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4961
250
50
200 /
Subthresh. BIN '" 1 MS
150
Cx
100 les.
50 ...
0 01 : 1 0 01 : 2
0.0 0. 1 0.2 0.3 0.0 0. 1 0.2 0.3
TIME (SEC) TIME (SEC)

Figure 7.16. A, B: Representative poststimulus time
histograms illustrating responses of two BPN single
units to stimulation of the forelimb motor cortex
(FLMCx, A) or forelimb sensory cortex (FLSCx, B).
Thin arrow, excitatory response; thick arrow, inhibitory
trough; asterisk, stimulus artifact. C: Poststimulus time
histogram illustrating the activity of a single BPN unit
following subthreshold stimulation of the sensorimotor
cortex. No excitatory response is apparent while the
inhibitory trough is present. D: Following ablation of
gray matter comprising a large portion of sensorimotor
cortex and stimulation of the underlying white matter
or the medullary pyramid, the inhibitory trough can
still be observed. E: Oscilloscope traces depicting the
responses of two BPN units to antidromic activation
after stimulation of the white matter of the cerebellar
paramedian lobule.

persisted when a large expanse of the sensori-
motor gray matter was carefully removed by suc-
tion and the subjacent white matter stimulated
(Fig. 7.l6D). Occasionally, single units responded
to stimulation of more than one cortical site with
the most frequent combination being forelimb
sensory and vibrissae sensory areas.
In several but not all cases, stimulating elec-
trodes were positioned in a cerebellar folium or
the brachium pontis to test for antidromic activa-
tion of BPN units. Neurons that exhibited the
characteristic responses to cortical stimulation
described above were also responsive to anti-
dromic activation (Fig. 7.16E). The antidromic
response appeared at a constant latency of 1 to
2 ms and was capable of following repetitive
stimulation up to 300 Hz.
In contrast to the unitary excitatory responses
to cortical stimulation, the response of single BPN
neurons to peripheral electrical stimulation of
the forepaw consisted of two excitatory peaks
characterized by a short burst of activity at 4 to
7. The Basilar Pontine Nuclei
WGA - HAP
Figure 7.17. Experimental protocol employed to de-
monstrate convergence of afferents originating in the
dorsal column nuclei (DorCN) and forelimb cerebral
cortex (FLSCtx) on a single BPN neuron that projects
to the paramedian lobule (PML) of the cerebellum.
6 ms, followed by a slightly longer burst beginning
at 12 ms and lasting for 10 to 30 ms (Fig. 7.15B).
Interestingly, both peaks persisted in experiments
that involved ablation of sensorimotor (forelimb)
cortex. Responses to electrical stimulation at vari-
ous points on the face occurred more rapidly
with latencies of2 to 5 ms. All neurons responsive
to peripheral electrical activation were also tested
using natural stimulation applied with a cotton
swab, blunt probe, forceps, or joint manipulation.
Approximately 81% of the cells tested responded
to light tactile stimulation of glabrous or hairy
skin, whereas 16% responded to deep pressure or
tapping and only 3% responded to joint manipu-
lation.
Also, as can be seen in Figure 7.15C, many
BPN units responded to stimulation of both the
periphery and the corresponding region of sen-
sorimotor cortex, in this case the forepaw peri-
pherally (electrical and light tactile brushing), and
forelimb sensory cortex. Of 110 units recorded,
42 (38%) responded to both peripheral and corti-
cal stimulation whereas 48 (44%) responded only
to cortical stimulation and 20 (18%) responded
only to peripheral stimulation.
Other BPN units responded to a combination
offorelimb peripheral and forelimb motor cortex
stimulation. Moreover, when cells were tested in
155
BPN
Boutons of the two afferent systems were labeled either
by the orthograde transport of WGA-HRP or by de-
generation after a lesion involving the DorCN. BPN-
PML projection neurons were labeled by the retrograde
transport of WGA-HRP.
combination with cortical and natural peripheral
stimulation, those units responsive to sensory
cortex stimulation were activated by light tactile
stimuli whereas those responsive to motor cortex
were activated by squeezing or tapping of the
forepaw and were unresponsive to light tactile
cutaneous stimulation.
Based on these findings, which demonstrate a
convergence of cortical and peripheral inputs
on single BPN neurons, two questions arise. The
first concerns the identification ofthe anatomical
substrate for this convergence and the second is
where in the cerebellar cortex do such BPN neu-
rons project. In response to the first question,
since it had been recently demonstrated that the
dorsal column nuclei (DorCN) project to the BPN
(Kosinski et aI., 1986; Swenson et aI., 1984) a series
of experiments employing the protocol illustra-
ted in Figure 7.17 was implemented. First, a large
ablative lesion was performed so as to involve the
entire extent of the dorsal column nuclei on the
right side of the medulla. Three to 5 days later,
WGA-HRP was injected into the left sensorimo-
tor cortex and the right paramedian lobule of the
cerebellum. After another 36- to 48-hour survival
period, the animal was sacrificed, the BPN sec-
tioned on a vibratome, and the tissue reacted to
visualize HRP. After stabilizing the HRP reaction
156
Figure 7.18. Electron micrograph illustrating the re-
sults of the experiment depicted in Fig. 7.17. In the
center of the field, a dendrite of a BPN-PML projection
neuron is identified by the presence of retrogradely
transported WGA-HRP. This dendrite receives a
product, the tissue was prepared for electron
microscopy (Kosinski et ai., 1988). The rationale
was to establish that an HRP-labeled pontopara-
median neuron received both cortical and DorCN
synaptic boutons and thus, the PML would be
identified as a target for the convergent inputs to
BPN neurons.
Upon electron microscopic examination ofthe
BPN in such an experiment, numerous HRP-
labeled dendrites of pontoparamedian neurons
were observed in synaptic contact with HRP-
labeled boutons or degenerative boutons. Al-
though a few examples were observed, it was
extremely rare to see both cortical and DorCN
boutons in contact with the same pontopara-
median neuron dendrite (Fig. 7.18). This was to
be expected, however, since corticopontine syn-
apses are known to be formed with distal por-
tions of BPN neuron dendritic systems whereas
G.A. MihailofT et al.
synapse from a degenerative DoreN axon (large arrows)
and a cortical bouton (small arrows) labeled by the
orthograde transport of WGA-HRP. (Reprinted with
permission from Kosinski et aI., 1988.)
DorCN boutons tend to contact primarily the
proximal and intermediate portions of the den-
dritic tree. Experiments to control for the possibi-
lity of spurious results due to the transneuronal
movement ofWGA-HRP included (a) injection of
WGA-HRP into only one ofthe three areas under
study in a single animal, and (b) a PML injection
of WGA-HRP combined with a lesion in either
cortex or DorCN. No evidence for the possibility
of either retrograde or anterograde transneuronal
movement of HRP was obtained at either the
light or electron microscopic level.
Stimulation of Cerebellopontine Afferents
In addition to cortical and peripheral stimulation,
single BPN units were tested for responsiveness
to stimulation of the cerebellar nuclei (DCN).
After electrical stimulation of the lateral cerebel-
7. The Basilar Pontine Nuclei 157

A B
200 " PG4534 200 pg4422

150 150

100 DeN 100 DeN

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50
., B IN : 2 MSEC
50

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.~ ~,.......... 0-
0.0 0.2 0,4 0.0 0.2 0.4
C
200 / SEC o SEC
PG4533 200 4751
",

150
DeN 150 BCtx
FLMCx FLMCx
100 100

50 50
-I '1 J
'* BIN : 2MSEC 111 BIN : l msec
0
0.0 0.2
~\, t' .. ~ ~~,.. .... ,w.1,
0.4
.,wII!II'''''''''''0.' ' 1"' ' ~0.2
'I'II• 0.3 .~''II~~''.
0.4 0.5
SEC SEC

Figure 7.19. Representative poststimulus time histo-
grams illustrating the response of BPN single units to
stimulation of the deep cerebellar nuclei (DCN). A:
BPN unit showing an initial excitatory response (thin
arrow) followed by inhibition (thick arrow). Asterisk
indicates stimulus artifact. B: BPN unit responding
only with inhibition. C: BPN single unit activity follow-
ing combined stimulation of forelimb motor cortex
(FLMCz) and DCN. Compared to A, the inhibitory
trough is enhanced. D: Following transection of the
brachium conjunctivum (BCtx) and stimulation of the
FLMCz, the inhibitory trough persists.

lar nucleus, single neurons in medial or ventral
BPN regions either exhibited spike activity
(Fig. 7.19A) at very short latencies (2.5 ms or less)
or their activity was suppressed (Fig. 7.19B) with-
out evidence of any preceding excitation. Since it
was not possible to move the stimulation site in
the DeN once the recording electrode had been
positioned in the BPN, whether or not a given
BPN neuron that was initially excited by DeN
stimulation might subsequently be inhibited (or
vice versa), could not be ascertained.
Although it has been shown that pontocerebel-
lar axons provide a very small number of colla-
terals to the DeN (Dietrichs and Walberg, 1987),
there are pontocerebellar axons coursing through
the white matter adjacent to the DeN that could
be incidentally activated by the current pulse
delivered through the stimulating electrode and
thus produce antidromic spikes in BPN neurons
that might be confused with the short latency of
orthodromic activation. To test for this possibi-
lity, if a BPN unit exhibited short latency spike
activity to DeN activation, the stimulus fre-
quency was increased in a stepwise fashion. If the
spike activity followed stimulus frequencies above
150 Hz, the response was interpreted as anti-
dromic. In fact, none of the recorded DeN-re-
sponsive units exhibited such following capability
and thus we are confident that the increased spike
activity recorded was the result of a short latency
excitatory input from the DeN.
DeN-responsive units in the BPN were also
tested to determine if they received cortical or
peripheral inputs. Although none received peri-
pheral somatosensory input, some were clearly
responsive to stimulation at various cortical sites,
158 G.A. Mihailoff et al.

with the combination of DeN and forelimb motor made it clear that the basilar pontine nuclei rep-
cortex being particularly effective. Interestingly, resent one of the most (if not the single most)
when the cortical and cerebellar nuclear activa- important cell group involved in the transmission
tion of BPN neurons was paired such that cere- of cortical information to the cerebellum. There-
bellar stimulation followed the cortical stimula- fore, it should be understood that the observa-
tion by 2.5 ms, the effect of the convergent inputs tions described in this chapter must not be
was to enhance the inhibitory trough (Fig. 7.19, interpreted as an attempt to minimize the im-
compare A and C). In addition, in two cases, the portance of the transfer of cerebral cortical
brachium conjunctivum (BC) was transected- information to the cerebellum as a fundamental
midway in the recording session after BPN units functional operation of the BPN. Rather, it is
that were responsive to combined DeN-motor intended that this chapter should point out addi-
cortex stimulation had been identified. After the tional functional attributes of the BPN by focus-
transection, BPN units were not responsive to ing attention on a) the newly identified BPN
DeN stimulation alone but continued to exhibit afferent systems and b) the recently described
spike activity in response to cortical stimulation. cellular characteristics and synaptic circuitry in-
The inhibitory trough observed after cortical trinsic to the BPN. Taken together, these two
stimulation was still present after Be transection features strongly suggest that the BPN function
but in an attenuated form. No BPN units have with much more diversity in their role as a source
yet been recorded that exhibited evidence of re- of cerebellar afferent signals than has previously
ceiving convergent peripheral and DeN input. been recognized.
The convergence of cerebellar and cortical in-
puts on single BPN neurons has also been con-
N oncortical BP N AfJerents
firmed with anatomical methods (Lee et aI., 1990).
Seven days after a series of 4 unilateral electrolytic
In survey studies of noncortical BPN afferent
lesions were made in the cerebellar nuclei, WGA- projections using the retrograde transport of
HRP was injected at 3 to 5 sites within the con- WGA-HRP, more than 50 subcortical cell groups
tralateral sensorimotor cortex. After an addi- were identified (Mihailoff et aI., 1988b). Because
tional 48-hour survival period, the animals were of this rather large number, one might question
perfused and processed for the stabilized HRP whether false-positive labeling due to injury-filling
reaction and electron microscopy, as described in ofaxons might account for some of the labeled
the studies of cortical and peripheral somato- cell groups. We suggest that this is unlikely be-
sensory convergence. These studies revealed the cause all injections were made by penetrating
presence of HRP-Iabeled cortical boutons and from the ventral surface ofthe BPN and no cases
degenerative cerebellar nuclear terminals in syn- were used if there was any evidence that the
aptic contact with a single BPN neuron dendritic micropipette extended into or dorsal to the fiber
shaft. systems (medial lemniscus, cerebral peduncle, late-
rallemniscus) forming the dorsal boundary of the
basilar pontine gray. Furthermore, although the
Discussion zone of effective HRP uptake cannot be defined
Over the past several years, a rather substantial accurately, it has been demonstrated that there
amount of information has been amassed regard- is little likelihood that WGA-HRP can be taken
ing the anatomical connectivity of the cortico- up and transported in sufficient quantity by un-
pontocerebellar system in the rat, cat, and monkey damaged fibers to produce false-positive somatic
(for review see Brodal 1982, 1987). These studies labeling (Brodal et aI., 1983). Thus, it seemed un-
have made it possible to appreciate better the likely that any of the axonal systems coursing
precision and complexity that characterizes this through the tegmentum adjacent to the BPN had
system. But perhaps of equal importance is the inadvertently been labeled.
fact that collectively, such studies have reempha- Another complication was considered, how-
sized the enormity of the cerebral cortical projec- ever, when it was noticed that several cell groups
tions to the cerebellum and, at the same time, labeled after BPN injections were known to have
7. The Basilar Pontine Nuclei
direct projections to the cerebellum. For several
of these cell groups, such as the locus coeruleus
or the lateral reticular nucleus, it was known that
at least some of their axons entered the cerebel-
lum by way of the superior or inferior cerebellar
peduncle but for certain other cell groups, the
route of cerebellar entry was unknown. Thus, the
possibility was entertained that some of these
cells were labeled because their axons entered the
cerebellum through the middle cerebellar peduncle
(brachium pontis) and thus were unknowingly
damaged and injury-filled at the injection site
within the BPN as they coursed toward the
brachium pontis. This possibility was negated,
however, by experiments that involved a multiple
array of WGA-HRP injections in the cerebellar
cortex which, in some animals, were preceded by
large bilateral lesions of the brachium pontis.
The pattern and number of retrogradely labeled
somata were essentially the same under the two
conditions, and thus it was reasoned that the cell
groups in question did project to both the cere-
bellar cortex and the BPN, and that such cells
did not use the brachium pontis to enter the
cerebellum, at least not in appreciable quantity.
Of the 50-odd afferent cell groups identified in
these studies, 22 had previously been demonstrat-
ed in a variety of orthograde tracing experiments.
Thus, approximately 28 of these cell groups re-
present previously unrecognized BPN afferents.
In some instances, the number of labeled cells in
a nucleus was quite sparse (Le., the central nucleus
of the amygdala and horizontal nucleus of the
diagonal band), and one might question whether
such connections are truly functional or might
represent a vestige of a developmental anomaly.
This question remains for future investigation,
although the presence of such connections in a
number of animals suggests that they are not the
remnants of truly aberrant development.
The number of labeled somata observed in
certain other cell groups was numerically more
substantial, but the implications regarding their
functional relationship to the BPN and cerebellum
are unclear. In this category are included the
midbrainperipeduncular nucleus, the dorsal teg-
mental nucleus, and the periaqueductal gray. Per-
haps somewhat less mysterious because of their
involvement with sensorimotor circuitry are the
labeled cells in substantia nigra pars reticulata
159
and pars lateralis, along with the pedunculopon-
tine tegmental nucleus and the spinal vestibular
nucleus. In addition, there are the afferent cell
groups that were shown in subsequent studies to
contain GABA-ergic components; these included
the lateral cerebellar nucleus, zona incerta, peri-
rubral cells, anterior pretectal nucleus, and cells
in the medullary reticular formation. Many BPN
projecting cell groups involved in visual system
circuitry are also of interest, as are the numerous
cell groups that, in addition to their BPN con-
nections, also project directly to the cerebellum.
Obviously, many ofthese connections are worthy
of additional investigation and their existence
serves to emphasize the diversity of afferent sys-
tems reaching the BPN.
Do BPN Local-Circuit Neurons Exist?
There is as yet no unequivocal answer to this
question, although the existing evidence suggests
an affirmative response. Several years ago, Golgi
and electron microscopic (EM) studies in the
monkey (Cooper and Beal, 1978; Cooper and Fox,
1976) and rat (Mihailoff et aI., 1981a; Mihailoff
and McArdle, 1981) illustrated BPN neurons with
complex dendritic appendages, multiple, locally
ramifying axonlike processes, flat and pleomor-
phic vesicle boutons, synapses between vesicle-
containing profiles, and evidence for the possible
existence of presynaptic dendrites. Several years
earlier, studies in the opossum had produced
similar observations (Mihailoff and King, 1975)
whereas quite a number of years previous to the
opossum report, Ramon y Cajal (1911) had illus-
trated a BPN neuron with a locally ramifying
axon. Although this collection of evidence was
quite suggestive, the question was by no means
resolved since there was no way to be certain with
the Golgi (and EM) method that the entire extent
of the presumptive locally ramifying axon was
being visualized. Moreover, existing electrophy-
siological studies of BPN neuronal activity were
contradictory. Single-unit recording by Sasaki
et al. (1970) in the cat provided strong evidence
for the existence of cortical and recurrent colla-
teral activated inhibition in the BPN. Conversely,
the intracellular studies of Allen et aI. (1977) indi-
cated the presence of weak inhibitory postsynaptic
potentials (IPSPs) that were only rarely observed,
160
and thus these authors essentially denied the
existence of a significant number of BPN local-
circuit neurons.
More recently, ICC studies have demonstrated
the presence of GAD- and GABA-immunoreactive
somata and axon terminals in the BPN ofthe rat
(Border and Mihailoff, 1985), cat (Brodal et aI.,
1988), and primate (Brodal et aI., 1988; Thier and
Koehler, 1987). Although it is clear, at least in the
rat, that some of the GABA-ergic terminals are
derived from cell groups extrinsic to the BPN
(Border et aI., 1986) one might presume that the
population of GABA-ergic neurons within the
BPN might also give rise to a portion of the
GABA-ergic axon terminals. So it is that ultra-
structural ICC studies in the rat (Border and
Mihailoff, 1990) indicate the presence of two
populations ofGABA-ergic boutons in the BPN.
In the monkey, observations from the present
study are also somewhat suggestive of the pres-
ence of two populations of GABA boutons. If,
eventually, only one population of GABA bou-
tons is shown to be present in the primate or
alternatively two types with one variety in great
abundance, one might speculate that the tremen-
dous increase in the number of BPN GABA
neurons is linked in some way to a decrease in,
or an elimination of, some or all the extrinsic
GABA-ergic BPN afferent systems. This course
of events could explain why one segment of the
GAB A bouton pool (the largest GAB A boutons)
appears to be relatively sparse in the monkey.
The present study also demonstrated in control
neuropil the existence of two types of synaptic
complexes (glomerular and nonglomerular) that
included GABA-Ir boutons. Each of these com-
plexes exhibited serial synaptic contacts involving
at least two vesicle-containing profiles and one
or several dendritic elements. In the glomerular
type synapse, a large central axon terminal is
contacted on its perimeter by several dendritic
structures that are postsynaptic to the central
bouton. In addition, one or more of the perimeter
profiles often contains pleomorphic synaptic
vesicles and appears to be presynaptic to an ad-
jacent dendritic profile and postsynaptic to the
central bouton. The triadic arrangement of these
structures, and the pale appearance of the pleo-
morphic vesicle bouton, are very similar to the
glomerular complexes described in the dorsal
G.A. Mihailoff et al.
lateral geniculate nucleus of the cat (Famiglietti
and Peters, 1972) and mouse (Rafols and Valverde,
1973), where it was demonstrated that the pale
bouton with pleomorphic vesicles was actually a
presynaptic dendritic appendage emanating from
a local-circuit neuron. An earlier EM study of
the primate BPN also illustrated a similar pale
presynaptic profile (Cooper and Beal, 1978) and
further, the presence of presynaptic dendrites in
our own material (Mihailoff and Border, 1990)
strongly supports the notion that local circuit
neurons are present in the BPN.
The nonglomerular complex also includes a
pale GABA-Ir synaptic profile; however, the
round vesicle bouton that is in a presynaptic
position is often a much smaller profile than the
central axon terminal of the glomerular synapse.
Also, the arrangement of the nonglomerular com-
plex is such that the pale profile is presynaptic to
a dendritic element that is not postsynaptic to the
participating round vesicle bouton. Usually no
more than three profiles are involved in the non-
glomerular complex and there is no glial invest-
ment of this group of boutons as there is with the
glomerular variety.
Although still in a preliminary stage, our
monkey ICC material also illustrates GABA-Ir
boutons that typically contain many synaptic
vesicles giving the bouton a darker appearance.
These boutons form relatively isolated one-to-
one synapses with essentially all somatic and
dendritic compartments but mostly small dendrit-
ic structures. They also form synaptic complexes
with GABA-Ir pale boutons. At present, we inter-
pret these observations to suggest that at least
some ofthe dark GABA-Ir boutons are of extrinsic
origin and further that these afferents interact
with the intrinsic GABA neurons represented by
the pale GABA-Ir boutons. An important question
to be resolved is whether the local-circuit neurons
have axons and if so, how can the synaptic boutons
of such axons be identified. It is clear that most
X-type interneurons in the lateral geniculate nu-
cleus of the cat exhibit conventional action poten-
tials (Ramos et aI., 1985; Sherman and Friedlander,
1988), but there is at least a suspicion that Y-type
interneurons might not give rise to an axon.
Once again it seems that the accumulated EM
and ICC observations in the monkey are not
absolutely definitive but argue strongly for the
7. The Basilar Pontine Nuclei
existence of a BPN local-circuit neuron. Although
the possibility that the BPN GABA-ergic neurons
actually project to the cerebellum should be con-
sidered, on the basis of double-labeling studies in
the cat (Brodal et aI., 1988) and preliminary experi-
ments in our laboratory, no such projections have
yet been identified. It remains, however, that such
projections might be quite sparse in number and/
or restricted in distribution so that their detection
would require a systematic and comprehensive
search. It seems that what is required is for future
studies to employ immunocytochemistry along
with intracellular recording and HRP-filling to
allow visualization of the entire axon and per-
haps in this way be able to definitively resolve
this question.
Current Observations on BPN
Functional Properties
Two particularly relevant observations can be
gleaned from the preliminary electro physiological
studies of the corticopontine system reported in
this chapter. First, in nearly every test situation a
BPN unit that responded to cortical stimula-
tion exhibited first an excitatory response (spike
activity) that was then followed by an inhibition
or suppression of activity that lasted for a variable
period oftime (50-80 ms). This inhibitory trough,
as it is referred to, was present even when cortical
stimulation intensity was lowered below the thresh-
old value required to produce an excitatory re-
sponse in the BPN neuron, and was also observed
after cortical gray matter ablation and subse-
quent stimulation ofthe underlying cortical white
matter. The latter two observations suggest that
a) the inhibition is an active process and not the
result of cortical disfacilitation and b) the inhibi-
tion is not a function of intracortical circuitry.
Whether an inhibitory pontine local circuit is
solely responsible for the inhibitory trough re-
mains for further investigation since in the rat, it
is possible that subcortical GABA-ergic afferent
projections might be activated by cortical stimu-
lation, thus resulting in inhibition within the BPN
with a similar latency.
A second relevant observation concerns the
convergent input to BPN neurons that involves
cortical afferents and peripheral somatosensory
161
input mediated by the DorCN, or the combina-
tion of cortical and cerebellar nuclear afferents.
Although previous studies have reported that
BPN neurons receive convergent input in various
combinations from a variety of different cortical
regions (Boyd and Aitkin, 1976; Oka et ai., 1975;
Potter et aI., 1978; Ruegg and Weisendanger,
1975; Ruegg et aI., 1977; Sasaki et aI., 1975), the
work of others tends to emphasize the selectivity,
restriction, or segregation of the various types of
cortical input to BPN neurons (Baker et aI., 1976).
In our own experiments, some BPN neurons
clearly received convergent input from different
portions of sensorimotor cortex such as forelimb
and face sensory cortices. However, it was our
intent to search for the potential convergence of
cortical and peripheral somatosensory input and
it was not at all uncommon to encounter single
BPN units that received peripheral somatosensory
input from the face region or the forepaw in
combination with an input from the correspond-
ing region of sensorimotor cortex. Similarly, BPN
neurons were encountered that, for example, re-
ceived convergent input from the lateral cere-
bellar nucleus and forelimb motor cortex. Curi-
ously, in both types of convergent input the ex-
citatory or spike-generating capacity of the two
inputs did not seem to summate but rather the
depth and duration of the inhibitory trough was
enhanced, apparently as a function of the conver-
gent inputs. In fact, stimulation of the lateral
cerebellar nucleus alone sometimes produced only
inhibition in the BPN. This might prove to be an
interesting correlation with the observation in rat
that a portion of the pool of lateral cerebellar
neurons that project to the BPN are GABA-ergic.
Equally curious is the finding that ifthe brachium
conjunctivum is transected, the inhibitory trough
is still present after cortical cerebral stimulation.
It seems. clear, however, that activation of some
cerebellopontine neurons leads to inhibition or
suppression ofBPN neuron activity. Exactly what
role is played by such an inhibitory input must
remain unanswered for the moment, although
such observations suggest that inhibitory opera-
tions may exist at several levels in the BPN. The
presence of both excitatory and inhibitory BPN
inputs from the DCN also correlates with previous
EM studies that suggested that the cerebellopont-
ine system consisted of more than one type of
162
neuron (Mihailoff et aI., 1981 b; Watt and Mihailoff,
1'983). These findings are consistent with double-
label experiments involving the combination of
ICC and retrograde transport of WGA-HRP,
which indicated that GABA-ergic (Border et aI.,
1986) and glutaminergic (Border and Mihailoff,
1991) neurons of the cerebellar nuclei project to
the BPN. Also, recent fluorescent double-label
studies have revealed that the cerebellopontine
system includes axon collaterals of neurons that
project rostrally to the thalamus as well as colla-
terals arising from axons that project to the inferior
olive (Lee et aI., 1989). Thus, the existing evidence
seems to indicate that the cerebellar projection
to the BPN is complex and consists of both exci-
tatory and inhibitory inputs that appear to arise
from at least two different populations of neurons.
In summary, the anatomical and electrophys-
iological observations that comprise this chapter
indicate that as a whole, BPN neurons receive an
unusually diverse set of afferent projections, that
some BPN neurons in fact receive convergent
inputs from different afferent sources that are
then transmitted to a specific locus in the cerebel-
lar cortex, and that the functional activity within
the BPN includes inhibitory operations that might
be accomplished at least in part by local-circuit
neurons. Taken together, these findings suggest
a more expansive, diverse, and integrative role for
the BPN than has previously been anticipated.
Much additional experimentation will be required
to address several of the new questions that have
emerged from the present studies.
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 Part 2
Electrophysiology of Purkinje
and Inferior Olivary Neurons

The first chapter in this part is an up-to-date review of the electrophysiologi-

cal properties of single Purkinje cells. It begins with a review of the many
experimental approaches which were brought to bear to dissect the wide
range of ionic conductances found in these neurons and to reveal the
distribution of the various channels over the somadendritic membrane.
This section of the chapter ends with the story of the "P" (for Purkinje)
channel. The second section, beginning with consideration of Purkinje cell
ligand-gated responsiveness, speaks to the question of the functional signi-
ficance of the wealth of conductances enjoyed by these cells.
The second chapter brings the glass closer and considers the electrical
properties of single channels in the soma of Purkinje cells in organotypic
culture. Patch clamp recordings using both cell-attached and outside-out
configurations were used to study voltage-gated sodium and potassium
currents; calcium currents were not identified. The sodium current had
two components (transient and sustained). The authors believe that one
channel may underlie this based on the elemental properties of the channels
as determined by fluctuation analysis and single-channel measurements.
Two types of K channel were identified using single-channel recordings;
one (small conductance channel) with properties similar to the delayed
rectifier, the other (large conductance) probably corresponding to the
gK.Ca' Purkinje cells do not respond to NMDA, but large inward currents
are elicited by non-NMD A glutamate agonists, and they respond to GAB A
with a large inward current. Soma apparently have a high density of
GABAergic channels and a low density of glutaminergic channels.
The last chapter in this section is concerned with the oscillatory properties
of 10 neurons and introduces the use of a computer-brain slice hybrid to
study this problem.

165
8
The Electrophysiology of the
Cerebellar Purkinje Cell Revisited
Rodolfo R. Llimis and Mutsuyuki Sugimori
The electro physiological properties of mamma-
lian Purkinje cells has been a subject of study
for more than 30 years (Eccles et aI., 1967) and
has produced a wealth of information relating to
the ionic conductances in mammalian central
neurons (cf. Llimis, 1988). Yet it is clear from the
many examples of intracellular recordings ob-
tained in vivo that the Purkinje cell is capable of
many functional states. Such complexity is the
expected product of the rich intrinsic electrical
properties of these cellular elements and of the
wealth of synaptic connectivity they receive.
Indeed, if we consider that the tertiary spiny
branch lets of mammalian Purkinje cells are con-
tacted by many as 200,000 parallel fibers (Fox
and Barnard, 1957; Fox et aI., 1967) and that,
unlike other central nervous system (CNS) spines
(Peters et aI., 1976), only one parallel fiber synapse
is made onto each spine head (Paley and Chan-
Paley, 1974), a large variation of dendritic-input
patterns and thus, an enormous variability in
the activation states, may be expected. Equally
surprising is the other main synaptic input, the
climbing-fiber system. In most adult cells only
one climbing-fiber afferent contacts a given
Purkinje cell. The contacts are made on groups
of postsynaptic spines arising from the smooth
surface of the primary and secondary dendrites
(Larramendi and Victor, 1967). Clusters of six to
eight spines are often organized in longitudinal
rows along the dendrite and are contacted by en
passant enlargements of the climbing fiber as it
progresses over the surface of the dendrites. As
many as 300 spines are contacted by a single
afferent fiber (Llimis et aI., 1969). Given this struc-
ture, it is not surprising that a stereotyped syn-
aptic response to climbing fiber activity, the com-
plex spike, is observed at all levels of cerebellar
phylogeny (Llinas and Hillman, 1969).
Purkinje Cell Electroresponsiveness
That the electrophysiology of Purkinje cells is as
baroque as their morphology (Fig. 8.1, see color
Plate 1) was established long ago when it was
discovered that these cells generate complex pat-
terns of dendritic spiking (Llinas et aI., 1968;
Llinas and Nicholson, 1971). These action poten-
tials were found to be generated by different
branches such that as many as six to eight different
dendritic spike-generating zones were revealed
by the presence of several all-or-none components
in each action potential. Later it was discovered
that these action potentials are calcium dependent
(Llinas and Hess, 1976; Llinas et aI., 1977; Llinas
and Sugimori, 1980a).
The detailed study of the ionic mechanisms
underlying Purkinje cell electro responsiveness
began with in vitro intracellular recordings in
cerebellar slices (Llinas and Sugimori, 1980a, b).
The results presented in those papers suggested
that, in addition to dendritic-calcium electrores-
ponsiveness, somatic electroresponsiveness may
be characterized by the presence of at least two
types of voltage-gated sodium conductances.
Ionic Conductances:
General Description
Although current-clamp studies showed that the
sodium and calcium conductances in Purkinje
cells were largely confined to the somatic and
167
168
dendritic membranes, respectively (Llinas and
Sugimori, 1980a, b), a more direct demonstration
of the distribution of these ionic conductances
was required in order to understand the complex
firing properties of these cells. Two types of experi-
ments were designed toward this end. The first
consisted of com paring the properties of the spike
waveform, as recorded intracellularly with the
extracellular field potentials simultaneously gen-
erated by the same neuron. To implement this
paradigm, Purkinje cells were recorded intracel-
lularly at soma while the extracellular current flow
was monitored using a currentometric amplifier
configuration and a patch pipette (Fig. 8.2A).
Depolarization to threshold was accompanied
11111
o E
1.1 '
Figure 8.2. Simultaneous ' intracellular and extracel-
lular recording from a Purkinje cell. In A, intracel-
lular recordings were obtained at the somatic level
while a patch electrode filled with normal Ringer solu-
tion recorded current transmembrane at the somatic
level. In 8, the upper trace is the intracellular somatic
potential trace. The second trace is the transmem-
brane current. Note that every spike in the intracel-
lular record is accompanied-by a sharp extracellular
negativity (inward current). The third trace figure indi-
cates the size and duration of direct current injections
that activated the cell. C: Similar recording as 8 , but
a higher current-injection pulse. The fast spikes were
accompanied by simultaneous extracellular negativity
Rodolfo R. Llimis and Mutsuyuki Sugimori
by a full set of sodium-dependent action potentials
at the somatic level, which generated clear in-
ward all-or-none action currents in the immediate
vicinity of the soma, with amplitudes on the order
of hundreds of pico amps (Fig. 8.2B, middle trace).
The negative deflection (inward current) indi-
cated that the sodium-dependent spikes were gen-
erated at the somatic level (Fig. 8.2C). With a
further increase in the depolarizing current in
addition to the fast sodium spikes, slower calcium-
dependent spikes were elicited (dots). These latter
spikes were seen by the somatic patch electrode
(middle trace) as positive deflections (outward
currents) (dots in Fig. 8.2C), indicating a spike
generation site remote from the soma. That the
~18OOpA
J 1
<--_ _
F
- ~.
I
CI 20mV
.6OOpA
2M
100m...,
while the high-threshold calcium spikes (dots) generated
extracellular positive deflections (outward current flow).
The calcium spikes are indicated by dots (the intracel-
lular and extracellular records). In D , the extracellular
electrodes moved from the somatic to the dendritic
level of the same cell. Experiments similar to those in
8 and C were performed. In E, the direct stimulation
of cells at the somatic level produced clear positive
potentials. Extracellularly (outward current) following
a stimulus increase (F), calcium spikes (dots) are seen
as negative current fields (inward currents) by the cur-
rent metric amplifier (Llimis and Sugimori, unpublished
obvervations).
8. Electrophysiology of the Purkinje Cell
direction of current flow at the somatic level
was the opposite for the sodium- and calcium-
dependent action potentials indicates that these
two distinct types of spikes were generated at
different sites in the neuron.
The patch electrode was then gently moved to
the dendritic level while the intracellular electrode
was kept in place (Fig. 8.2D). In this configuration,
the patch electrode recorded extracellular cur-
rents with precisely the opposite polarity ofthose
recorded when the electrode was near the soma.
That is, when fast action potentials were activated,
the patch electrode near the dendrites recorded
outward current transients (Fig. 8.2E), but when
the slower calcium spikes were generated, clear
inward current flow was detected at the dendritic
level (Fig. 8.2F).
In a second type of experiment, designed to
test further the hypothesis that voltage-gated
channels are segregated over the plasmalemma,
intrasomatic recordings were made in the pres-
ence of tetrodotoxin (TTX) and the absence of
extracellular calcium (Fig. 8.3A). Calcium or
c E
B o Co", TTl
20mV
2n.o.
I
-----12n.o.
'j(j"$
Figure 8.3. Intrasomatic recording from a Purkinje
cell in the presence of TTX and zero extracellular
calcium. In A, the intracellular recording obtained at
the somatic level following outward transmembrane
current pulses before electrophoretic barium applica-
tion to the dendritic tree. Three levels of transmem-
brane current injection are shown in mid-trace. In C,
barium is electrophoretically applied (note lowest trace)
at the dendritic level. During the iontophoretic injec-
169
barium was then iontophoresed into the bath
and full spikes were observed as soon as either
of these divalent cation species reached the surface
of the dendritic tree of the impaled cell. The res-
ponse to BaCl 2 iontophoresis is shown in Figures
8.3C and 8.3D. Indeed, current injection of in-
creasing amplitude produced subthreshold
responses at low levels and prolonged barium-
dependent all-or-none depolarization at high cur-
rent levels. The middle traces in Figure 8.3C illus-
trate the amplitude and duration of the trans-
membrane current injected, and the lower trace
shows the onset and duration of the iontophoretic
BaCl 2 injections. Direct observation at the ion-
tophoretic electrode made it possible to direct the
barium solution to different regions of the Purkinje
cell dendritic tree. The traces in Figure 8.3D are
shown at a slow sweep speed to demonstrate the
prolonged spikes typically recorded following
barium superfusion over the dendritic tree. When
the barium electrode was placed immediately next
to the soma (Fig. 8.3E), no obvious electro-
responsiveness was observed following direct
80"
n~="",,:
F
=========
20mV
I 2n.o.
800n.o.
tions the soma is directly depolarized and a large
response is obtained. This response is seen at slower
sweep speed in D, where six stimuli similar to those
shown in C are presented at short intervals. In E, the
barium electrode is moved from the dendritic region
to the immediate vicinity of the soma and the experi-
ment was repeated. Under these conditions no barium
action potential was observed (F) (Llinas and Sugimori,
unpublished observations).
170 Rodolfo R. Llinas and Mutsuyuki Sugimori

depolarization of the soma (Fig. 8.3F). This was Dendritic Conductances

the case although the barium injection was the
Because Purkinje cell smooth dendrites in mam-
same and the voltage steps were higher than those
mals, as well as in some lower vertebrates, are
used in Figures 8.3C and 8.30. These experiments
large enough to allow intradendritic penetration
indicate once again that the somatic membrane
without cell injury, a direct study of the electro-
of the Purkinje cell has a very low density of
physiology of Purkinje cell dendrites has been
voltage-gated calcium channels.
possible in several species. Intradendritic record-
In addition to the electrophysiological investi-
ings were first made in reptilian Purkinje cells,
gation ofthe properties of the Purkinje cell soma,
where it was discovered that dendrites are capa-
a supplementary set of experiments was designed
ble of voltage-dependent electro responsiveness
using fluorescent TTX (Sugimori et aI., 1986) to
(Llimis et aI., 1968; Llinas and Nicholson, 1971).
localize the sodium channels over the Purkinje
In a latter set of studies on avian Purkinje cells
cell (Fig. 8.4, see color Plate 2). The fluorescent
it was further demonstrated by intradendritic re-
derivative ofTTX (7-diethylaminocoumarin-TTX)
cording that these processes can support calcium-
used in these studies (Angelides, 1981) was ex-
dependent electroresponsiveness (Llinas and
cited at 380 nm and had fluorescent emission at
Hess, 1976). Both of these studies also showed
480 nm. Aliquots of fluorescently labeled TTX
that dendritic spikes are quite different from
(10-100mM) were added to the bath at a final
axonic action potentials in that they are not con-
concentration of 10 to l00nM. The slices were
ducted continuously. In vitro recordings from
then observed via a 6, 25, or 50 x objective, using
Purkinje cells in guinea-pig cerebellar slices
a silicone-intensified television camera (MTI
(Llinas et aI., 1977; Llinas and Sugimori, 1980b)
Model 65) fiber-coupled to a double-microchannel
revealed that Purkinje cell dendrites are charac-
plate (I. T.T). The images obtained by this method
terized by two distinct types of electroresponsive-
were stored and digitized using a polyproces-
ness: so-called plateau potentials and compound
sor (Hamamatsu system) that allowed us to
dendritic spikes. The difference between these
quantify the amount of fluorescence omitted from
two types of dendritic activation, in our opinion,
the tissue. Slices were photographed in pseu-
reflects the distribution of calcium channels over
docolor using an image processing system
the dendritic arbor rather than the presence of
(Oeanza Inc.).
different species of voltage-gated conductances.
At low magnification (Fig. 8.4A) TTX binding
Also associated with dendritic electroresponsive-
sites were clearly visible in the axonal and somatic
ness was the activation of voltage- and calcium-
portion of the Purkinje cells but were not seen in
activated potassium channels (Llinas et aI., 1968;
the dendritic tree, except for the very basal portion
Llinas and Nicholson, 1971) and calcium- activated
of the main arbor. Note that whereas the labeling
chloride channels (Llano et aI., 1991).
was most prominent in the white matter and in
the granular and Purkinje cell layers, it was faint
in the molecular layer, probably because the
parallel fibers were transversely cut. At a higher
Plateau Potentials
magnification (Fig. 8.4B) the somata of Purkinje The plateau potentials of Purkinje cells are char-
cells could be clearly seen as fluorescent, whereas acterized by prolonged all-or-none events of
the dendritic tree, which was clearly seen in white rather low amplitude that may last for tens
light, did not show any TTX fluorescence. The or even hundreds of milliseconds (Llinas and
presence of nonspecific binding was investigated Sugimori, 1980a, b). This is shown in Figure 8.5,
by adding unlabeled TTX, which reduced the where the impaled dendrite was directly activated
fluorescence to close to autofluorescent levels, via the recording electrode. The plateau responses
indicating that the fluorescence of the labeled generated by injected current pulses (lower traces)
TTX was specifically linked to TTX binding sites. were totally separable from the somatic fast spike
Blocking of sodium action potentials following and the dendritic spike potentials. Indeed, as illus-
application of labeled TTX was demonstrated trated in Figure 8.5, a small dendritic depolariza-
by intransomatic recording. tion may produce different amplitude plateau

Figure 8.1. Intracellular fluorescence image of a
Purkinje cell injected with Lucifer yellow in an iso-
lated cerebellar slice from the adult guinea pig. This
image indicates the size and distribution of the differ-
ent portions of the Purkinje cell in the absence of
Plate 1
fixation. Note that the diameter of the primary, sec-
ondary, and even the tertiary dendrites is large
enough to be impaled intracellularly (somatic diame-
ter 45 lIm).
Plate 2
Figure 8.4. Fluorescence microscopy demonstrating the
distribution of TTX binding sites in cerebellar slices.
Note that the fluorescent-label Trx in A is clearly
present in the cerebellar white matter as well as at the
granular layer and the Purkinje cell layer, but is not
Figure 8.7. lliustration of different levels and locations
of [Ca2 +]j during spontaneous dendritic spiking in a
Purkinje cell. The body of the Purkinje cell has been
drawn to mark its location with respect to the fura-2
signal. A: Fluorescent image of the neuron after remov-
ing the intracellular electrode. B: Subtraction of back-
ground fluorescence from continuous recording show-
ing no increase in [Ca2 +]j at the resting potential. C:
[Ca2 +1i at the beginning of the burst before dendritic
action potential generation, the so-called plateau poten-
pre!,ent at the Purkinje cell dendritic tree. In B, high
magnification of this fluorescence demonstrates the
somatic distribution of TTX binding sites in Purkinje
cells (Sugimori, Lliruis and Angelides, unpublished ob-
servations) .
tial. D-H: Fluctuations of fluorescence in the dendritic
tree during activity. E-G: Full dendritic spikes occurred
and were accompanied by increased [Ca2 +]j in the main
dendrites. H: Intermediate time between spikes. I: Re-
turn of [Ca2 +]j to the baseline level after termination of
dendritic spike burst. In this and other figures, increases
in [Ca2 +]j are indicated by the color bar on the right:
Green indicates low and red indicates high [Ca2 +]j
(modified from Sugimori and LIinas, 1990b).
 Plate 3

•
.,
I
() 0

,.
I
.,
CI
0

LL

•·
0 CI 0
Q

" a
Q

CO I
••
.,=·
0

.,"
Plate 4
Figure 8.8. Distribution of [Ca2+]j after iontophoretic
application of glutamate to the dendrite of a Purkin-
je cell following fura-2 loading. A: The fluorescent
cell (arrowhead marks the position of the ion-
tophoretic electrode). B: Iontophoresis producing a
plateau potential (left-pointing arrowhead) after the
initial ligand-dependent depolarization. (The den-
dritic tree was drawn in white to mark its location
with respect to the fura-2 signal.) During the plateau
potential, a distinct increase of [Ca2+]j was observed
in the absence of a plateau potential. C: The plateau
potential was followed by full dendritic spiking. Note
that the [Ca2+]j increase, although widespread, was
highest in the three lower main dendrites . Calibration
was 30 m V, 5 ms (modified from Sugimori and
Llimis, 1990b).
Figure 8.9. Distribution of intracellular
calcium in Purkinje cells due to ligand
activation of a metabotropic glutamate
receptor. A: Purkinje cell filled with
fura-2. B: Fura-2 response following fir-
ing of the Purkinje cell. C: The response
of the cell after removing extracellular
calcium. D: Response of Purkinje cell to
glutamic acid injection at the somatic
level to demonstrate the release of calci-
um from intracellular stores following
activation of metabotropic glutamic acid
receptor at the soma and dendrites of the
Purkinje cells (Llinas and Sugimori, un-
published observations).
8. Electrophysiology of the Purkinje Cell
.~.--------------------
20mv
2 nA
1 the dendrites. This produced well defined ligand-
50msec
Figure 8.5. Intradendritic recording from a Purkinje
cell in the presence of TTX. Direct depolarization at
this site generated initially a passive response. At an
intermediate level calcium-dependent plateau poten-
tials were observed. At a higher level of depolarization
dendritic spikes were generated (Llinas and Sugimori,
unpublished observations).
potentials, some of which may be large enough to
trigger a family of dendritic calcium spikes.
Plateau potentials were abolished by calcium
channel blockers or by the removal of extracel-
lular calcium (Llimls and Sugimori, 1980a, b).
Furthermore, in the presence of potassium chan-
nel blockers such as tetraethylammonium, the
plateau voltage often overshot the resting poten-
tial. This suggests that the amplitude of these
A Normal B
Ringer
-~
Figure 8.6. Intradendritic recording following direct
application of glutamic acid in the immediate vicinity
of the recording site. In A, iontophoretic glutamate
pulses of different amplitudes (A) generated prolonged
responses consisting of slow depolarizations followed
by calcium-dependent plateau responses. At higher
levels of glutamate injection the trans dendritic depo-
171
plateaus represents an equilibrium state in which
the inward calcium current is balanced by an
outward potassium current flowing across the
dendritic membrane. The dendritic calcium cur-
rent has recently been described at the single
channel level using patch clamping of Purkinje
cell dendrites (U sowicz et aI., 1991).
A detailed description of the voltage-gated
calcium conductances was obtained following
iontophoretic application of glutamic acid onto
dependent potentials in the dendrites on which
voltage-gated events were observed (Fig. 8.6A).
Thus, glutamic acid iontophoresis produced a
depolarization whose amplitude was related to
the amplitude of the iontophoretic current
(Fig. 8.6A). The traces in Figure 8.6A show the
responses to six stimuli of increasing amplitude
under control conditions. A similar set of stimuli
were delivered after calcium removal and in
the presence of TTX (Fig. 8.6B). Note that
in Figure 8.6A the response consisted of three
distinct components. The lowest iontophoretic
injection elicited a smooth triangular depolariza-
tion that reached a p~ak at the end of the injection.
A slightly larger injection generated a longer de-
polarization and a plateau potential that out-
lasted the iontophoretic pulse. Note that the
plateau potential began after the termination of
the iontophoretic injection, had a distinct peak,
Ca' free
20 mV
100 nA
100 msec
larization generated fast small spikes, indicating somatic
firing ofthe Purkinje cell and, finally, longer and more
prolonged dendritic calcium spikes. In B, a similar set
of glutamic iontophoretic injections generate smooth
potentials in the presence ofTTX, and no extracellular
calcium (Llinas and Sugimori, unpublished obser-
vations).
172
and a very slow falling phase. This is particularly
clear when compared to the pure ligand-dependent
potential shown in Figure 8.6B. With further in-
creases in iontophoretic pulse amplitude, the firing
level for the soma was reached producing small
(3-4 mV), fast sodium-dependent spikes that were
electrotonically conducted to the dendritic record-
ing site. Note also the prolonged plateau potential
that followed this initial response. Finally, the
largest two injections, in addition to plateau poten-
tials and somatic sodium spikes, generated large
dendritic calcium spikes (20-30 m Vat the record-
ing site). Figure 8.6B illustrates the responses
elicited after addition of TTX and removal of
calcium from the bath. Note the smooth character
of these potentials, indicating that at even the
lowest level of glutamic acid injection a voltage-
gated calcium current adds to the ligand-dependent
polarization. These results also demonstrate the
clear difference between plateau potentials and
dendritic calcium spikes.
In an attempt to understand better the mech-
anism underlying the dendritic plateau potentials,
calcium entry was visualized directly and meas-
ured using the dye, fura-2. In earlier Ca-sensitive
dye studies the intracellular calcium concentra-
tion changes were visualized using the ratio method
for fura-2 (Tank et aI., 1988), with activation at
340 and 380 nm (Tsien and Poeni, 1986), or using
arsenazo II (Ross and Werman, 1987). Both sets
of measurements demonstrated a clear increase
in calcium concentration in the dendritic tree
during the generation of calcium spikes. During
the plateau phase of the potential the dendritic
branchlets were the first structures to demonstrate
calcium entry while full action potentials occurred
concomitantly with an increase in calcium entry
into the main dendritic branches (Tank et aI.,
1988). These results supported the view that cal-
cium influx occurs at the dendritic branchlets and
is recorded at the main dendrites and at the soma
as plateau potentials or spikes, depending on the
number of dendritic branches that are activated.
As shown in Figure 8.7 (see color Plate 3),
Purkinje cells injected with fura-2 could be imaged
with good spatial resolution with excitation at
380nm (Sugimori and Llinas, 1990b). Figure 8.7B
represents the image obtained after on-line sub-
traction of the fluorescence of the injected Purkinje
cell (Fig. 8.7A) from the averaged (n = 16 frames)
Rodolfo R. Llimis and Mutsuyuki Sugimori
background fluorescence (obtained during an
electrically silent period just before activation).
Entry of calcium during the plateau potential
that preceded dendritic calcium spike activation
was most prominent in the periphery of the den-
dritic tree (Fig. 8.7C), whereas increased [Ca 2 +]j
was not seen in the main dendrites until the initial
peak of a calcium spike (Fig. 8.70). Subsequent
action potentials occurred in different branches
of the Purkinje cell as shown in Figure 8.7E
through 8.7G. The image in Figure 8.7H was
taken at a time of intermediate activity between
dendritic spikes. In Figure 8.71, dendritic spiking
abruptly ceased and [Ca2+]j returned to control
levels. Note that the increase in [Ca 2 +]j occurred
initially at the fine dendritic branches and that
during repetitive activity some dendritic branches
showed larger signals than others (Sugimori and
Llinas, 1990b).
Dendritic Glutamate Iontophoresis:
Voltage-Dependent and
M etabotropic Responses
The distribution of [Ca2+]j following iontopho-
retic application of glutamate to the Purkinje cell
dendrites is shown in Figure 8.8 (Sugimori and
Llinas, 1990a, b, see color Plate 4). The arrowhead
in Figure 8.8A marks the position of the ionto-
phoretic electrode with respect to the fura-2-
loaded cell. As shown in Figure 8.8B, iontophoresis
of glutamate elicited a plateau potential (left-
pointing arrow) during which distinct calcium
entry was observed at the level of the dendritic
branchlets (right-pointing arrow). Whenever the
ligand-dependent depolarization did not generate
a plateau potential (e.g., waveform indicated by
the dots in Fig. 8.8B), no calcium entry was ob-
served. When the plateau potential elicited full
dendritic spiking (Fig. 8.8C), widespread calcium
entry was particularly prominent in the main
dendrites (yellow and green).
These results indicate that glutamate ionto-
phoresis activates the spiny branch lets that gene-
rate maximal amplitude responses via the opening
of voltage-gated calcium channels in these fine
processes (Sugimori and Llinas, 1990a, b). It is
these responses that were observed at the somatic
level as plateau potentials. When a sufficient
number of branchlets were depolarized to bring
8. Electrophysiology of the Purkinje Cell
the plateau potential to the threshold for spike
initiation, the secondary dendritic branches gen-
erate full spikes. These spikes propagated ortho-
dromically toward the soma and antidromically
into other distal dendritic branchlets producing
an image such as that shown in Figure 8.8C.
The results further suggest that, in vivo, once
enough spines are activated through the parallel
fiber activation, the branchlets respond in a close-
to-unitary manner and that at the junction between
the spiny branch lets and the main dendrite, acti-
vation of the voltage-dependent calcium and then
the potassium conductance and chloride curtails
the high amplitude of these responses. Further-
more, it must be remembered that dendritic
inhibition, which is extraordinarily effective in
blocking both plateau potentials and dendritic
action potentials, acts directly on the smooth
portion of the dendritic tree (Llinas et ai., 1968)
and serves as a remote inhibitory mechanism
capable of regulating the transmission of dendritic
activity down to the soma.
Besides the activation of voltage-gated calcium
channels, iontophoretic injection of glutamic acid
can activate intracellular calcium release in the
absence of extracellular calcium and in the pres-
ence of ethylene glycol tetraacetic acid (EGT A)
(1 mM). This metabotropic response, which seems
to induce calcium release from intracellular stores,
is demonstrated in Figure 8.9 (see color Plate 4.)
following fura-2 injection into a Purkinje cell.
Note that the cell, which was loaded with fura
in the somatic and dendritic compartments
(Fig. 8.9A), demonstrated a clear fura-2 response
at the dendritic arbor during spike activation
(Fig. 8.9B). Following removal of extracellular
calcium and addition of EGT A to the bath, acti-
vation of the cell did not trigger a fura-2 response
(Fig. 8.9C). By contrast, iontophoretic washing
of glutamic acid onto the body and dendrites
following a clear increase in [Ca 2 +1 was seen. In
this case, the intracellular response was not limited
to the dendrites but was also strongly visible at
the somatic level (Fig. 8.9D), which would be
expected where Ca is released from intracellular
stores.
These results have suggested to us that a
mechanism that, regulates intracellular calcium
concentration such as inositol triphosphate (IP3)
may be activated by the metabotropic receptors
173
in Purkinje cells. Indeed, since the phosphoinosi-
tide-derived second messenger IP3 has been shown
to be a potent mobilizer of internally stored calcium
in numerous tissues, including the CNS, (Berridge
and Irvine, 1989; Ferris et ai., 1989), we expected
that the increase in free intracellular calcium may
be mediated by IP3. Since the activation of the
phosphoinositide cycle is in response to stimula-
tion with neurotransmitters, hormones and growth
factors have been extensively characterized in a
number of tissues (Berridge and Irvine, 1984).
Following binding of the agonist to the cell surface
receptor protein, an as yet unidentified G protein
is activated, which in tum activates a phospho-
inositide-specific phospholipase C. The phospholi-
pase C hydrolyzes phosphatidylinositol-4,5-bis-
phosphate to yield two putative second messengers,
IP3 and diaccylglycerol (DG). Inositol triphosc
phate has been shown to mobilize calcium from
internal endoplasmic reticulum stores (Berridge
and Irvine, 1984), whereas DG is thought to acti-
vate a phospholipid- and calcium-sensitive protein
kinase (Nishizuka, 1984). There is also evidence
to suggest that a metabolic product of IP3,
inositol-l,3,4,5-tetrakisphosphate (IP4), may play
a role in maintaining elevated calcium concentra-
tions (Berridge and Irvine, 1984).
More specifically, phosphoinositide turnover
and the generation of second messengers in the
CNS results from the actions of a number of
neurotransmitters (Nahorski et ai., 1986), includ-
ing glutamic acid. Blackstone et ai. (1989) dem-
onstrated that glutamate and its analogues,
quisqualate and ibotenate, were able to induce
accumulation of inositol phosphates in rat cere-
bellar slices. The effect of phosphoinositides was
not secondary to release of other neurotransmit-
ters or to calcium influx. In animals with Purkinje
cell degeneration, glutamate was ineffective. The
calcium-transporting properties ofthe IP3 receptor
protein purified from cerebellum (Supattapone
et ai., 1988) have been demonstrated in reconsti-
tuted lipid vesicles (Ferris et ai., 1989) and the
protein has been localized to the Purkinje cell
endoplasmic reticulum (Ross et ai., 1989). Al-
though stimulation of phospho in osit ide turnover
by glutamate and the mobilization of radioactive
calcium by IP3 have been studied in these cells,
mobilization of calcium by glutamate has never
been characterized.
174
In short, then, two types of calcium-dependent
siow conductances can be observed at the den-
dritic level by glutamate iontophoresis: one is
voltage-dependent and the other is metabotropic.
Dendritic Action Potentials
The second eiectroresponsive property of Purkinje
cell dendrites to be studied was the ability ofthese
processes to generate full action potentials. Per-
haps the most intriguing characteristic of their
initial description was the fact that dendritic
action potentials comprise several distinct all-or-
none components (Llinas and Nicholson, 1971;
Llinas and Hess, 1976). Thus, rather than the
simple waveforms observed, for example, in a
motoneuron, where one soma dendritic com-
ponent may be observed (Terzuolo and Araki,
1961; Eccles et aI., 1966a), Purkinje cell spikes
may have as many as six all-or-none components,
as initially demonstrated in reptiles (Llinas and
Nicholson, 1971). This implies that the action
potentials are probably generated in the main
branches of the Purkinje cells. Specific dendritic
branches may, in fact, be activated independently
c o
FTX+TTX
r--->_____________
B FTX
______________
s---!~
100 ms
Figure 8.10. A: Intracellular recording from a Purkinje
cell shows fast Na + -dependent spikes and Ca 2 + -de-
pendent action potentials (arrows). B: Five minutes
after application of FTX, Ca 2 + spiking disappeared,
and a prolonged Na + -dependent plateau potential is
observed that far outlasts the duration of the stimu-
lation (lower trace). C: Addition of TTX to the bath
Rodolfo R. Llim'ts and Mutsuyuki Sugimori
of one another during parallel fiber activity. As
with the plateau potentials, dendritic action poten-
tials are totally abolished by pharmacological
manipulations that block calcium channels or by
the removal of extracellular calcium. In contrast,
in the presence of extracellular BaH powerful
dendritic responses may be generated that seem
to obliterate the differences between dendritic
spikes and plateau potentials. In short, the charac-
teristics of Purkinje cell dendritic excitability lead
us to conclude that different parts of the dendritic
tree are independently electro responsive and that
the two categories of response, the plateau poten-
tials and dendritic spikes, differ only in that they
are generated by different compartments of the
Purkinje cell dendrites that, in addition, have
different complements of potassium channel types.
The P Channel
Beyond concluding that a single class of calcium
channel underlies dendritic electro responsiveness,
we concluded further that this calcium channel,
present in Purkinje cells, is not among those de-
scribed by Nowycky et aI., in dorsal root ganglion
TTX
E
TTX +FTX
120 mV
120 mV
11 nA
2 nA
20 ms
blocks the fast action potentials and the plateau poten-
tials shown in B. D: Ca 2 + -dependent slow action poten-
tial is generated by direct stimulation of the Purkinje
cell in the presence of TTX. E: Addition to FTX to the
bath produces a blockage of the voltage-dependent
Ca2+ electroresponsiveness (Llinas and Sugimori, un-
published observations).
8. Electrophysiology of the Purkinje Cell
cells (1985). That is, it is not an L channel on the
grounds that it is not blocked by dihydropyri-
dines; not an N channel in that it is not blocked
by il-conotoxin and does not inactivate; and not
a T channel in that it is activated at - 45 m V and
does not inactivate. Recently, we have demon-
strated that calcium-dependent responses in
Purkinje cells are blocked by funnel-web spider
toxin (FTX). The effect of FTX on the electrore-
sponsive properties of Purkinje cells is illustrated
in Figures 8.10A and 8.10B.
In Figure to.8A, a Purkinje cell demonstrates
oscillatory behavior upon direct intracellular sti-
mulation. The arrows indicate the calcium spikes.
After bath application of FTX (Fig. 8.l0B), the
oscillation was blocked and a prolonged activation
of the persistent sodium conductance (Llinas and
Sugimori, 1980a; Sugimori et a!., 1986; Gahwiler
and Llano, 1989) produced a prolonged plateau
potential. This plateau was blocked by the addition
of TTX to the bath (Fig. 8.10C). The opposite
experiment, where the sodium conductance was
initially blocked by TTX and a voltage-gated
calcium spike was elicited by direct stimulation,
is illustrated in Fig. 8.l0D. Following the addition
of FTX to the bath, the prolonged calcium spike
was blocked (Fig. 8.10E). Funnel-web spider toxin,
which has been shown to be a polyamine (Cherksey
et aI., 1989), was effective in the micromolar range
and was capable of blocking both plateau poten-
tials and-dendritic action potentials, supporting
the view that one type of channel underlies these
two responses.
In addition, the characteristics of single chan-
nels isolated from guinea-pig cerebellum have
been determined after their incorporation into
lipid bilayers (Cherksey et a!., 1989; Llinas et aI.,
1989). The current-voltage relation is similar to
that of the macroscopic current observed in
Purkinje cells with patch clamp (Llinas et aI.,
1989) and has the same sensitivity to FTX as
Purkinje cells studied in brain slices (Llinas et aI.,
1989). The average open time, which is voltage-
dependent, varies from 1 to 3 ms. The single-
channel conductance, measured using an extra-
cellular divalent anion concentration of 80 JlM,
was on the order of 8 to lOpS for barium and 6
to 8 pS when calcium was the divalent anion.
This characterization of the P calcium channel
clearly distinguishes it from the three types of
175
calcium channels studied in dorsal-root ganglion
and other cells (see Miller, 1987).
More reccently, histochemical studies, based
on the development of a polyclonal antibody
against the P channel, yielded a rather compre-
hensive picture of the spatial distribution of the
P channels in the CNS (Hillman et a!., 1991).
Moreover, the distribution of antibody binding
sites agrees well with the electro physiological
findings relating to the distribution of P calcium
channels. Indeed, a dense reaction in Purkinje
cell dendrites indicates antigen localization dis-
tally on the dendritic arbor, especially at bifurca-
tion points. Ultrastructural studies of the immuno-
response further demonstrated membrane reaction
occurring as patches on the main dendrites, spiny
branches, and spines (Hillman et a!., 1991). The
latter, perhaps the most surprising finding, de-
monstrates that the head, and occasionally the
neck, of the spine have clear immunoreactive
sites at the plasmalemma, indicating that spines
themselves may be capable of electro responsive-
ness.
Functional Properties of the
Synaptic Input
The Ascending Axons of the Granular
Cells and Fractured Somatotopy
It has long been established that the mossy fiber
system can elicit, via the granule cell relay, well-
defined regions of Purkinje cell activity that are
restricted to the immediate vicinity ofthe granule
fiber terminals and that do not correlate with the
horizontal distribution of the parallel fibers over
the molecular layer (Eccles et al., 1966b; Oscarrson
and Sjolund, 1977a, b; Shambes et a!., 1978). This
finding, which was recently confirmed using im-
aging probes linked to c-fos and with 2-deoxy-
glucose (Sharp et a!., 1989), is at odds with the
view that parallel fiber stimulation can generate
activity in Purkinje cells located along the whole
length of the parallel fiber system. A possible
solution for this problem was proposed some
years ago with suggestion that the ascending
portion ofthe granular cell axon was responsible
for this behavior (Llinas, 1982).
Following this proposal, intracellular studies
in this cerebellar slices (100-150 Jl) (Fig. 8.11 A)
176
A C
B 2
1 20 mV
50 msec
Figure 8.11. Recording of intracellular potentials in
Purkinje cells by stimulation of the ascending axons
of granule cells. A: In vitro preparation consisting of a
very thin (approximately 100 11m) cerebellar slice that
is stimulated at the granular cell level while the Purkinje
cell, located immediately above the stimulation site, is
intracellularly recorded. In B, stimulation to the gran-
ular layer generates distinct steps of synaptic activa-
tion with monosynaptic latency. The synaptic poten-
tial may be large enough to reach threshold for spike
initiation and to generate full-size action potentials as
have demonstrated that the activation of granule
cells can produce, even in this very restricted
slice, clear synaptic potentials and full activation
of the Purkinje cells (Figs. 8.11 B, C). The sche-
matic diagram in Figure 11 A illustrates that with
very thin slices, much of the surviving granule cell
axon is represented by its ascending portion. The
graded nature ofthis synaptic response to granule
cell activation is shown in Figures 8.11 Band 8.11 C
(0 m V) as a set of synaptic potentials of increasing
amplitudes. The nature of this response is de-
monstrated in Figure 8.11 C, where a diminution
and reversal of this synaptic potential was ob-
tained with membrane depolarization, while the
Rodolfo R. Llimis and Mutsuyuki Sugimori
18
14 ---~ ==
12 'v-
10
8
r
Jl
nA
o
-2 __ --'1'-..___ I 60 mV
50 msec
shown in Band C. The gradation of these graded
synaptic events occurs in distinct steps as indicated by
the distinct components of the rising phase. In C, the
responses are shown at lower gain to demonstrate their
synaptic nature. When the cell is hyperpolarized by
injection of - 2 nA to the recording electrode, all action-
potential activity ceases. As the cell is depolarized, the
amplitude of the synaptic potential decreases and finally
reverses at an injection level of 10 nA, demonstrating
the chemical nature of this potential (Llimis and
Sugimori, unpublished observations).
potential increased with membrane hyperpolari-
zation. These results demonstrate the chemical
nature of this response.
The possible functional effect of the ascending
axon has been questioned on quantitative ana-
tomical grounds. Indeed, it was demonstrated
that whereas Purkinje cells in the rat may receive
as many as 150,000 parallel fibers, only a small
percentage (3%) are actually generated by the
ascending axons (Napper and Harvey, 1988). It
was further estimated that one ascending axon
may contact a particular Purkinje cell some 17
times and that the total number of synapses
between a Purkinje cell and the granule cells
8. Electrophysiology of the Purkinje Cell
lying immediately under its dendritic arbor (274
on the average) would provide no more than
4700 synapses on the Purkinje cell (Napper and
Harvey, 1988). However, this may be quite a
large number when interpreted from a functional
viewpoint. Indeed, since a climbing fiber requires
about 300 junctions to generate a "most power-
ful activation of the Purkinje cells" (LIiil<ls et aI.,
1969), the important variable may not be the
total number of synapses contacting a structure.
Indeed, 4700 contacts may easily represent the
synaptic connectivity of 10 or so climbing fibers.
Another issue that Napper and Harvey (1988) did
not seem to consider is that the timing of synaptic
activation is very important in determining syn-
aptic effectiveness. Indeed, activation of the mossy-
fiber input to a patch of granular cells produces
well-defined activity within that patch producing
synchronous and repetitive activation of a parti-
cular set of granular cell axons that converge and
synapse on a Purkinje cell (more than 4000) on
their way toward their final bifurcation in the
molecular layer.
From a functional point of view the implica-
tions of the numbers given above are quite signi-
ficant, making the ascending axons of the granule
cells an important component in the activation
of Purkinje cells via the mossy fiber-granular cell
input. It may be important to remember that in
addition to the powerful excitability observed
under anesthetized conditions (Eccles et aI., 1966b),
the parallel fibers may produce a modulating
effect where even small excitatory inputs may be
sufficient to modulate the firing rate of a Purkinje
cell that is close to its firing level.
GABAergic Responses
and Dendritic Inhibition
A more complete view of the many functional
states that each cell may have can be envisioned
if one considers that at least two types of inhibi-
tion play directly on the soma and dendrites of
this neuron. Because the descending axons of the
basket cells wrap around the somata and come
together over the axon to form the so-called
Pinseau Terminal (Ramon y Cajal, 1904), the
177
inhibition produced by the release of gamma-
aminobutyric acid (GABA) from these terminals
is capable of blocking spike initiation at the axon
hillock (Andersen et aI., 1964). This is especially
powerful since a Purkinje cell may receive as
many as 10 to 20 such terminals (Paley and Chan-
Paley, 1974). Less well known, on the other hand,
is the fact that the basket cells also have ascend-
ing axon collaterals that ramify on the Purkinje
cell arbor to produce powerful dendritic inhibi-
tion capable of modifying dendritic spiking
(Llinas et aI., 1968). In addition, the stellate cells,
which are located in the molecular layer, send
fine presynaptic terminals that cover most of the
Purkinje cell dendritic tree. These cells are an
order of magnitude more numerous than the
basket cells and the fields of their terminal axonic
arborizations are smaller than the dendritic span
of any given Purkinje cell (Paley and Chan-Paley,
1974). They clearly play an important role in
dendritic integration within a single Purkinje cell.
Iontophoretic injection of GABA at the den-
dritic level produces clear membrane hyperpo-
larizations that can be reversed by membrane
hyperpolarization. The sensitivity of these inhibi-
tory postsynaptic potentials (IPSPs) to bicculine
(Sugimori and LIinas, 1985) indicates an under-
lying GABA type-A receptor. Furthermore, in
addition to decreasing the excitability of the soma
and dendrite by the activation of a chloride con-
ductance, GABA has a direct effect on the calcium
conductances at the dendritic level. This is illus-
trated in Figure 8.12, where iontophoresis of
glutamate (Fig. 8.l2A) elicited sodium- and cal-
cium-dependent action potentials in the soma
(Fig. 8.12B). Under similar conditions, applica-
tion of GAB A to the dendritic tree was accom-
panied by a reduction of calcium electrorespon-
siveness (Fig. 8.l2C). Thus, as shown in Figure
8.12C, following the iontophoresis of GABA at
the dendritic level, the rapid firing of sodium
spikes was unchanged, whereas the powerful cal-
cium response seen before the GABA injection
was abolished. Equally interesting is the fact that
even when GABA receptors are blocked pharma-
cologically by picrotoxin, application of GABA
still reduces the calcium response (not shown)
178
Figure 8.12. A: Intracellular recording from soma of
a Purkinje cell and dentritic iontophoretic application
of glutamic acid and GABA. In B, the glutamate ion-
tophoretic injection produces full sodium-dependent
action potentials and calcium spikes as observed by
(Llimls and Sugimori, unpublished results). These
findings suggest that GABA may have a direct
effect on calcium channels that is independent of
its action on GABA A receptors. Inhibitory syn-
apses would then have three mechanisms of action:
through membrane hyperpolarization, by shunt-
ing active somatic and dendritic, and by reducing
the excitability of the dendritic tree by acting
directly on calcium channels.
Summary
In summary, the main characteristic of mam-
malian Purkinje cells is their electrophysiological
versatility, which is due largely to the quite specific
corifiguration of channels in its some-dendritic
membrane. Indeed, at the dendritic level, calcium
channels seem to be the main voltage-gated con-
ductance, and these are distributed differently
in the fine and large dendrites, whereas at the
Rodolfo R. L1inas and M utsuyuki Sugimori
2n'"
20mv
150n'"
200mlK
oscillatory events. In C, application of glutamic acid
at the dendritic level leads to a total reduction of
calcium spikes without affecting the somatic firing
of neurons (L1inas and Sugimori, unpublished obser-
vations).
soma, sodium channels predominate. In addition,
the firing of a Purkinje cell in response to parallel-
fiber or climbing-fiber activation is dictated by
the types and distributions of synaptic input that
the cell receives, as well as to their intrinsic elec-
trophysiology.
Parallel-Fiber Activation
Parallel fibers activate primarily the spiny branch-
lets and do so either directly through the verti-
calor ascending portion of the granular cell axon
and through the horizontal compartment of the
parallel fibers. A Purkinje cell receives as many
as 200,000 parallel fibers, making this quantita-
tively the richest synaptic input. The postsynaptic
depolarization is restricted to the fine terminals
of the dendritic branches and has been demon-
strated to activate the so-called simple spikes,
which fire at a frequency of 10 to 20 Hz (cf. Llimls
and Simpson, 1981). This response begins as a
8. Electrophysiology of the Purkinje Cell
depolarjzation of the spiny branchlets and is
boosted by local calcium plateau potentials, which
may begin as early as the postsynaptic spine
(Hillman et aI., 1991). The dendritic plateau poten-
tials in turn activate a low-threshold, sodium
plateau potential in the soma that serves to trigger
the full sodium spikes that travel down the axon
to release transmitter at the Purkinje cell presyn-
aptic axon terminals, in the deep cerebellar nuclei.
The presence of two plateau potentials between
the synaptic input and somatic firing allows for
an extraordinarily subtle modulation of firing
since the plateau potentials may be regulated by
anyone or a combination of three mechanisms:
a) voltage- and calcium-gated potassium conduct-
ances that have very different time courses, b) the
activation of GABAergic receptors that increase
the conductance to chloride, which may reduce
calcium flow at the dendrites, and c) shunting
somatic currents to block both the plateau poten-
tial and the full action potential. The latter is
known to be the case, as a large shunt is observed
at the somatic level during the activation of basket
cells and dendritic-level stellate cells. Thus, paral-
lel fiber- Purkinje cell synapses and the associated
interneurons represent the circuits that ultimately
modulate Purkinje cell simple spike firing.
Climbing-Fiber Activation
As first shown in mammals, activation of climbing
fibers produces a burst response known as a
"complex spike" (Eccles et aI., 1966a). This com-
plex spike produces different patterns of action
potentials in the Purkinje cell axon, depending
on the membrane potential (Eccles et aI., 1966a),
and may also produce powerful calcium-de-
pendent plateau potentials (Llimls and Sugimori,
1980b). The climbing fibers activate both dendritic
spikes and plateau potentials with large levels of
current flowing longitudinally into the cell soma.
In contrast, this is prevented during parallel fiber
activation by the large load imposed by the
Purkinje cell dendritic tree. However, since climb-
ing fibers cover primarily the smooth portion of
the dendritic tree, which they depolarize in a
more or less synchronous manner, the loss of
current in the dendrites is minimal and thus the
Purkinje cell soma and axon can fire action poten-
tials at a high frequency (Llimls and Sasaki, 1989).
Such firing may be as high as 400 Hz, producing
179
fast and powerful IPSPs at the postsynaptic target
cell terminals, as demonstrated in the isolated
cerebellum brain stem preparation (Llim'is and
M iihlethaler, 1988).
In short, then, the electro responsive properties
of the Purkinje cell to activation by each of its
afferent systems seem to be related closely to its
integrative properties. The mossy fiber-parallel
fiber system produces integration that may modu-
late cerebellar nuclear cells for a long time. This
is in contrast to the climbing-fiber system, which
elicits well-defined, short-lasting, powerful inhi-
bitory potentials in deep nuclear cells. The ulti-
mate relation of these two forms of Purkinje cell
activation to cerebellar function has been the
subject of a great deal of research. Our own view
is that climbing- and parallel-fiber systems time-
share the Purkinje cell but are not completely
independent of one another functionally.
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9
V oltage- and Transmitter-Gated Channels
in Purkinje Cells from
Organotypic Cerebellar Cultures
Isabel Llano, Beat H. Gahwiler, and Alain Marty
The Use of Advanced Biophysical
Techniques to Study the Membrane
Properties of Central
Nervous System Neurones
The Technical Challenge
Classical membrane biophysics has been devel-
oped in carefully selected preparations that bear
no direct relation to the mammalian central ner-
vous system (CNS). This is easily understand-
able. Tissue derived from mammalian CNS is
not only difficult to maintain and manipulate,
but it also contains nerve cells with intricate den-
dritic processes that render the use of voltage-
clamp techniques hazardous. Furthermore, their
cellular surface is not easily accessible to experi-
mental manipulations. All these sources of diffi-
culty have been recognized for a long time. As a
result, new technical developments such as the
patch-clamp technique (Hamill et ai., 1981) have
received relatively few applications in the CNS,
in contrast to the wealth of information gathered
with this experimental approach in peripheral
tissues. Much ofthe CNS work has concentrated
on preparations such as the neuroendocrine cells
of the pituitary gland, which can be isolated and
studied in vitro relatively easily. These cells have
a simple shape that is suited to voltage-clamp
studies and they can be identified before recording
by using the noninvasive hemolytic plaque assay
(e.g. DeRiemer and Sakmann, 1986).
The Search for a Suitable CNS Preparation
Up to this date, most patch-clamp studies of CNS
tissue have been carried out either with acutely
182
dissociated adult neurons or with cultures of dis-
sociated neurons of neonatal or fetal origin.
Whereas the former approach yields a prepara-
tion of nerve cells devoid of any synaptic contacts
with other neurons, nerve cells in dissociated cul-
tures usually suffer from insufficient cellular dif-
ferentiation and organotypic organization. As a
result, individual neurons cannot be identified
easily in the living state on the basis of their
characteristic cytoarchitecture or cellular loca-
tion.
One way to overcome some of these difficulties
is to turn to slice preparations, which contain the
normal anatomical neuronal relationships of the
tissue of origin. Furthermore, many brain areas
are organized in a laminated pattern, so that
appropriately oriented slices will preserve many
ofthe synaptic connections. Recently, a procedure
has been described allowing to "clean" the sur-
face of neuronal cells in acute brain slices and
thus making them suitable for patch-clamping
(Edwards, et ai., 1989). This new technical devel-
opment opens the way for detailed studies of
identified neuronal preparations without the use
of culturing procedures.
We report here an alternative approach that
involves cultivation of slices derived from young
animals. Despite thinning down to monolayer
thickness, organotypic slice cultures (Giihwiler,
1981 a, 1984) retain the basic structural and con-
nective organization of the tissue of origin. Easy
access to nerve cells not only facilitates the use of
standard electro physiological techniques, but also
makes these cultures amenable to patch-clamp
methods without the necessity of cleaning the
surface of the tissue. In the following sections we
9. Voltage- and Transmitter-Gated Channels
describe the morphological and physiological
properties of the preparation that has been used
in our studies, "organotypic" cultures of rat cere-
bellum, and compare the functional results ob-
tained with those that have been gathered using
similar electrophysiological approaches in other
preparations of mammalian cerebellum.
Cerebellar Organotypic Cultures
Preparation of Cultures
In the present studies, cerebellar cultures were
prepared from newborn (0 to I-day-old) rats.
Early postnatal age is a good compromise since
at least part of the cytoarchitecture is already
present and a high degree of neuronal survival
can be achieved. Cerebella are dissected ascepti-
cally and quickly cut with a McIlwain chopper
into 400-.um thick sagittal slices. The slices are
placed individually on glass coverslips and em-
bedded in a plasma clot formed by chicken plasma
and thrombin. To optimize aeration and feeding
of the cultures, the coverslips bearing the cultures
are inserted into plastic test tubes to which 0.5 ml
of medium are added, placed in a roller drum that
is kept in a dry incubator at 36°C, and slowly
rotated at 10 revolutions per hour. Cultures are
fed at weekly intervals with a medium consisting
of horse serum (25%), basal medium (Eagle, 50%)
and Hanks' balanced salt solution (25%), sup-
plemented with glucose to a final concentration
of 6.5 gil. To reduce the number of nonneuronal
cells, the fresh ex plants are subjected to moderate
doses of ionizing irradiation (x ray, 250 kV, 600-
1200 rads), and the cultures are treated during
one day with the antimitotic agent cytosine
arabinoside. The slices are used for electrophysio-
logical experiments within 2 to 5 weeks after
explanations.
Cellular and Tissue Differentiation
After about 10 to 15 days in vitro, the majority
of cells and fibers damaged during the slicing
procedure have degenerated; as a result, the cere-
bellar cultures have flattened from the original
400.um into a monolayer preparation. This
spreading greatly facilitates visualization of indi-
vidual living neurones with contrast-enhancing
optics.
183
The overall cytoarchitecture of cerebellar tissue
with cortical and deep cerebellar nuclei can still
be recognized after several weeks in vitro. Because
of continued neuronal migration, Purkinje cells
are no longer aligned in single rows as in situ, but
rather clustered in folialike structures. In contrast,
deep cerebellar neurones form distinct nuclei that
are located near the base ofthe cerebellar cultures.
Living Purkinje cells can be identified by their
large size, their characteristic dark cytoplasm
(viewed with phase contrast microscopy), and
their relative location within the slices. Recently,
a more stringent identification of Purkinje cells
has been carried out using antibodies to the 28-
KDa Ca-binding protein, calbindin. Figure 9.lA
illustrates the distribution of calbindin-positive
Purkinje cells in cerebellar cultures. Whereas
some calbindin-positive Purkinje cells were ap-
parently scattered within the slices, others formed
organized folialike structures. Purkinje cell axons,
again identified by calbindin immunohisto-
chemistry, are observed to myelinate during the
second week in vitro. They grow toward and ter-
minate within the deep cerebellar nuclei (Fig. 9.1A).
Interestingly, the shape of dendritic arborizations
greatly depends on the relative location of Purkinje
cells within the slices. Whereas Purkinje cells that
have migrated away from the laminae display
relatively short, randomly oriented multipolar
dendrites, Purkinje cells within laminae have
dendrites that are clearly oriented within the
molecular layer (Fig. 9.1 B). All primary and
secondary dendrites are densely covered by spiny
processes.
It is evident that the dendritic trees of Purkinje
cells never achieve the full arborization reminis-
cent of their adult in situ conterparts, but resem-
ble, as far as multipolar dendrites are concerned,
those observed in the experimentally, degranu-
lated cerebellum (Altman and Anderson et aI.,
1972; Crepel et aI., 1980; Herndon et aI., 1971;
Sotelo, 1975). Well oriented Purkinje cells located
within laminae have dendritic arbors resembling
those of Purkinje cells in approximately 2- to
3-week-old rats (Hendelman and Aggerwal, 1980).
The reasons for the failure of Purkinje cells to
grow a fully arborized dendritic tree are not com-
pletely understood, but it is likely that the out-
of-phase course of parallel fibers in these two-
dimensional cultures plays a decisive role (Blank
and Seil, 1982).
184 Isabel Llano, Beat H. Giihwiler, and Alain Marty

Figure 9.1. A: Center of a cerebellar culture after the culture presented in A, viewed with interference
30 days in vitro. The culture has been stained with contrast optics after staining with antibodies against
aI)tibodies raised against the 28 KDa Ca-binding calbindin. Bar = 500/lm for A and 50/lm for B.
protein calbindin. B: Laminae of Purkinje cells from
9. Voltage- and Transmitter-Gated Channels
In newborn rats, only Purkinje cells with small
filapodia and a short axon can be visualized
(Gahwiler, 1981 b). The intricate dendritic and
axonal arborizations of Purkinje cells in cerebel-
lar slice cultures derived from newborn rats clearly
demonstrate that maturation continues in the
in vitro environment after the tissue has been
isolated. The electrophysiological data to be dis-
cussed in the next section will show that the same
holds true for the functional properties of cultured
Purkinje cells. The observation of morphological
and physiological maturation in vitro is particu-
larly important in view of recent reports sug-
gesting that differentiation may stop at the time
of explantation if the tissue is to be derived from
older animals. In those cultures (Jaeger et aI.,
1988; Kapoor et aI., 1988), survival of cultured
cerebellar neurones from 8 to II-day-old rats
apparently did not differ appreciably from those
taken at earlier stages. Differentiation and matu-
ration ofneurones, however, varied considerably.
These studies (Jaeger et aI., 1988) in fact showed
that only maturation attained in vivo could be
maintained in the in vitro environment, and that
little morphological or physiological maturation
occurred after the tissue had been isolated. It will
have to be determined whether these results are
solely due to the age of the donor animals or to
the particular culture conditions used.
Cerebellar-Olivary Cocultures
In cultured slices, afferent fibers are severed during
the slicing procedure, rapidly degenerate, and
can therefore no longer be stimulated. Cocultures
derived from anatomically remote brain areas
provide an interesting alternative approach for
studying functional properties of neuronal pro-
jections in an in vitro preparation. To achieve
this goal, two slices are embedded side by side on
a glass coverslip at a distance of about 1 mm
and then cultured for several weeks. During this
period, fibers originating in cells of the origin of
the projection grow toward and into the neigh-
boring slice and form functional synaptic connec-
tions with the target neurones.
We have made use of this approach to establish
an in vitro analog of the olivocerebellar projec-
tion. Within the first 2 weeks in vitro, olivary
fibers bridge the gap separating the two explants
and grow into the adjacent cerebellar slice to
innervate cerebellar Purkinje cells. Electrophysio-
185
logical studies using extracellular (Gahwiler,
1978) and intracellular (Mariani et aI., 1991)
recording techniques have clearly shown that
olivary stimulation results in Purkinje cells in the
generation of complex responses that closely
resemble the characteristic climbing fiber re-
sponses as observed in situ and in acute cere-
bellar slices.
The co culture approach offers not only unique
opportunities for studying the functional pro-
perties of cerebellar afferents, but may also allow
characterization of the transmitter used by climb-
ing fibers. Moreover, such a preparation may be
instrumental in elucidating mechanisms involved
in target search and recognition and may provide
important information about the role played by
trophic substances in the establishment of syn-
aptic connections.
Ionic Conductances ofPurkinje Cells
Voltage-Gated Conductances
of Purkinje Cell Somata
Intracellular recordings from Purkinje cells in
cerebellar mammalian slices (Llimls and Sugimori,
1980a) have yielded a detailed description of the
various firing patterns that characterize the elec-
troresponsiveness of these neurones and have
suggested the existence of several membrane con-
ductances as underlying their electrical behavior.
Specifically, these studies have demonstrated that
the Na conductance plays a role not only in the
generation of the fast potentials, but also in the
generation of prolonged or "plateau" potentials,
which are prominent after the blockade of voltage-
dependent Ca conductances. The effects of intra-
cellular injections of the K channel blocker tetra-
thylammonium (TEA) on the level of the Na-
dependent plateau depolarization as well as on
the duration of action potentials have further
indicated that K conductances contribute to
shaping the electrical signals. Finally, the com-
parison of somatic recordings with direct den-
dritic intracellular recordings (Llimls and Sugimori,
1980b) has led to the suggestion that most of the
voltage-dependent Ca conductance of Purkinje
cells is localized at the dendritic arborization of
these cells.
As in explant cultures (Gruol, 1983; Gruol and
Franklin, 1987) and in dissociated cerebellar cul-
186
B c
12.5 nA
20 ms
Figure 9.2. A: Recording of the membrane potential
obtained under whole-cell current-clamp conditions
from a Purkinje cell perfused with KCl saline (con-
taining, in mM: 130 KCl, lOEGTA-K, 10 Hepes-K;
pH: 7.2). The cell resting membrane potential was - 56
mY: spontaneous synaptic potentials as well as over-
shooting actions potentials are observed. The horizontal
line marks the OmV membrane potential level. Unless
otherwise noted, data shown in this and subsequent
figures have been obtained from slices bathed in an
external saline containing (in mM): 140 NaCl, 2.5 KCl,
1 CaCI 2 , 1 MgCI 2 , 10 Na-Hepes, and 5.5 glucose (pH:
tures (Hirano and Ohmori, 1986) prepared from
fetal rat cerebellum, Purkinje cells in organotypic
cultures exhibit in current-clamp whole-cell re-
cording conditions spontaneous synaptic poten-
tials and overshooting action potentials (Fig. 9.2A),
indicating that they have developed in vitro a
set of membrane conductances. When examined
under voltage-clamp, depolarizing steps to K +
perfused neurones elicit large inward currents
with fast activation and inactivation time course,
which are followed by prominent outward cur-
rents (Fig. 9.2B). When the Na and K conduc-
tances are blocked by the addition of tetrodotoxin
(TTX) and TEA to the external solution, and by
Isabel Llano, Beat H. Giihwiler, and Alain Marty
10 mV I
100 ms
10 ms
7.2). B: Whole-cell membrane currents recorded under
voltage-clamp conditions in response to a depolarizing
step to - 20 m V. Holding potential: - 70 m V. The cell
was perfused with KCl saline. The record has not been
corrected for either leak current or capacitive currents.
C: Whole-cell membrane currents elicited by a depolari-
zation to - 20 m V from a holding potential of - 90 m V,
in a cell perfused with CsCI (in mM: 120 CsCl, 10 Cs-
EGTA, 10 Cs-Hepes; pH: 7.2). The external solution
contained 500 nM tetrodotoxin (TTX) and 2 mM tetra-
ethylammonium (TEA). The record has been corrected
for leak current and capacitive transients.
replacement of K + with Cs + in the recording
pipette, several nA of inward currents can be
obtained upon step depolarizations; these cur-
rents present a transient as well as a sustained
component (Fig. 9.2C). These three types of cur-
rents can be ascribed to the activation of Na, K,
and Ca conductances, respectively.
However, the use of the whole-cell recording
technique for the characterization of voltage-
dependent conductances is hindered in Purkinje
neurones by the complicated morphology of these
cells, which renders it impossible to achieve a
spatially homogeneous control of membrane
potential. In fact, lack of control of the membrane
9. Voltage- and Transmitter-Gated Channels 187

potential was evident in whole-cell recordings
from various anomalies in the current-voltage
relations and in the time course of the voltage-
activated currents. Thus, the identification of the
specific ionic channels that underlie the voltage-
dependent conductance changes and the descrip-
tion of their biophysical properties must be carried
out using other experimental techniques. The cell-
attached and cell-free membrane patches that can
be obtained using patch-damp techniques offer
alternative approaches. Owing to its noninvasive
nature, the cell-attached recording configuration
can be especially useful for an initial screening
of the various types of membrane conductances
present, whereas the cell-free configurations, which
allow a strict control of membrane potential and

Test pulse
(mV)
30

50

70

Figure 9.3. Cell-attached record-

ings from the soma of Purkinje cells.
Each set of traces corresponds to
single sweeps obtained on voltage
steps, the amplitude of which is
indicated at the left. The pipette
holding potential was set at 30 m V,
which brings the resting patch poten-
tial to approximately -90mV. The
recording pipette was filled with the
standard ex ternal saline. In this and
other figures concerning voltage-
activated currents, individual re-
cords have been corrected for leak
and capacitive transients. Filter
cut-off frequency (fc): 3.5 kHz. In
this and subsequent figures upward
arrows mark the onset of the voltage
pulse, downward arrows the return
to holding potential.
188
of the solutions to which the patch of membrane
is 'exposed, are best suited to carry out a formal
description of these conductances. In the following
sections we summarize the properties of the
somatic voltage-gated conductances of Purkinje
cells that we have observed using the cell-attached
and the outside-out configurations of the patch-
clamp technique.
Cell-Attached Recordings
In cell-attached recordings of Purkinje cells ob-
tained with pipettes filled with a standard external
saline, depolarizing voltage steps reveal inward
current fluctuations that are rare for depolariza-
tions of 30 mV and become prominent at higher
membrane potentials (Fig. ,}.3). These current fluc-
tuations are characterized by a fast activation
B ~5PA
5 ms
Isabel Llano, Beat H. Giihwiler, and Alain Marty
time course and are followed, during depolarizing
steps of 70 m V or higher, by outward current
fluctuations that last throughout the duration
of the pulse. The addition of 5 mM TEA to the
recording pipette eliminates the outward compo-
nent of the current (indicating that it is due to the
activation of K conductances), and allows us to
study the inward current fluctuations. As illustra-
ted in Figure 9.4A for a depolarizing step of 50 m V,
the voltage-dependent inward currents activate
rapidly upon depolarization (the time to peak of
the averaged current, last trace, being 0.65 ms)
and partially inactivate during the ensuing 10 ms.
Inward currents are observed for depolarizing
pulses higher than 30mV, the maximum current
being obtained for pulses of70mV. Under these
recording conditions, all cell-attached patches
tested displayed inward currents with similar
Figure 9.4. A: Cell-attached recordings
from Purkinje cell somata, obtained with
a pipette filled with standard external
saline to which 5 mM TEA had been
added. The three upper traces correspond
to single sweeps ofNa current fluctuations
elicited by 20-ms depolarizations of 50 mV;
the last trace shows the average current
calculated from 16 pulses. The patch
membrane potential was hyperpolarized
by 30 mV relative to its resting potential.
.fc = 3.5 kHz. B: Cell-attached recordings
from Purkinje cell soma, obtained with
a pipette containing 55 mM BaCl 2, 85 mM
TEA-Cl, 10mM TEA-Hepes (pH: 7.2).
The three upper traces correspond to
single sweeps elicited by 20-ms depolar-
izations of 50 m V; the last trace shows the
average current calculated from 20 pulses.
The patch membrane potential was hyper-
polarized by 30 m V relative to its resting
potential. fc = 3.5 kHz.
9. Voltage- and Transmitter-Gated Channels 189

kinetics and voltage dependence. As will be dis-
cussed below, these properties correspond well
to those that we have described for the voltage-
dependent Na channels in this preparation using
outside-out patches from Purkinje cell somata
(Gahwiler and Llano, 1989).
Hirano and Hagiwara (1989) have reported
that in Purkinje cells from dissociated cultures of
fetal rat cerebellum, two types of Ca channels
(steady and transient) are present in Purkinje cell
somata. In contrast, we find that the somata of
Purkinje cells in organotypic cultures do not
present any channel activity that corresponds to
that expected of voltage-gated Ca channels. Thus,
in--conditions that optimize the recording of Ca
channel activity (Ba- and TEA-containing solu-
tions in the recording pipette, Fig. 9.4B), there
was no measurable channel activity for depolar-
izing pulses ranging from 30 to 120 mV in any of
the patches studied. These results indicate that,
in organotypic cultures, most of the voltage-
dependent Ca conductance, which is evident from
the capacity of these cells to produce Ca action
potentials, and voltage-activated Ca currents in
whole-cell recording conditions, must be located
at the dendritic arborization, as is the case for
Purkinje cells in acute slices of adult mammalian
cerebellum (Llimls and Sugimori, 1980b). Our
results contrast clearly with those of Hirano and
Hagiwara, who find that, although a number of
somatic patches had little Ca channel activity, a
third of the patches displayed Ca currents with a
peak current of 50 pA or more. Also in contrast
with our results, these authors report that many
patches did not show any Na channel activity. In
our studies, Na channel activity has been systema-
tically observed on both the cell-attached and the
outside-out configuration (see below), provided
that the membrane holding potential is held suf-
ficiently hyperpolarized to prevent inactivation.
These results presumably have to be ascribed to
the fact that culture conditions can determine the
functional expression and/or distribution of specific
channel types within a neuronal cell. In fact, stu-
dies carried out on dissociated cerebellar cultures
from new-born rat cerebellum have shown that
the electro responsiveness of Purkinje cells in cul-
ture depends drastically on the type of culture
medium used (Bossu et aI., 1989).

Figure 9.5. A: Na currents recorded

from an outside-out patch in response
to 300-ms step depolarizations
to -40mV. Holding potential:
-90mV. The two upper traces cor- ____--J110 pA
respond to single sweeps, the lowest 50 ms
trace to the average current calcu-
lated from 21 depolarizations, applied I r '

at intervals of 4 s. The peak of the

mean current is 24.2 pA and the
sustained mean current is 0.4 pA. fc =
2.5 kHz. The external bath was sup- B
plemented with 1 mM TEA, and the
pipette solution contained, in mM:
120CsF, lOCsCI, 1 NaCI, 10 Cs-
EGTA, and lO Cs-Hepes. B: Bursts
of single-channel activity selected
from the final 200 ms of six 300-ms
step depolarizations to -40mV,
from the same patch as illustrated in
A. Holding potential: - 90 mV, fc =
2.5 kHz. (Adapted with permission
from Giihwiler and Llano 1989.) 5 ms
190
Outside-Out Patches
The N a Channels
The studies of Llimls and Sugimori (1980a) have
shown that Purkinje cells exposed to Co 2 + gen-
erate both fast Na-dependent spikes and a slow
depolarization, which tends to maintain the
membrane potential near - 20 mV. These plateau
potentials are sensitive to TTX and are absent
when extracellular Na + ions are removed from
the external solution. These observations have
been interpreted by the authors as indicating the
existence of two distinct Na conductances: a fast
Na conductance and a slow, non-inactivating
Na conductance. Our studies have led us to pro-
pose that a single type ofNa channel can underlie
these two types of electrical phenomena.
The first indication to suggest the latter inter-
pretation comes from the observation that the
inward current elicited by long depolarizing steps
in outside-out patches obtained with CsF-filled
pipettes displays two distinct components. As
illustrated in Figure 9.5A for a depolarization to
- 40 m V, the initial response to the change in
membrane potential is characterized by a surge of
inward current fluctuations, which reach their
peak amplitude within 0.6 ms and subsequently
decline. After this initial surge of current, bursts
of single-channel openings interspersed with
periods of silence are observed throughout the
duration of the depolarizing step. This type of
channel activity gives. rise to a macroscopic cur-
rent that presents a maintained component (see
Fig. 9.5A, lowest trace). Since both early and late
components of the inward current are blocked by
the specific channel blocker TTX, they can be
identified as being the result of the activity of Na
channels.
The question as to whether both components
of the Na current are due to one single type of
Na channel or whether two distinct channel types
need to be invoked was answered by comparing
the elementary properties of the channels that
underlie both phases. As is seen in Figure 9.5B,
wh'ere one of the bursts of channel openings is
shown at an expanded time scale, single-channel
events can be clearly detected during the bursts
of activity. The analysis of their amplitude and
open time distributions yields a single-channel
Isabel Llano, Beat H. Giihwiler, and Alain Marty
current value of 2 pA and a mean open time of
0.37 ms for depolarizations to -40mV. The sus-
tained bursts of channel openings can be observed
only in membrane patches that have peak inward
currents of relatively large amplitudes (greater
than 10 pA at - 40 mV), and in which the detec-
tion of single-channel events during the early
phase of the current is accordingly not possible.
We have thus used fluctuation analysis to estimate
the single-channel current and the number of
active channels in those patches that had "macro-
scopic" Na currents. The result of such analysis
has provided estimates of the elementary current
that are very close to those obtained for the
channels active in the late phase ofthe Na current.
In membrane patches containing a low number
of channels, individuals channel openings can be
clearly discerned during the early component of
the current. These channels open with very brief
latency upon depolarizing steps (Fig. 9.6A) and
have an elementary current amplitude at - 40 mV
of 2 pA (Fig. 9.6B), which is in accord with both
the estimates from fluctuation analysis in patches
containing many channels and with the analysis
of single-channel parameters of the channels res-
ponsible for the late bursts of channel openings.
Also in accord with the latter are the measures of
mean open time for the early channels (0.3-0.5 ms,
depending on the patch studied).
The comparison of single-channel properties
during both phases of the Na current indicates
that a single type ofNa channel is responsible for
the totality of the Na current observed upon
sustained depolarization. Can this channel ac-
count for the plateau depolarizations reported by
Llinas and Sugimori? The main requirement for
this is that the channels present a voltage depend-
ence such that they would give rise to a sustained
activity for a membrane potential near - 20 m V.
In fact, the sustained component is clearly voltage
dependent; it is observed only for membrane
potentials between - 60 and - 20 mV, its peak
amplitude being reached at -40mV. The exis-
tence of this component is reflected by a remark-
able feature of the steady-state properties of the
macroscopic Na current: there is a significant
overlap between the activation and inactivation
curves between - 60 and - 40 m V, which implies
that the Na channels do not inactivate completely
in this voltage range. This type of behavior has
9. Voltage- and Transmitter-Gated Channels
A B
t
-
I/J
. 5 80
0
a..
as
1;j
c 40
~2PA
5 ms
Figure 9.6. A: Single Na channel events activated by
20-ms steps to -40mV. Holding potential: -90mV.
The lowest trace corresponds to the averaged current
calculated from all the sweeps that had active channels
within a set of 25 depolarizations. fc = 2.5 kHz. External
and internal solutions were the same as in Fig. 9.5.
B: Upper panel: Histogram of elementary current ampli-
tudes for the patch shown in A. The solid line corre-
sponds to the best fit of the data values to a single
also been reported for the rat brain sodium chan-
nel type II cDNA clone expressed in oocytes
(Stiihmer et aI., 1987). As will be described below,
the K conductance of Purkinje cell somata has
an activation threshold of - 20 mV. The combined
effect of the maintained Na current and of the
absence of K current in the voltage range com-
prised between - 60 and - 30 m V will be to clamp
the membrane potential near -30 or -20mV,
as observed by Llinas and Sugimori (1980a).
The K Channels
Under physiological conditions, the interplay
between the Na channels and the K channels
should determine the firing properties of the
191
120
o 1 2 3 4
Amplitude (pA)
0.5 <'
-
a.
CD
"0
1.5 :E
a.
• E
• • • c:(
2.5
gaussian distribution. The value of single-channel cur-
rent obtained from the fit is - 2.06 pA (standard devi-
ation: 0.27). Events near 4 pA correspond to double
openings. Lower panel: Single-channel current-voltage
relation for the patch shown in A. Elementary current
values were obtained from fits of the elementary current
histograms to a gaussian distribution, as shown in the
upper panel. (Reprinted with permission from Giihwiler
and Llano, 1989.)
Purkinje cell soma. In somatic patches from
Purkinje cells in organotypic cultures, the K
conductance is quite prominent, giving rise to
outward currents as large as 100 pA (for depolar-
izations to 20 m V) in some of the patches tested.
Figure 9.7 presents an example ofthe macroscopic
K currents recorded from an outside-out patch
with a pipette filled with KCl. The K conductance
activates in a voltage-dependent manner the time
to peak of the current decreasing from 4 to 1.4 ms
between - 20 m V (the activation threshold for
the outward current) and 50 m V. The current
dacays during the pulse, reaching values of 0.2 to
0.1 of its peak amplitude, with time constants of
the order of6 ms, the value of which is not voltage
dependent. The macroscopic K currents are quite
192
J -40 mY
~25
10 ms
variable from patch to patch, especially with res-
pect to their time course, the extent of the current
decay during depolarizing pulses, and their sensi-
tivity to the value of the holding potential.
The variability of the macroscopic features of
the K current arises most likely from the existence
of various types of K channels, with a contribu-
tion to the total current that varies from patch
to patch. Whole-cell recordings (Bossu et aI.,
1988) and cell-attached recordings (Hirano and
Hagiwara, 1989) of the macroscopic K currents
of cultured Purkinje cells have already shown
that the K conductance ofthese neurones has two
components, a rapidly inactivating, "transient,"
component and a "sustained" component. The
features of the "transient" component, especially
its sensitivity to the holding potential (Bossu et aI.,
1988; Hirano and Hagiwara, 1989) and to 4-
aminopyridine (Bossu et aI., 1988), have led both
groups to consider this "transient" component of
the K conductance as an A current. At the single-
channel level, Bossu et al. have identified a non-
inactivating, TEA-sensitive K channel with a 22
pS elementary conductance as the main channel
type contributing to the maintained portion of
Isabel Llano, Beat H. Giihwiler, and Alain Marty
+50 mY
+30 mY
Figure 9.7. K currents obtained from
an outside-out patch maintained at
a holding potential of - 90 mV. The
o mY outward current amplitude increases
as the membrane is stepped to test
potentials of - 40, - 20, 0, 30, and
-20 mY SOmV, as indicated to the right of
each trace. Each trace is the average
of 10 depolarizing steps of 40 ms
duration. fc = I.S kHz. The solid lines
on each record correspond to single
exponential fits of the decay phase of
pA the current. The external bath con-
tained 200 nM TTX and the pipette
KCI.
the K conductance. Single-channel recordings
corresponding to the "transient" K conductance
increase have not been reported.
We have identified, using single-channel re-
cordings, two types of K channels that differ in
their elementary conductance and their kinetic
properties (Giihwiler and Llano, 1989). Activity
corresponding to these channels is illustrated in
Figure 9.8A, which corresponds to a steady-state
depolarization to 30 m V. Two discrete current
levels can be discerned, with elementary currents
of 2.6 pA and 10.4 pA, respectively (Fig. 9.8B).
Both ofthese K channels are blocked by low con-
centrations of TEA (2 mM). The channel with
lower elementary current presents a linear single-
channel current-voltage relation (elementary
chord conductance of 28 pS), activates with brief
voltage-dependent delays upon depolarizing steps,
and is subjected to a slow inactivation process.
Its properties are similar to those of the delayed
rectifier K channel, which has been described for
skeletal muscle (Standen et aI., 1985) and for the
giant squid axon (Llano et aI., 1988b). This channel,
which is probably identical to the 22 pS channel
of Bossu and collaborators, contributes a large
9. Voltage- and Transmitter-Gated Channels
Figure 9.8. A: Selected portions of K single-
channel events recorded from an outside-out
patch during a steady-state depolarization to
30 m V. fc = 1.5 kHz. Same external and internal
solutions as in Fig. 9.7. B: Histogram of event
amplitudes for the single-channel events recorded
during a maintained depolarization to 30 mV.
Only single step transitions were included in the
histogram. The data were fitted by the sum of
two gaussian distributions, with single-channel
current amplitudes of 2.66 pA (standard devi-
ation: 0.20) and 10.38 pA (standard deviation:
104), respectively. (Reprinted with permission from
Giihwiler and Llano, 1989.)
fraction of the macroscopic K current recorded
upon step depolarizations in Purkinje cell somata.
The large conductance (90 pS) K channel is
most likely the same as the 70 to 100 pS channel
reported for Purkinje cells in explant cultures
(Yool et aI., 1988). This channel probably cor-
responds to a Ca-dependent K channel, called
BK channel, as described in various neuronal and
nonneuronal preparations (reviewed by Latorre
et aI., 1989). The existence of Ca-dependent K
channels in Purkinje cells has already been sug-
gested by intracellular recordings, which show a
prominent after hyperpolarization following Ca
spikes (Uimis and Sugimori, 1980a).
193
B ~10PA
20 ms
60
U)
'E
Q)
~
30
o +,L..D...r.L_:a...c~AL:~';1.
.. ,
1
o 8
Amplitude (pA)
16
Transmitter-Activated Conductances
A formal identification of the neurotransmitter
mediating the synapse between climbing fibers and
Purkinje cells is still lacking; however, the situation
is clearer for the input arriving to Purkinje cells
from both the parallel fibers of the granule cells
and the axons of basket and stellate cells. Several
lines of evidence suggest that glutamate mediates
the excitatory action of granule cells on Purkinje
cells, whereas gamma-amino butyric acid (GABA)
mediates the inhibitory action of basket and stel-
late cells on Purkinje cells. Our studies have led
us to describe some properties ofthe specific ionic
194
channels that are activated by glutamatergic
agonists and by GABA in Purkinje cells, and have
provided further support for the spatial segrega-
tion of functional receptors on these cells (Llano
et aI., 1988a).
Differential Sensitivity of Purkinje Cells
to Neuroactive Amino Acids
Figure 9.9A presents whole-cell recordings of the
responses of a Purkinje cell to local applications
of GAB A and of the three main types of glutama-
tergic agonists, N-methyl-D-aspartate (NMDA),
quisqualate, and kainate. The cell has been bathed
in Mg2 + -free external saline to prevent the voltage-
dependent block of NMDA channels by Mg2 +
ions (Nowak etaI., 1984); TTX and Cd2+ were
added in order to inhibit synaptic transmission
and decrease the possibility of synaptic inter-
actions. Under these conditions, the Purkinje
cell responds with large inward currents to the
two non-NMDA agonists, but fails to respond to
NMDA. This differential sensitivity to glutama-
A
100llM NMDA 10 IJM Kai
10 pM Gly
B
2IJM GABA
25fJM Bic
r, i Iflr
Figure 9.9. A: Whole-cell current responses to gluta-
matergic agonists and GABA. Holding potential, - 60
m V. Agonists were applied through a fast perfusion
system during the time indicated by the bars above
each record. The slice was bathed in Mg2 + -free saline,
which was supplemented with 200 nM TTX and 250 J1M
Cd 2 +. Internal solution, KCI. (Adapted from Liane
Isabel Llano, Beat H. Giihwiler, and Alain Marty
tergic agonists was observed systematically in all
Purkinje cells that we have studied, even though
the experimental conditions (absence of extracel-
lular Mg2 + and presence of glycine in concentra-
tions which have been shown to potentiate the
NMDA response; Johnson and Ascher, 1987) were
designed to optimize the activation of NMDA
receptors. These results are in accord with pre-
vious studies that have shown that adult Purkinje
cells in acute slices have a low sensitivity ofNMDA
(Crepel et aI., 1982). It must be pointed out that
under similar experimental conditions, pyramidal
cells from organotypic hippocampal cultures res-
pond with large conductance increases to NMDA
(Llano et aI., 1988a). We thus consider that the
insensitivity of Purkinje cells to NMDA does not
reflect nonspecific effects of culture conditions on
the development and/or maintenance ofNMDA
receptors, but rather an intrinsic property of
Purkinje cells, which is expressed in organotypic
cultures as it is in vivo.
The response to GABA in this particular cell
is relatively small (Fig. 9.9A), most likely due
2pM Quis 5 IJM GABA
1nA
2).lM GABA 2 IJM GABA
i r
"J 11'
et aI., 1988a.) B: Whole-cell current responses of a
Purkinje cell to short applications of GABA in control
solution (left trace), in the presence ofbicuculline metho-
chloride (center trace), and after washing ofbicuculline
(right trace). Holding potential, - 60 m V. The external
bath contained 200 nM TTX. Internal solution was
KCI.
9. Voltage- and Transmitter-Gated Channels
to the f~ct that external Cd2+ partially inhibits
GABA receptors. In the absence of Cd 2+ , 2 pM
GABA elicits large inward currents, which can be
reversibly decreased by the GABA-A receptor
antagonist bicuculline (Fig. 9.9B).
In the course of characterizing the whole-cell
responses to neuroactive substances, it was system-
atically observed that although GABA responses
reversed at the membrane potential expected from
the equilibrium potential of Cl- ions (the charge
carrier through the GABA channel under our
recording conditions), the responses to both q uis-
qualate and kainate reversed at membrane poten-
tials 20 to 40 m V more positive than that expected
for cation-selective channels (0 m V). These obser-
vations suggested that the whole-cell currents for
the glutamatergic agonists were arising from a
10 11M Quis
50
.,'"
E
.,>
......
o
ci
Z
25
2 PAL
Figure 9.10: Single-channel activity elicited by quis-
qualate in a somatic outside-out patch held at a mem-
brane potential of - 90 m V. The traces have been selec-
ted to illustrate the various current levels that are found
upon agonist application. fc = 1.5 kHz. The bath con-
tained Mg2 + -free saline, with 200 nM TTX; pipette
195
region of the cell that was not under accurate
potential control. In accord with this possibility,
comparisons of the responses to GABA and to
glutamatergic agonists in the whole-cell and the
outside-out recording configuration have indi-
cated that Purkinje cell somata have a large den-
sity GABA-ergic channels but a very low density
of glutamatergic channels, the large whole-cell
currents observed in response to excitatory amino
acids being most likely of dendritic origin (Llano
et aI., 1988a). These results are of interest since
they demonstrate that Purkinje cells in organo-
typic cultures attain the spatial segregation of
functional amino acid receptors, which is expected
of their adult in vivo counterparts from binding
assays and electrophysiological studies (e.g., Chan-
Palay, 1978; Crepel et aI., 1982).
0~--~~--------,- ______3 -____- ,
o 1.25 2.5
10 ms
Amplitude (pA)
solution, CsC!. Right panel: Corresponds to the dis-
tribution of elementary currents for the ensemble of all
openings elicited during a long agonist application in
the same patch. The data were fitted by the sum of
three gaussian distributions (continuous curve) with
single-channel current values of 0.86, 1.37, and 1.65 pA.
196 Isabel Llano, Beat H. Giihwiler, and Alain Marty

Properties of Amino Acid-Activated The latter hypothesis has been put forward by
Channels Jahr and Stevens (1987) and by Cull-Candy and
Usowicz (1987), based on their observations that
Glutamatergic Channels in outside-out patches obtained from dissociated
On the basis of their differential sensitivity to hippocampal neurones and from cultured cere-
pharmacological agents, three types of glutamate bellar neurones, NMDA and non-NMDA agonists
receptors have been described for the vertebrate produce channel openings to the same subcon-
nervous system (see Mayer and Westbrook, 1987 ductance states and that transitions between the
for review). These receptors are preferentially acti- various states occur. In contrast to this view,
vated by the glutamatergic agonists NMDA, quis- Ascher and Nowak (1988) advocate that the ability
qualate, and kainate, respectively. Whether the of non-NMDA agonists to elicit NMDA-like
different agonists act on individual receptor-ionic openings arises from a weak agonist action of
channel complexes or whether one single type of these compounds on NMDA receptors and favor
ionic channel coupled to three different receptor the hypothesis according to which NMDA and
subtypes mediates all the glutamatergic actions non-NMDA agonists activate distinct receptor-
has been the matter of considerable debate. ionic channel complexes. Because Purkinje cells

50 J.lM Kai

100 ms

1
....>.
.....
til
t::
Q)
q
-....
t':S
1-4
0
Q)
0.1
Figure 9.11. Current fluctua-
tions recorded from a somatic
p.. outside-out patch upon the
en application of kainate, starting
0.01
about 70 ms after the beginning
of the trace. Holding potential,
- 60 m V. External and internal
solutions as in Fig. 9.10. Lower
0.001 panel: The power spectrum
for the kainate-induced current
noise was fitted with a single
Lorentzian function (solid line),
I I I I II I I I '" I I I I I I II
which yielded a time constant of
10 100 1000 1.04 ms. (Adapted from Llano
Frequency (Hz) et ai., 1988a.)
9. Voltage- and Transmitter-Gated Channels
are insensitive to NMDA, they provide an oppor-
tunity to test the responses to non-NMD A agonists
in a preparation that is devoid of NMDA recep-
tors. Figure 9.10 presents typical examples ofthe
single-channel events that are activated by appli-
A 10 pM GABA
J 0.55
50pA
B
21lMGABA
~2PA
20 ms
3.0
Figure 9.12. A: Response of a somatic outside-out patch
to the application of GAB A, during the time indicated
by the bar above the record. The membrane potential
was maintained at - 60 m V.Ie = 1.5 kHz. External and
internal solutions as in the two previous figures. The
solid line corresponds to the fit of the decay phase of
the current to a double exponential function plus a
baseline component. Fit parameters: 446 ms, - 57 pA;
80 ms, - 51 pA; baseline: - 4.5 pA. B: Selected portions
197
cation of quisqualate to an outside-out patch
from the soma of a Purkinje cell. The amplitude
distribution of the quisqualate-activated events
shows three peaks at current values of 0.9,
1.4, and 1.7 pA, corresponding to elementary
1.5 o
Amplitude (PA)
of the current events elicited by GABA in another
outside-out patch. Holding potential: -80mV. The
right panel presents the distribution of elementary cur-
rent amplitudes for the GABA-activated channels in
this patch. The solid line corresponds to the fit of the
data to a single gaussian function, with a single-channel
current of - 1.6 pA. Bins of amplitude lower than
-1.1 pA were excluded from the fit.
198
conductances of 9.5, 15, and 18 pS, respectively.
Absent from such records were the large conduct-
ance (50 pS) events that characterize NMDA-
induced openings.
NMDA-like openings were also absent upon
application of kainate in all outside-out patches
studied, an example of which is shown in Figure
9.11. The spectrum of the current fluctuations
induced by kainate is described by a single Lorent-
zian with a time constant of 1 ms (Fig. 9.11, lower
panel). Analysis of the relation between the mean
current and the variance yields a value of 2.5 pS
for the elementary conductance.
The results show that responses to non-NMDA
agonists differ in Purkinje cells, which are totally
insensitive to NMDA, and in cultured CNS neu-
rones, which are sensitive to all three types of
glutamatergic agonists. Our finding that NMDA-
like openings are totally absent from the former
responses gives support to the hypothesis of
Ascher and Nowak and casts serious doubts on
the single-channel type hypothesis.
GABA-ergic Channels
As mentioned earlier, outside-out patches from
the soma of Purkinje cells have a large density of
GABA-sensitive channels. A typical example of
the currents obtained upon activation of these
channels is presented in Fig. 9.12A. Application
of 10 JlM GABA leads to an inward current with
a peak value close to 100 pA, which declines close
to baseline values even though agonist application
is maintained. The time course of the current
decay is described by two exponential components
with time constants of 80 and 446 ms. Desensiti-
zation of GABA-activated currents takes place in
both spinal cord neurones (Hamill et aI., 1983)
and chromaffin cells (Bormann and Clapham,
1985), and it is described as well by two exponen-
tials. However, the time course of desensitization
we find for Purkinje cell GABA-ergic channels is
much faster than that of peripheral cells. Time
constants of2.8 and 20.8 s have been reported for
the decay time of the response to 20 JlM GABA
in chromaffin cells. A complete study of the desen-
sitization process of the GABA channel from
Purkinje cells will be needed to determine whether
these results reflect a basic molecular difference
in the receptor types involved.
Isabel Liano, Beat H. Gahwiler, and Alain Marty
In a few outside-out patches that happened to
have a small number of active channels, an analy-
sis of single-channel events was carried out. An
example of the channel activity induced by 2 JlM
GABA is presented in Figure 9.12B. The most
frequent conductance state ofthese channels cor-
responds to 28 pS (see histogram fit), but openings
to a level of 20 pS are observed as well. Multiple
conductance states are characteristic of GABA
channels, and have been studied in detail in mouse
spinal cord neurones in culture (Bormann et aI.,
1987). The most frequent conductance state in
those neurones is 30 pS, a value close to the 28 pS
level we report for Purkinje cells.
Conclusions
The Promises of a Patch-Clamp
Approach to the CNS
The long resistance to introduce patch-clamp
methodology to the CNS is in part due to techni-
cal difficulties. As we are witnessing now, some
of these difficulties can actually be overcome.
There is, however, another more fundamental
objection against such an approach. This line of
thought is advocated by people who underscore
the gap existing between the properties of single
molecules and the behavior of complex neuronal
networks. The number and diversity of single ele-
ments (ion channels, or even individual synapses)
is such that it can appear as a delusion to try to
explain "interesting" properties of the CNS by
combining such elements. Therefore, it could
appear more informative to concentrate on com-
paratively integrated information (e.g., by study-
ing current-clamp responses at the cellular level),
at least up to the point where major neuronal
types will be characterized from that point of
view.
There are undoubtedly good reasons for caution
in these criticisms. Thus, it is certainly necessary
to integrate single-channel results at the cellular
level as one goes on. This is particularly difficult
with neurones because of somatodendritic differ-
entiation and because of the lack of spatial voltag!!
control in whole-cell recordings. In Purkinje
cells, we have tried to circumvent this difficulty by
recording "macroscopic" somatic currents from
large outside-out patches, but other, better suited
9. Voltage- and Transmitter-Gated Channels
techniques will have to be designed to study den-
dritic conductances as well. In general, however,
we feel that the analytical approach represented
by the patch-clamp technique offers a promising
lead to the study of the CNS. The study of syn-
aptic transmission should greatly benefit from the
identfication of the specific neurotransmitters and
ion channels involved. The study of voltage-
dependent ion channels should help in the under-
standing of the origin of the spiking properties of
individual neurones. Generally, we are of the
opinion that a detailed analysis of ion channels
can simplify the interpretation of cell properties.
Finally, one decisive advantage ofthe patch-clamp
technique is to gain access to the cell interior. This
will permit the study of synaptic transmission
involving intracellular messages, an area of enor-
mous potential that has been left practically un-
touched in the CNS.
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10
Electroneuronal Hybridization:
A Novel Approach to Investigate
Rhythmogenesis in the Interior Olivary Nucleus
YosefYarom
Detailed studies of the structure and function
of neurons of the inferior olivary nucleus carried
out over the last two decades have pointed out some
unique features ofthese cells. These neurons have
an immensely dense dendritic tree (Scheibel and
Scheible, 1955; Scheibel et aI., 1956) and in addi-
tion to multiple spinelike appendages (de Zeeuw
et aI., 1989a), they have a unique synaptic organi-
zation in which excitatory, inhibitory, and electri-
cal synapses are meaningfully organized along
the dendritic tree (Sotelo et aI., 1974; de Zeeuw
et aI., 1989b).
Physiological studies have also indicated several
unique prQperties. The exceptionally low firing
rate (Armstrong et at, 1968; Armstrong and
Rawson, 1979; Bell and Grimm, 1969; Leonard
et aI., 1988; Llim'ts and Sasaki, 1989; Sasaki et aI.,
1989), the electrical coupling (Llinas et aI., 1974;
Llinas and Yarom, 1981a), the olivary reflexes
(Eccles et aI., 1966), and the powerful synaptic
connection between these neurons and the cere-
bellar Purkinje cell (Eccles et aI., 1966; Llinas and
Sugimori, 1980) are just a few of the properties
that distinguish these neurons from others in the
central nervous system.
This chapter focuses on the electroresponsive
properties of individual olivary neurons and the
emergent electrical properties of the nucleus as
revealed by in vitro experiments combining the
conventional intracellular technique with a new
experimental approach based on use of a hybrid
system: the neuron in the slice preparation to-
gether with an analog model of the nucleus.
Electroresponsive Properties
of Olivary Neurons
In the last decade, with the development of intra-
cellular techniques permitting stable, reliable re-
cording from neurons of the mammalian central
nervous system, it has become evident that the
type and density of ionic channels and their
distribution over the somadendritic membrane
determine the electroresponsive properties of
central neurons (Llinas, 1988). The uniqueness of
olivary neurons arises from the diversity, rela-
tively high density, and almost complete segrega-
tion of their ionic channels. The different ways in
which these channels can coactivate or interact
endow the olivary neurons with the capability to
generate a variety of patterns of electrical activ-
ity ranging from a single robust action potential
through a repetitive pacemakerlike activity to
subthreshold synchronized membrane potential
fluctuations (Llinas and Yarom, 1981a, b, 1986a;
Yarom, 1989; Yarom and Llinas, 1987).
The intracellular records, which were obtained
through the use of the brain slice technique
(Fig. lO.1), typify the various electrical activities
detectable in olivary neurons. In Figure lO.1A a
supra threshold positive current pulse delivered at
resting potential level elicited a complex neuronal
response characterized by a broad potential, a
prolonged afterhyperpolarizing potential, and a
prominent rebound response. In previous work
(Llinas and Yarom, 1981 a, b) it was demonstrated
that the broad action potential results from the
201
202 Yosef Yarom

A B

J---
JvA--
120mv~
'"----
~25mv
..-r-l1--_--===!lnA
100msec 0.5 sec
C D

IlnA
---1.----------'
---I 2 sec
Figure 10.1. The different types of electrical activities
encountered in intracellular recordings from olivary
neurons. A: At resting potential level, olivary neurons
respond to a positive current pulse with a complex
neuronal response that reflects the sequential activation
of various types of ionic channels. B: The repetitive
activation of the low-threshold Ca spike by negative
current pulses of different intensities (top to bottom:
increase in current intensity) reveal the oscillatory
nature of this conductance. The absence ofNa-dependent
400msec
response is due to the presence of TTX (10 - 5 M) in the
physiological solution. C: Prolonged bursts of activity
after DC hyperpolarization of the membrane. Upper
trace obtained at lower current and demonstrates shorter
train of activity. The current intensity is shown at the
lowest trace. The duration of the burst of activity varies
in different preparations and probably represents dif-
ferent neuronal populations size within the particular
slice (Lampel and Yarom, unpublished observations,
see also Llinas and Yarom, 1981, 1986).

sequential activation of Na channels located at is completely inactive at resting potential and

the cell body level and Ca channels that are likely since it has a distinct threshold that is close to or
to be distributed over the dendritic membrane. at the resting level, it effectively governs the
The activation ofCa-dependent K channels, which electrical behavior of neurons (Gutnick and
are probably distributed all over the somaden- Yarom, 1989; Llimis, 1988).
dritic membrane, generate the prolonged after- As shown in Figure lO.1B, a negative current
hy~rpolarizing potential. The rebound response, pulse delivered at resting potential level evoked
on the other hand, reflects the activation of a a repetitive activation of this conductance even
low-threshold Ca conductance located mainly at under conditions where Na conductance was
the cell body and characterized by its low voltage blocked by tetrodotoxin (TTX). An increase in
of activation and relatively fast voltage-dependent the current intensity (top to bottom) resulted
inactivation. Since the low-threshold conductance in an increase in the duration of the repetitive
10. Electroneuronal Hybridization
activation. The repetitive activation of the low-
threshold conductance results from the relatively
slow inactivation process and the afterhyperpo-
larization that follows each individual response.
Thus, the low-threshold Ca conductance has an
intrinsic tendency to oscillate.
Under normal conditions, a much longer burst
of activity could be occasionally recorded. As
shown in Figure 10.1 C, these bursts of activity
are triggered by the release from a prolonged
negative current that held the membrane at a
hyperpolarizing level. The burst duration depends
on the preceding hyperpolarizing level and always
terminates with a short series of low-threshold
Ca spikes (LTS) resembling those obtained in the
presence ofTTX (Fig. 10.1 B). These observations
have led us to hypothesize that olivary neurons
can be regarded as limit cycle oscillators (Llinas
and Yarom, 1986a). Indeed, in about 10% of the
experiments the membrane potential of olivary
neurons undergoes sinusoidal-like oscillations.
The examples shown in Figure 10.1 D demonstrate
the waveforms of these oscillations and their
voltage dependence. The most important features
revealed by this experiment are that the frequency
ofthe oscillations is independent ofthe membrane
potential whereas the amplitude decreases as the
membrane is hyperpolarized (top to bottom).
In an attempt to explore the mechanisms under-
lying the oscillatory activity of inferior olivary
neuronal ensembles, a novel experimental ap-
proach was developed. According to the ratio-
nale underlying this approach, if an analog model
(constructed according to the working hypothesis
assumptions) successfully replicates the phenom-
ena observed in the neuronal system, and if the
analog system can be merged into the neuronal
system and the hybrid system can still produce
the same phenomena, the working hypothesis is
then strongly reinforced. A brief description of
the approach and the main results obtained
through its use is given in the following sections.
The Simulator
The simulator consists offour identical intercon-
nected oscillating units; a schematic illustration
ofthe system is given in Figure 10.2. The heart of
each oscillating unit (OSC 1-4) is an operational
amplifier with a notch filter, tuned to a given
203
frequency, with controllable gain in its feedback
loop. Such a unit will generate sustained oscilla-
tions at the given frequency, provided that the
closed loop gain is equal to or greater than 1. Each
unit receives its input from a point that sums the
activity in all the other oscillating units (I: 1-4)
as well as from a triggering source. The activity
in this system is determined by the "unit gain,"
which determines the feedback loop gain of each
unit, and the "coupling gain," which determines
the extent ofthe coupling between the units. Trigger
signals to the electrical units were generated by
a rate-controllable trigger source that distributes
a constant trigger signal either randomly to all
the units or at a constant rate specifically to any
given unit. A set of switches (Sj, St, So) controls
the internal connections between the units. In this
system sustained oscillations are generated if the
equivalent overall gain (the feedback loop-gain
in each unit plus the contributions to the loop
from the other units) exceeds a certain level ( + 1).
Figure 10.2 also demonstrates the connection
of the olivary neuron to the simulator. The out-
puts of all four units were summed, translated
into current, and injected into the neuron through
the intracellular microelectrode. The neuronal
response (E), as recorded by the microelectrode,
was introduced into the summing points of each
of the oscillating units. With this arrangement,
the connections to and from the olivary neuron
are similar to those of any of the oscillating units.
Properties of the Simulator
The properties of the simulator are shown in
Figure 10.3. The increased oscillations induced
by increasing the unit gain in an isolated oscillating
unit are demonstrated in Figure 10.3A. Oscillations
were evoked by a single triggering pulse (the first
negative deflection in each trace) delivered to the
same unit at four different levels of unit gain. As
the gain increases (top to bottom), the decay time
of the oscillations increases; as a result, the ampli-
tude and duration of the oscillations increase.
The effect of increasing the coupling level
between the simulator units is demonstrated in
Figure 1O.3B, C. Simultaneous recordings of the
electrical activity in all the units, at two levels of
coupling, are presented. The three coupled units
(three lower traces) were mutually connected,
204
RATE ~~.-__~T~R~IG~GpE~R~S~O~U~R~C~E______~
GAIN
COUPLING
OUTPUT
VOLT AGE TO CURRENT
OLIVARV NEURON
Figure 10.2. Schematic presentation of the simulator
illustrates the organization of the hybrid system. Trig-
ger source, A rate-controlled trigger source that distri-
butes a constant trigger signal either randomly to all
the units or specifically to any given unit. St, A toggle
switch connecting any given unit to the trigger source.
OSC 1 to 4, The four oscillating units, whose gain is
simultaneously controlled. Sj, A toggle switch connect-
ing the input into the oscillating units. S 1 to 4, Summing
points preceding the inputs of each of the oscillating
whereas the input to unit 1, which received the
triggering signal, was unconnected; thus, unit 1
serves as the source but does not receive the
feedback signal. As the coupling between the units
increases, the oscillations in the coupled units
increase both in amplitude and duration. It should
be mentioned that in this system the load effect,
expected in a coupled system, does not exist.
Therefore, even when the coupling was increased,
the source, unit 1, remained unchanged. Further-
more, since each of the coupled units responds
actively to the input signal and since all of them
are interconnected, at the high coupling level
(Fig. lO.3C), the oscillations in the coupled units
outlast the response in unit 1.
YosefYarom
I I
units. The couping gain (coupling) that is determined
after the summing point is also simultaneously con-
trolled. So. A toggle switch connecting the output of any
given unit to the summing points of all the other units.
The outputs from all the units are summed, translated
into current, and injected into an olivary neuron whose
output (E) is fed back into the summing points of each
of the units. (Adapted with permission from Yarom,
1989.)
Generation of Sustained Oscillations
By interconnecting the entire system and deliver-
ing a trigger signal to one of the units, sustained
oscillations could be generated, provided that the
unit gain and the coupling level were properly set.
This behavior is demonstrated in Figure 1O.3D,
which shows the activity induced by a triggering
signal delivered to unit 1 (upper trace) and re-
corded simultaneously in all four units. The build-
up of sustained oscillations is characterized by an
initial phase during which the amplitude of the
response in unit 1 decreases, while the amplitude
of the responses in the coupled units increases
until the activities in all the units are locked in
10. Electroneuronal Hybridization
A B C of one or more of the units from the system. The
_Af'-~ __ ... - - - --A.rv--_. -.
400 msec
D settings where a single trigger signal to unit 1
Figure 10.3. Properties of the simulator. A: Increased
oscillations induced by an increase in unit gain. The
activity of unit 1 during progressive increases in the
unit gain. In each trace the unit gain was set to a different
level, increasing from top to bottom. Note that the unit
gain affects the duration of the oscillations more than
their amplitude. B-C: The activity in all the units is
shown at two different levels of coupling gain. Only
unit 1 (upper traces) was triggered. All the units were
mutually connected except for the input to unit 1
(Switch Sj of unit 1 was open; see Fig. 10.2). Note
that increased coupling level did not affect the signal
in unit 1 and that, at the highest coupling gain (C), the
signals in the coupled units outlast that of unit 1. D:
Sustained oscillations generated by the electrical simu-
lator with a threshold setting of the unit and coupling
gains. The activity recorded in all four units after a
triggering signal to unit 1. Note that the sustained
oscillations develop simultaneously in all units and
that the amplitude is larger than that induced by the
triggering signal. (Adapted with permission from Yarom,
1989).
phase and amplitude. From this time on, a pro-
gressive increase in amplitude in all the units is
evident. The process then develops into its final
form, and sustained oscillations of constant ampli-
tude are generated. As shown in this figure, accu-
rate synchronization of the units seems to be a
prerequisite for the generation of sustained oscil-
lations.
Once sustained oscillations are generated they
will be maintained if no interference is introduced;
interference is defined either as a reduction in
coupling level or unit gain or as a disconnection
205
ability to produce sustained oscillations depends
on the unit gain and the coupling level in such a
way that a system with a high coupling level and
low gain behaves similarly to a system with a low
coupling level and high unit gain. In fact, once a
set of "threshold" parameters (a level of gain
evoked sustained oscillations) was selected, they
could be modified in a reciprocal way and the
system would still maintain the same properties.
Therefore, a system comprising coupled oscil-
lating units, each of which can generate damped
oscillations and responds actively to input signals,
can generate sustained oscillations. In such a
system in-phase activity of the oscillating units
is a necessary condition for the development of
sustained oscillations. The ability to reach this
condition, which is an intrinsic property of the
system, depends on the extent of the coupling
between the electrical units. On the other hand,
maintenance of the oscillations depends on the
integrity of the system.
The Hybrid System
In order to determine to what extent the mech-
anism underlying the generation of oscillations
in the olivary nucleus behaves similarly to the
analog simulator, a hybrid system, consisting of
the analog simulator and an olivary neuron in a
slice preparation, was constructed. If such a hybrid
system were to retain the properties of the analog
simulator by itself, and if an inferior olivary neuron
behaved similarly to each of the electrical units
and contributed actively to the generation of
rhythmic activity within such a system, then the
olivary neuron would also appear to act as a
generator of damped oscillations. Moreover, it
would not be unreasonable to suggest that the
oscillating inferior olivary nucleus itself operates
similarly to the analog model.
Sustained oscillations induced in the hybrid
system by a single triggering pulse are demons-
trated in Figure 1O.4A. The top two traces are the
activity in units 1 and 3 (units 2 and 4 were also
connected but are not illustrated). The third trace
is the activity in the olivary neuron as recorded
206
unit
olivary
neuron
B
unit #1
unit #3
olivary
neuron
~------------------~~~________====~IO.5nA
i O.5sec
Figure 10.4. The activation and termination of sus-
tained oscillation in the hybrid system. Simultaneous
recording from units 1 and 3 (two upper traces) and
from an olivary neuron (lower trace). Units 2 and 4 are
also connected but are not shown. A: A continuous
recording illustrates the development of the oscillations
after a single triggering signal delivered to unit 1 (arrows
denote the triggering time). Note the phase shift of the
response ofthe neuron in relation to the electrical units
during the first three waves and the accelerated rising
phase of the waves recorded from the neuron just
before the development of full size oscillations. B: As
in A, continuous recordings of the activity in units 1
by the intracellular microelectrode. A trigger sig-
nal was delivered to unit 1, recorded as a fast
negative deflection (arrow). As with the simulator
by itself, in the hybrid system the sustained oscil-
lation evoked by the triggering signal is also
characterized by an initial phase, during which a
slow build-up of the oscillation was observed.
During the sustained oscillation, the amplitude
of the olivary wave was in the range of25 to 30 mV
and, as a result, the peak of the oscillatory wave
occasionally reached threshold and action poten-
tials were generated. Although the sustained
YosefYarom
O.5sec
\20IllV
/V"'J''J'.r.,."....,---
and 3 and the olivary neuron are shown. The lowest
trace indicates the changes in the DC holding current.
Oscillations were invoked by releasing -0.3 nA DC
holding current in the neuron (first arrow). Oscillations
were terminated by hyperpolarizing the neuron (second
arrow). Note that the release of the holding current was
followed by a train of LTSs, and that the last LTS that
occurred in phase with the electrical oscillations (de-
noted by *) seems to be the immediate trigger for the
development ofthe sustained oscillations. The neuron
was treated with harmaline (5 mg/ml) and CsCI (5 mM)
in order to reduce various rectifications.
oscillations in the olivary neuron followed the
frequency dictated by the electrical system, the
waveform was deformed and did not follow the
sine wave generated by the electrical system.
A careful examination of the initial phase re-
veals that immediately after the triggering signal,
a prominent phase shift exists between the wave
in the olivary neuron and the electrical units. TQis
phase lag is actually expected whenever a sinusoi-
dal current is irttroduced into an electrical circuit
composed of a resistor and a capacitor (RC
circuit) such as provided by the neuron. During
10. Electroneuronal Hybridization
the initial phase, a progressive decrease in the
phase shift of the olivary oscillations was obser-
ved. This decrease in phase lag was accompanied
by increasing deformation of the waveform in the
olivary neuron. The deformation was character-
ized by an accelerated rising phase, indicating the
presence of an active process. Once a phase-locked
condition was achieved, a state of sustained oscil-
lations was quickly reached in the entire system.
Therefore, as in the simulator by itself, synchron-
ization between the different elements of the
hybrid system appears to be a necessary pre-
requisite for the generation of sustained oscil-
lations.
According to the above description, a regen-
erative response in the olivary neuron contri-
buted to the reduction of phase lag, thereby
insuring complete synchronization between the
olivary neuron and the electrical units. The pro-
perty of olivary neurons that most likely explains
the phase advance leading to synchronization is
the low-threshold Ca conductance. Since both
the activation and inactivation of this con-
ductance occurs within a rather small potential
range around the resting level, it generates graded
regenerative responses as the result of relatively
small depolarizations, provided that they are pre-
ceded by hyperpolarization (Llinas and Yarom,
1981a). Thus, the hybrid system, which oscillates
about the resting potential, provides favorable
conditions for the activation of this low-thresh-
old Ca conductance.
The contribution of the low-threshold Ca re-
sponse to the generation of sustained oscillations
was examined by shifting the membrane potential
to either more negative or less negative values
(relative to the resting level). Since the LTS has a
distinct threshold and since it is completely inac-
tive at potentials less negative than the resting
level, a DC voltage shift in either direction will
prevent the generation of the LTS. The effect
of DC hyperpolarization on the sustained oscilla-
tion of the hybrid system is demonstrated in
Figure lOAR. The experiments were performed
in the presence of harmaline (5 mg/ml) and CsCI
(5 mM) which, by blocking several types of recti-
fication, induce linear current-voltage relations
(Yarom and Llinas, 1987). As in Figure 10AA,
only the olivary activity (lower trace) and the
activity in two of the coupled units (upper traces)
207
are shown. The neuron was held at a hyper-
polarizing level by - 0.3 nA DC current injected
through the microlectrode. Releasing the holding
current (arrow) generated an LTS that was large
enough to trigger a sodium-dependent spike and
to activate the electrical oscillators. Since the
frequency generated by the olivary neuron was
in the order of 5 Hz (while the tuned frequency of
the electrical system was 9 Hz), the electrical acti-
vity during the first second after release from
hyperpolarization appears chaotic and the ampli-
tude of the oscillation varied from wave to wave.
This situation persisted until the rhythm gener-
ated by the olivary neuron subsided and the fifth
LTS (marked by *) coincided with the sine wave
in the electrical units. From that time on, a state
of sustained oscillation was quickly reached.
Reintroducing the -0.3 DC current into the
olivary neuron (second arrow) induced, in addition
to the drop in membrane potential, a dramatic
reduction in the amplitude of the olivary neuron
oscillations. As a result, the oscillations in the
coupled electrical units slowly declined. Thus, it
is clear that most of the oscillatory response in
the olivary neuron is generated by a voltage-
dependent conductance, most likely the low-
threshold Ca conductance, which appears to be
essential for the generation of sustained oscilla-
tions.
Preferred Frequencies of Oscillations
Even if the hybrid system does not accurately
simulate the conditions in the inferior olivary
nucleus, it provides a new tool with which the
complex neuronal properties postulated to exist
in olivary neurons can be investigated. The follow-
ing example demonstrates the use of the hybrid
system to explore the behavior of olivary neurons
in the frequency domain.
Since, as already demonstrated, the LTS of the
inferior olive neuron actively participates in the
generation of sustained oscillations by the hybrid
system, it is expected that the ability of the hybrid
system to generate sustained oscillations will be
frequency dependent. The frequencies where the
low-threshold Ca conductance is most readily
activated will be the frequencies "preferred" by
the olivary neuron. At these preferred frequencies,
minimum "gains" should be needed to evoke sus-
tained oscillations.
208
COl •• •
..........
CJ
Q)4
.........
III
d
....0
~2
s..
:=;j
'0
0
2 4 6 B
frequency
Figure 10.5. Frequency-duration curve of the hybrid
system reveals that the ability to generate sustained
oscillation is frequency dependent.l nsets: Intracellular
recordings from the olivary neuron illustrating the
responses of the hybrid system at four different fre-
quencies. The simulator gain settings were constant
throughout the experiment, which was carried out in
In order to determine the preferred frequencies,
the gains of the system were set to given values,
usually just below threshold for oscillations at the
lowest frequency. Then the duration of the oscilla-
tions induced by a single triggering pulse (given
to one of the electrical units) at different frequen-
cies (set at the electrical units) was measured and
plotted as a function of the frequency. The results
of such an experiment are shown in Figure 10.5.
The four recorded traces, which were obtained at
the frequencies indicated by the arrows, represent
the activity ofthe olivary neuron within the hybrid
system after a single triggering pulse (first negative
deflection) delivered to one of the electrical units.
These records clearly demonstrate that the ability
of the hybrid system to generate sustained oscilla-
tions, at a given set of gains, is frequency depen-
dent. The rapidly decaying oscillatory responses
YosefYarom
20mV
0.5 sec
the presence of TTX (10 - 5 M). All the responses were
elicited by a single trigger pulse delivered to unit 1. The
duration was measured from the first negative deflection
to the point where the amplitude was reduced to twice
the size of the noise. Note the discontinuity of the
duration axis (the uppermost data points represent
sustained oscillation).
triggered at low (3 Hz) and high (8.7 Hz) frequen-
cies appear to comprise two components: the
passive response to the sinusoidal input current
and the regenerative low-threshold spikes. At
these frequencies the L TSs were activated only
by the first one or two waves. In fact, a failure to
generate an LTS was immediately followed by
fast decay of the passive oscillatory response,
reflecting the decay of the current input. The
full-size oscillations observed at 6.4 Hz, however,
lasted 2 s but displayed a continuous decrease in
amplitude. This decrease eventually led to an
abrupt failure to generate an LTS, which was
followed by a rapid decline of the oscillations. At
intermediate frequencies (5.1 Hz), the same trig-
gering signal evoked sustained oscillations with
constant amplitude that persisted as long as the
system remained intact and the gains were un-
10. Electroneuronal Hybridization
changed. The results of these experiments are
summarized in the "tuning curve" shown, which
describes the duration ofthe oscillation as a func-
tion ofthe frequency. Only at three close frequen-
cies (4.7, 5.l, and 5.7) did sustained oscillations
develop. At other frequencies the oscillations
lasted only a few seconds. Although this apparent
high sensitivity (narrow-band tuning curve) could
result from the method of quantification, it reveals
that the olivary neuron preferred to operate at
certain frequencies. In other experiments the pre-
ferred frequency ranged between 4 and 6 Hz, with
an average of 5.2 (n = 7).
It should be mentioned that in a series of control
experiments, including the examination of the
responses ofthe same neuron with the same setting
of unit and coupling gains both with and without
octanol (0.01%), it has been verified that the fre-
quency dependence of the hybrid system reflects
the properties of the neuron and not those of the
electrical units.
Discussion
Functional Role of Olivary Subthreshold
Oscillations
Even though the present study did not address the
question of the functional role of the subthresh-
old oscillations, it is clear, however, from the very
essence of their nature that the oscillations can
serve as an internal time reference (Llimis, 1989).
According to this concept, the activity in a group
of olivary neurons whose size is determined by
the extent of the coupling is associated with a
rhythmic activity that takes place in the motor
system. It is not unlikely that this rhythm is in
fact the source of the physiological tremor that
occurs in a muscle or in a group of muscle units
during motor behavior (Llimis, 1984).
The ability of the olivocerebellar system to
determine not only the size of the group of coupled
olivary neurons (through the inhibitory pathway,
originating- in the deep cerebellar nucleus and
terminating next to the olivary gap junction;
Llimis and Sasaki, 1989; Sotelo et aI., 1986) but
also its functional state (quiescent or oscillating)
offers a unique mechanism to associate a variable
number of muscle units with a variable number
of olivary neurons. It is also possible to use the
209
same neurons in different constellations, thereby
increasing the versatility of the system.
It is important to determine if this oscillation
is the source of the motor rhythm or just its ref-
lection. The difference between these two possibi-
lities reflects a fundamental difference in our
interpretation of olivary function. If the olive is
the source (Lamarre, 1979, 1984; Llimis, 1984),
its role is to determine the basic rhythm of muscle
activity that underlies all motor performance
(Llinas, 1984). If, on the other hand, it is just a
reflection of motor rhythm, it might serve as a
correcting relay station (or "phasic motor control
system"; see Llinas, 1970) and the subthreshold
activity could then be a device that ensures the
most efficient correcting procedures (Schoner and
Kelso, 1988).
If the olive is the drive for motor activity, how
can subthreshold oscillation, which by definition
is an internal phenomenon, determine the rhythm
of muscle activity? It is possible that in order to
maintain a rhythm only one or a few olivary
neurons within the defined (coupled) group have
to be active at any given time. Thus, the firing of
an olivary neuron, which under normal physio-
logical conditions appears to be unrelated to any
behavioral parameter (with the exception of the
neurons ofthe dorsal cap), is in fact following an
accurate timing schedule (Sasaki et aI., 1989).
According to this scheme, when movement is
disturbed or sensory inputs report an unusual
circumstance, a massive, synchronous activation
of the group of coupled neurons will reset the
motor rhythm. If this is true, measuring the acti vity
of a single neuron might lead to a wrong conclu-
sion regarding its functional role.
The basic assumption underlying the second
possibility is that the subthreshold oscillations
reflect motor rhythm rather than drive it. Accord-
ing to this view, the subthreshold rhythm in an
olivary neuron will function only during the
performance of a routine motor task. As in the
previous possibility, the olivocerebellar system
determines the size of the group of coupled neu-
rons, relative to the number of muscles involved
in that motor task, as well as the functional state
of the group relative to the onset of the movement.
The difference is that in this case the subthreshold
oscillation will sum with any synaptic input to
elicit an output at a precise time as determined
210
by the phase of the oscillations. It is not unlikely
that such precise timing would be needed for the
system to respond in the most effective way to
incoming signals (Llinas, 1984; Marshall and
Walsh, 1956).
Although there is no single compelling reason
to reject either of these possibilities, there are
several observations and considerations that
argue against each of these hypotheses. If olivary
subthreshold oscillations are the source of motor
rhythm, all cells should be active all the time, as
is the physiological tremor. The precise control
of the group size seems to be less important.
Furthermore, if only a few neurons within a group
of coupled neurons fire at any given time, the
convergence of olivary neurons onto a single neu-
ronal structure has to be postulated. Alternatively,
the group of coupled olivary neurons might drive
another less efficient oscillator.
On the other hand, the observation that physio-
logical tremor disappears after either a lesion
of the inferior olivary nucleus or in the presence
of alcohol (Sinton et al., 1989), a suppressor of the
low-threshold calcium conductance (Llinas and
Yarom, 1986b), is a strong indication that the
inferior olive is the source of motor rhythm.
Electroneuronal Hybridization
A new experimental approach to investigate the
complex electrical behavior of neuronal assembly
was introduced. This approach was used to point
at a possible mechanism that underlies the rhyth-
mic activity of inferior olivary neurons.
Most of the electrophysiological phenomena
that have been observed in biological systems,
such as the mammalian central nervous system
(CNS), were attributed to complex interactions
between several elements (e.g., neurons) or sub-
elements (e.g., channels). When these phenomena
were investigated, the following approach was
commonly used: the subelements were functional-
ly isolated, their specific properties characterized,
a comprehensive theoretical model was then con-
structed, and its predictions were compared to
the actual physiological phenomenon. Failure to
predict accurately the behavior of the biological
system indicates that a subelement or one of its
properties was inadequately presented in (or even
missing from) the model. As these models increase
Yosef Yarom
in size (due to many elements each having several
subelements), the number of poorly defined pa-
rameters increases, producing conditions where
practically any phenomenon could be simulated.
In the approach presented here, a working
hypothesis is first formulated and a "simulator,"
which is a hardware representation of this hypo-
thesis, is then constructed. The ability of this
hardware representation to interact with the bio-
logical system is then examined. The controllable
part in such a "hybrid system" serves as a unique
tool to examine the behavior of the biological
system under conditions that were postulated to
exist by the working hypothesis. The biological
part of the hybrid system, on the other hand,
limits the number of assumptions and thereby
the degrees offreedom; both an excessive number
of assumptions and degrees of freedom tend to
reduce the usefulness oftheoretical models. There-
fore, this approach tends to fill the gap between
pure theoretical modeling and conventional
electrophysiological experiments.
The working hypothesis in the present study
was formulated to account for the subthreshold
membrane potential oscillation Qf olivary neurons.
According to this hypothesis, each olivary neuron
is a generator of damped oscillations. Since these
oscillators are electrotonically coupled, b)! inter-
acting they can generate synchronized sustained
oscillations. The simulator in this case comprises
an interconnected set of oscillating units, each
capable of generating damped oscillations; when
interacting with each other, they can generate
sustained oscillations. The hybrid system com-
prises the simulator and an intracellularly impaled
olivary neuron in a slice preparation. It has been
demonstrated that the activity in such a hybrid
system is similar to that observed in the olivary
nucleus.
The three most important conclusions derived
from the behavior of the hybrid system are:
1. In-phase activity of the neurons is essential in
order for such a system to generate sustained
oscillations.
2. The L TS serves as a phase advance mechanism
that compensates for the phase lag introduced
by the RC circuit of the neurons.
3. Olivary neurons exhibit a preferred frequency
of oscillation that reflects the interplay between
several ionic channels.
10. Electroneuronal Hybridization
If a mechanism similar to that found in the
hybrid system operates in the inferior olivary
nucleus, several conclusions can be drawn that
provide a substantial contribution to our under-
standing of olivary activity. For example, from
the behavior of the hybrid system it is clear that
a sufficiently large group of olivary neurons has
to be coupled in order to generate sustained oscil-
lations. On the other hand, the frequency of these
oscillations is independent of the group size.
Thus, the coupling between olivary neurons
should be considered not just as a device that
synchronizes spikes but as the main control
that dictates the functional state of the internal
clock mechanisms. Accordingly, the dense gamma-
amino butyric acid (GABA)-ergic innervation of
the olivary nucleus (Sotelo et aI., 1986), which
is postulated to modulate the coupling between
these neurons (Llimis, 1974; Llinas et aI., 1974;
Llinas and Sasaki, 1989; Llinas and Yarom, 1981a),
determines not only how many neurons will be
synchronously active, but also whether the under-
lying timing device will be functional.
Acknowledgement. This work was supported by
a grant from the United States-Israel Binational
Foundation.
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 Part 3
Electrophysiology of Movement

The first chapter in this part documents a series of experiments (1) describing
the main characteristics of the spontaneous saccades in the rat in light and
darkness .(saccades have relatively high-peak velocity and large ability
while gaze holding ability is poor) and (2) the effect oflesioning the 10 and
two cerebellar areas known to be involved in the saccadic performance
(lobules VI, VII, VIII of the dorsal vermis-no significant change, and the
flocculus and paraflocculus-same as the monkey in that there is a
reduction in the time constant of the neural integrator, but different in that
fast postsaccadic drift is larger than in the monkey; 40% compared to 15%).
In the rat, the flocculus/paraflocculus are important to improve poor gaze
holding ability of the oculomotor system. 10 is essential for maintaining
leakiness of the neural integrator within physiological range and for gain
of the pulse to step transformation at the proper value. Effect is quanti-
tatively similar as that following floccular/parafloccular lesion. The condition
is that the 10 controls ocular motility through action on floccular/parafloc-
cular Purkinje cells.
In the third chapter, the authors ask whether there is meaning in the
geometry inherent in the CF zones and the orthogonally directed parallel
fibers. Answer: probably. AChE staining (delineates white matter compart-
ments with presumptive projections to different parts of the vestibular
nucleus complex), which provides an independent delineation of floccular
zones, invited these experiments to combine anatomical and electrophysio-
logical techniques to determine the patterns of eye movements evoked by
stimulation of each compartment. Results are considered in conjunction
with effects of floccular stimulation on the six VOR pathways. The major,
short latency components of the eye movements evoked here are in good
agreement with predictions from combining stimulation ofVOR paths with
measurements of pulling directions of individual muscles. Consequently the
classes of eye movements found in the present study differ in some ways
from those found earlier. For example: abduction of ipsi eye corresponds
to horizontal class in the old description. This difference can be explained
by known influence of the flocculus on the horizontal canal pathway to
lateral and medial recti muscle.
A possible connection between the mossy and climbing fiber system at
precerebellar level is explored in the next chapter. Extracellular recordings

213
214 Electrophysiology of Movement

-in the decerebrate cat and awake Rhesus monkey were taken. In the cat,
it was found that the CF system can be modulated phasically and tonically
in response to passive movements of the extremity. Short and rapid
movements are most effective, but the authors conclude that CF is able
to transmit all parameters of the movement, especially at which position
the movement has been performed. The results for the monkey are similar
to the cat, but during volitional movement, motor commands seem to
have preference upon the 10 with respect to the sensory feedback produced
by the passive movement. There is a relationship between SS and CS. The
inverse relationship has not necessarily to be produced by the effect of
the CS on the SS. Both inverse and parallel relationships were observed.
The fourth chapter is a review of recent work on the CF's heterosynaptic
action on PC, and the authors present the hypothesis that the CF are most
critical for real time operations performed by the cerebellum during motor
execution. They demonstrate a specific short-term action of CF on SS
responses in passive paradigms as well as in multiple, sagittally organized
PC during perturbed locomotion. At least part of the action of the CF
system is related to its capacity to produce a short term enhancement of
the PC's SS responsiveness to the MF-GC-PP input.
The authors in the fifth chapter use single-unit recording and transient
multicell inactivation in conscious trained animals to answer questions
concerning the relation between the cerebellar circuit and function as seen
by the research scientist and neurologist. They test two hypotheses; (l)
within the cerebellum there are multiple maps (one for each major division
and thus deep nucleus) of the body and multiple modes of motor control;
(2) there is a common function across maps and modes of movement that
is consistent with the physiological experiments and the anatomical circuitry.
That function is the coordination of movement (as originally proposed by
Fluorens in 1824).
In the concluding chapter, recent work points to a need to reappraise
the role of the cerebellum in behavior (e.g., the cerebellum regulates both
voluntary and involuntary movements). Once this is recognized, we can
reevaluate why voluntary movements are so susceptible to cerebellar
damage. "One can conclude that the purpose ofthe cerebellum is to ensure
that motor nuclei respond with strength proportional to the response
evoking properties of the eliciting stimuli as determined by their associative
strength, intensity and biological significance" (see p. 332).
11
Cerebellar Control of Saccadic Eye Movements
in the Pigmented Rat
Piergiorgio Strata, Leonardo Chelazzi, Filippo Tempia,
Ferdinando Rossi, and Mirella Ghirardi
As for most body movements, the cerebellum
is not necessary for the generation of ocular
saccades, but it is important for their correct
performance. Lesion, stimulation, and recording
experiments show that different regions of the
cerebellum contribute to this performance (see
Carpenter, 1988; Leigh and Zee, 1983, for reviews).
By contrast, little is known about the importance
of the inferior olive in saccadic control. Such
knowledge is important for a better understanding
of the role of the cerebellum in motor perfor-
mance. By comparing the different effects of the
lesion of the inferior olive and of localized areas
of the cerebellum on saccadic activity, it is also
possible to contribute to a better understanding
of the role of the olivocerebellar system in cere-
bellar operation.
We present here a review of recent data ob-
tained in our laboratory aimed at describing the
main characteristics ofthe spontaneous saccades
in the pigmented rat in light and in darkness
(Chelazzi et aI., 1989). In addition, we describe
the effects of lesioning the inferior olive and two
cerebellar areas that are known to be involved in
the saccadic performance: lobules VI, VII, and
VIII of the dorsal vermis and the flocculus and
paraflocculus (Chelazzi et aI., 1990; Strata et aI.,
1990).
The use ofthis experimental paradigm has two
advantages. On one hand, in the rat it is possible
to destroy almost entirely and selectively the in-
ferior olive by means of an intraperitoneal injec-
tion of 3-acetylpyridine (Balaban, 1985; Desc1in
and Escubi, 1974). On the other hand, the saccadic
system provides a suitable model to study motor
behavior, since saccadic parameters can be easily
and accurately measured. In addition, the neural
mechanisms that contribute to their performance
are rather well known (Carpenter, 1988; Fuchs
et aI., 1985; Leigh and Zee, 1983).
The Saccadic System
Saccades are fast eye movements that shift the
sight line. They are generated by a short-lasting
high frequency discharge of the ocular moto-
neurons impinging on the extraocular muscles.
Such a burst has been defined as pulse of inner-
vation and is responsible for rotating the eyeball
to a new position in the orbit. This phasic moto-
neuron activity is followed by a prolonged tonic
discharge at a lower frequency, which has been
defined as step of innervation. The latter is respon-
sible for holding the eyeball in the new acquired
position against the viscoelastic forces that pull
the eye toward the center ofthe orbit (Fuchs et aI.,
1985; Robinson, 1975). An adequate matching
of the pulse and of the step is therefore necessary
for this purpose. It is maintained that the phasic
discharge is generated by a burst of synchronized
activity of neurons located in the paramedian
pontine reticular formation (pulse generator) and
transmitted to the ocular motoneurons to pro-
duce the saccadic gaze shift (GS) (see Fuchs et aI.,
1985, for references). It is assumed that the pulse
generator conveys its signals also to a neural
integrator that transforms the phasic activity in
a tonic one. Also, this tonic activity is then trans-
mitted to the motoneurons and is responsible
215
216
for holding the eye position reached by the GS
(Robinson, 1975, 1989).
The tonic activity tends to decrease with time
and, therefore, in the darkness the eye presents a
slow postsaccadic drift toward the central position
in the orbit. This drift is attributed to an intrinsic
leakiness of the neural integrator (Robinson,
1974). By measuring the time constant of this
slow drift it is possible to determine the character-
istics of the integrator. In the light, the eye position
reached by the GS is maintained because the step
is kept constant by the superposition of an opto-
kinetic reflex. In other words, the leakiness of the
neural integrator is compensated by the visual
information elicited by the image slip on the retina,
when the eye tends to deviate from the position
reached by the GS.
Saccades performed in the light in the absence
of a lesion often show a fast postsaccadic drift,
which usually is of less than 10. It may appear
either as a forward or backward postsaccadic
drift and is due to a nonperfect matching of the
pulse and the step of innervation (Bahill et aI.,
1975a; Chelazzi et aI., 1989; Optican and
Robinson, 1980). Significant alterations of such
a matching are reflected by a corresponding in-
crease of postsaccadic drift amplitude. Thus, the
measure of the drift, particularly in pathological
conditions, may be taken as an index of the under-
lying neural abnormalities.
Since most of the saccadic parameters in the
rat have not been described in the literature, we
have first characterized the spontaneous saccades
in seven intact rats (Chelazzi et aI., 1989). Control
values are reported below as a reference, together
with those obtained in the rats with different types
of lesion. In five rats the inferior olive was des-
troyed by means of 3-acetylpyridine (68-75 mg/
kg IP) (Balaban, 1985; Desclin and Escubi, 1974).
In two other groups of five rats each, the floccu-
lus-paraflocculus or the posterior vermis (lobuli
VI, VII, and VIII) was ablated by suction. Record-
ing of spontaneous ocular movements has been
performed in the head-restrained condition 4 weeks
to 6 months after the inferior olive lesion and 1
week to 6 months after cerebellar lesions. Eye
position has been recorded by means of a phase-
detection, search coil system (Kasper et aI.,
1987).
Piergiorgio Strata et al.
Effect of Inferior Olive Lesion
In the intact rat, the slow postsaccadic drift re-
corded in the dark (Fig. 11.1 E) has an exponential-
like time course that is better defined for more
eccentric saccades. To calculate the time constant
of such a drift in the dark, we followed two dif-
ferent methods: a) We averaged the time constant
values determined on single randomly selected
saccades of different amplitudes and eccentricities.
In this case the time constant was 1567 ms (± 829
SD); b) We averaged 20 spontaneous saccades
starting from the midline and ending at 10° of
eccentricity. With this method the value was
4228 ms. These values are considerably shorter
than that of more than 20 s determined in higher
mammals including man and monkey (Becker
and Klein, 1973; Robinson, 1974; Zee et aI., 1981).
This fact indicates that in the dark the rat has a
poor ability to hold the eye in a lateral position.
Following inferior olive lesion, the small, fast,
postsaccadic drift becomes pronounced and is
always in the backward direction. In the dark it
is followed by the slow drift (Fig. 11.1 F) (Hess
et aI., 1988; Tempia et aI., 1989). The time constant
of the slow drift calculated with the first method
showed a significantly shorter value of 561.8
(± 211.4 SD) (Student's t test, p < .001). Also the
value calculated with the second method was
remarkably reduced, to 895 ms. From these data
we may conclude that the integrity of the inferior
olive is essential to maintain the time constant
of the neural integrator within its physiological
range.
Saccades recorded in the light from intact rats
usually show a good postsaccadic stability of the
gaze (Fig. 11.1 A). Fast exponential postsaccadic
drifts, when present, are of a very small amplitude
and directed either onward or backward. Their
time constant had an average value of 113 ms
(± 33.6 SD). Following inferior olive lesion, on
the contrary, every saccade presented a very large
fast postsaccadic drift, always in the backward
direction (Fig. 11.1 B), as previously reported at a
qualitative level by Hess et ai. (1988). Its time
constant was 101.3 ms (± 40.7 SD) and it was not
significantly different from that of the intact rat.
The fact that following inferior olive lesion the
fast postsaccadic drift was in the backward direc-
11. Cerebellar Control of Saccadic Eye Movements 217

LIGHT DARKNESS
A E

Control

10 lesion

C G

V lesion

D H

F-PF lesion
_t----

Figure 11.1. Eye position records in the light (left side)
and in the dark (right side) from a normal rat (A and
E), from a rat with inferior olive lesion (B and F), and
from animals with a lesion of the dorsal cerebellar
~200
5s
vermis (C and G) or of the flocculus-paraflocculus (D
and H). In F and H, the initial fast postsaccadic drift
and the following slow centripetal drift are easily re-
cognizable.

tion indicates that there is a pulse-step mismatch,
where the pulse is larger relative to the step. The
increased leakiness of the neural integrator, by
shortening the time constant of the slow postsac-
cadic drift, may also contribute to the amplitude
of the fast drift, particularly for centrifugal saccades
with large eccentricities. However, such a mech-
anism cannot account for the entire phenomenon,
since a large postsaccadic drift was also present
when the saccades were centripetally directed.
In order to ascertain whether the mismatch is
due to a larger pulse or to a smaller step, we have
218
compared the average amplitude ofthe GS of the
intact rats with that of the rats with lesion of the
inferior olive, both in the light and in the dark.
In the intact rats the average values were, res-
pectively, 13.2° (± 2.2 SD) and 9.20 (± 2.0 SD).
In the animals with the lesion of the inferior olive
such values were, respectively, 14.4° (± 2.5 SD)
and 11.8° (± 3.3 SD). Statistical analysis revealed
no significant difference between the two groups.
The gaze position held in the light relative to
the position before the saccade may be defined as
gaze-sustained deviation (GSD). In the intact rats
the average value was 13.4° (± 1.6SD) and it was
significantly different (p < .001) from that of 8.l °
(± 1.9 SD) measured after the lesion of the inferior
olive (Fig. 11.2). The steady position after a sac-
cade, which is maintained by visual feedback sig-
nals, depends on the step amplitude and on the
leakage of the neural integrator. When the sac-
cades end near the midline, however, such a posi-
tion does not depend on the leakage. Since these
saccades also presented a large postsaccadic drift
in the backward direction, the step amplitude
must be smaller relative to the pulse amplitude.
'""'
till
Q) 20 I:=:J GSD
"0
'-'
I.Ll
0
:J 15
t:
....J
Q..
10
~
z
<C
I.Ll 5
:E
ROL
PARAF
I F RJOR
IV
Figure 11.2. Mean GS and GSD amplitudes of sac-
cades recorded in the light from control rats (control)
and after lesion of the inferior olive, of the flocculus-
paraflocculus, and of the posterior vermis. The GSD
Piergiorgio Strata et al.
Therefore, the integrity of the inferior olive is
important for the pulse-step matching of the rat.
The next step was to study the quantitative
relationship between the pulse and the step of
innervation in the rats with lesion of the inferior
olive for saccades of different amplitudes and
eccentricities. The GS amplitude represents fairly
well the pulse, whereas more difficult is the deter-
mination of the amplitude of the step, which is
represented by the initial eye position value ofthe
slow component of the postsaccadic drift in the
dark. However, such a position is difficult to
determine in the rat with lesion of the inferior
olive because of the presence of the fast post-
saccadic drift of large amplitude, which is well
evident for more than 200 ms. Therefore, in order
to ascertain the step amplitude we have used
different approaches.
1. In the first experiment, we averaged 10 sac-
cades recorded in the dark starting from the mid-
line and ending at 10° of eccentricity. Since the
time constant of the fast postsaccadic drift is
about lOOms, its contribution to the postsaccadic
drift is over after 400ms. We then extrapolated
~ GS
FLO
POSTERI R
V RMI
amplitude is significantly reduced after lesion of the
inferior olive and of the flocculus- paraflocculus (p <
.001). (Reprinted with permission of Oxford University
Press from Chelazzi et aI., 1990.)
11. Cerebellar Control of Saccadic Eye Movements 219

,/

/
25 /.
•
/ •

./
...
/
.
::: ..... .
Figure 11.3. Relationship between the GSD /'
15 •• •
....:: :......
:. .. .
and the saccadic GS for one rat with lesion /'
of the inferior olive. The dashed line repre-
sents the ideal relationship with an angular
coefficient of 1; sacca des of normal rats, not
/.~:
~...
••
/
..• • ••
. •

I •. . ••
displayed, are well fitted by this line. The 5 /.: .::-:
solid regression line which is the best fitting •••
•
for this animal, crosses the abscissa at
a positive value and has a slope of 0.72.
(Reprinted with permission of Oxford 5 15 25
University Press from Strata et aI., 1990.) G (deg

the entire time course of the slow postsaccadic
drift from that part of the postsaccadic drift start-
ing at 400 ms. From this extrapolation, we found
that the eye position from which the slow post-
saccadic drift would start at the end of the GS
was 7.6°. Therefore, we may conclude that for
these saccades of 10° of amplitude and eccentri-
city, the gain of the pulse to step transformation
is 0.76 and therefore the contribution of the mis-
match to the postsaccadic drift is 2.4°. We also
averaged similar saccades performed in the light
and the GSD was 5.2°, with a consequent ampli-
tude of the postsaccadic drift of 4.8°. Therefore,
the contribution of the leakage of the neural inte-
grator to the postsaccadic drift is also 2.4°.
2. A second experiment was aimed at deter-
mining the gain of the pulse to step transforma-
tion and the relative contribution of the leakage
of the neural integrator to the postsaccadic drift
in the light as a function of the amplitude and the
eccentricity of the saccades.
Figure 11.3 illustrates the relationship between
the GSD and the GS for saccades of different
amplitudes and eccentricities in one rat. Their
ratio indicates the gain of the postsaccadic posi-
tion in the light (Strata et ai., 1990). In the intact
rats, this gain is near 1, since the postsaccadic
drift amplitude is very small; it is represented by
the interrupted line having an angular coefficient
of 1 and crossing the zero of the cartesian coordi-
nates. Following inferior olive lesion, the regres-
sion line (continuous line) presented an angular
coefficient of 0.72 and crossed the abscissa at a
positive value. These results show that a linear
correlation between these two parameters is main-
tained after the lesion. However, since the more
eccentric saccades are also those with a higher
amplitude, it is not clear how much of the post-
saccadic drift depends on the amplitude and how
much on the eccentricity.
Therefore, we studied a) the relationship be-
tween the postsaccadic drift and the saccadic
amplitude for classes of saccades with similar
eccentricities, and b) the relationship between the
postsaccadic drift and the eccentricity for classes
of saccades of similar amplitudes.
In the first case there was a linear relationship
between the drift amplitude and the GS amplitude
(Fig. 11.4A-E). Since in each plot the eccentricity
is constant, the average slope of all the regression
lines represents how much the postsaccadic drift
increases with the increase of the GS amplitude.
Such a slope had an average value of 0.21 and
therefore the gain of the pulse to step transfor-
mation was 0.79. This value is almost the same
as that obtained in the previous experiment.
Figure 11.4A-E shows another interesting
result. When the saccadic amplitude tends to
zero, the component of the postsaccadic drift due
to the pulse-step mismatch also tends to zero.
Therefore, the Y intercepts provide an indication
ofthe amount ofthe postsaccadic drift that is due
to the leakage of the neural integrator. The values
of the Y intercepts increased linearly with the
 220 Piergiorgio Strata et aI.

A B C
ECC 1-3 ECC 4-6 ECC 7-9
8: .6 b: .26 r: .93 8: 1.78 b: .2 r: .95 8: 1.89 b: .21 r: .87
15 15 15

10 10 10

5 5 5

0 0
10 20 30 0 10 20 30 0 10 20 30

0 E F
ECC 10-12 ECC 13-15
r: .77 3.07 b: .2 r: .79 6
8: 3.16 b: .16 8:

15 .- 5
15
~

10 -°
.-

c
(/)
c:J
4

-
(/) 10 °0 3 " "
a..
III
C
2
5 5 (/)
a.. 1
"
0 0 0
0 10 20 30 0 10 20 30 0 5 10 15 20
GS (0) Eccentricity (0)
Figure 11.4 A-E: Postsaccadic drift (PSD) in the light the Y intercept "a," and the correlation coefficient "r,"
as a function of GS amplitude for different classes of together with the eccentricity range, are reported on
saccades with similar eccentricities after inferior olive each plot. Note the increase of the Y intercept (extra-
lesion. Each class includes all the saccades within 3° of polated PSD for hypothetical saccades of 0° of GS)
eccentricity (ECC). The best fitting regression lines along with the increase of eccentricity as plotted in F.
have been calculated for the plotted mean drift ampli- (Reprinted with permission of Oxford University Press
tude values. Each triangle represents the mean (± 1 SD) from Strata et aI., 1990.)
of at least three saccades. The angular coefficient "b,"

increase of the eccentricity, with a slope of 0.21
(Fig. 11.4F). This means that the amount of post-
saccadic drift due to the leakage of the neural
integrator increases 0.21 for each degree of eccen-
0
tricity. This value also coincides with that ob-
tained in the previous experiment.
Figure 11.5A-G shows that there was a linear
correlation between the postsaccadic drift ampli-
tion of the mismatch to the postsaccadic drift is
the same for all the saccades of the same class.
Thus, the increase of the postsaccadic drift with
the increase of the eccentricity is due to the leakage
of the neural integrator. Such a contribution was
also linearly related to the eccentricity with an
average slope of 0.17. This means that for each
degree of eccentricity, there is 0.17 of drift due 0

tude and the saccadic eccentricity for classes of to the leakage.

saccades of defined amplitudes. Since in each plot From Figure 11.5 it is clear that the Y intercepts
the saccadic amplitude is constant, the contribu- provide an indication of the value of the post-
 11. Cerebellar Control of Saccadic Eye Movements 221

A B c
GS 1-3 GS 4-6 GS 7-9
8:.5 b: .34 r:.9 8: 1.93 b: .11 r: .74 8: 2.41 b: .18 r: .97
15 15 15

10 10 10

5 5

o~----~---------- o~----~----------~
o 10 20 30 o 10 20 30

D E F
GS 10-12 GS 13-15 GS 16-18
8: 3.58 b: .14 r: .78 8: 4.18 b: .08 r:.4 8: 3.88 b: .21 r: .72
15 15 15

10 10 10

5 5

o~----~----~--~ o~----~----------~ O+-----~----~----

o 10 20 30 o 10 20 30 o 10 20 30

G H

---
GS 19-21
6

-...
8: 5.18 b: .14 r: .5
°

/
15 >- 5

-
'u
4

---°
c: A/
10 CD
()
()
W
3 /
-
Q
UJ 'b 2
a.. 5
as
Q 1
UJ
0 a.. o~--~----~--~--~
0 10 20 30 o 5 15 20
Eccentricity (0)

Figure IISA-G: Postsaccadic drift (PSD) amplitude
in the light as a function of eccentricity for different
classes of saccades with similar amplitudes after in-
ferior olive lesion. Each class includes all the saccades
regression lines have the angular coefficient "b," the Y
intercept "a," and the correlation coefficient "r" re-
ported above each graph, together with the GS range.
H: Extrapolated PSD values of saccades of 0 of eccen-
0

within 3 ofGS. Each triangle represents the mean drift

0
tricity as a function of GS. (Reprinted with permission
amplitude (± 1 SD) of at least three saccades. The of Oxford University Press from Strata et aI., 1990.)
222
saccadic drift due to the mismatch. Since the
values of the Y intercepts increased linearly with
the increase of the amplitude with a slope of 0.24
(Fig. l1.5H), we may conclude that the contribu-
tion of the pulse-step mismatch to the postsac-
cadic drift is 0.24° for each degree of amplitude.
In other words, the gain ofthe pulse to step trans-
formation is 0.76 at all saccadic amplitudes. This
value also fits with the values calculated in the
two previous experiments.
The above series of experiments shows that
following inferior olive lesion the saccades per-
formed by the rat have a post saccadic drift with
an amplitude that depends both on a reduced gain
(about 0.77) of the pulse to step transformation
and on an increased leakiness of the neural inte-
grator. The latter contributes linearly, with a slope
of 0.21 to 0.24.
Finally, we studied the peak velocity and the
duration of saccades as a function of the GS am-
plitude. Such functions, called the main sequence
(Bahill et ai., 1975b), were not affected by the
inferior olive lesion either in the light or the dark.
Effect of the Lesion of the
Posterior Vermis and of the
Flocculus-Paraflocculus
Following lesion of the posterior vermis, there
were no significant changes in the parameters that
we investigated (Fig. 11.1 C, G). By contrast, fol-
lowing lesion of the flocculus-paraflocculus
(Fig. I1.1D, H), there were remarkable abnorma-
lities in the spontaneous saccades, which are strik-
ingly similar to those observed after inferior olive
lesion.
In fact, in the rats with lesion of the flocculus-
paraflocculus the spontaneous saccades performed
in the light showed a fast post saccadic drift in the
backward direction (Fig. 11.1 D). Following this
type of lesion, the average amplitude of the GS
in the light (12S, ± 4.0 SD) is not significantly
different from that obtained in the other three
experimental conditions, whereas the amplitude
of the GSD (7.4°, ± 2.9 SD) was significantly re-
duced with respect to that of the intact rats and
of the rats with dorsal vermis lesion (p < .001 in
both cases; Fig. 11.2). However, the GSD was not
significantly different from that calculated in the
Piergiorgio Strata et al.
rats with lesion of the inferior olive. Also the
amplitudes of the GS in the dark were not signifi-
cantly different in the four groups of rats.
As far as the time constant of the slow postsac-
cadic drift is concerned, its value in the animals
with vermal lesions was 1787 ms (± 1246 SD)
when determined on randomly selected saccades,
whereas when calculated on 20 averaged saccades
of 10° of amplitude and eccentricity, its value was
4188 ms. These values are not significantly dif-
ferent from those of the intact rats. In contrast,
following the lesion to the flocculus-paraflocculus
the value obtained with the first procedure was
675.7ms (±208.8SD) and significantly reduced
(p <.001). Also, the value obtained with the second
method was remarkably reduced, to 888 ms.
However, there was no significant difference be-
tween the time constant measured in the rats with
lesion of the inferior olive and of the flocculus-
paraflocculus.
In the rats with flocculus-paraflocculus lesion
we also attempted to define the relative contri-
bution of the pulse-step mismatch and of the
leakage of the neural integrator to the postsac-
cadic drift. To this aim, we performed a similar
series of experiments; the results may be sum-
marized as follows: The gain of the pulse to step
transformation was 0.79 at all saccadic ampli-
tudes. In addition, the contribution ofthe leakage
of the neural integrator to the postsaccadic drift
was 0.13 0 to 0.23° for each degree of eccentricity.
These findings are also very similar to those ob-
tained following inferior olive lesion.
Finally, we found no significant alterations in
the saccadic main sequence both in the light and
in the dark following flocculus-paraflocculus
lesion.
Discussion
Our experiments show, first of all, that the intact
head-restrained pigmented rat has a remarkable
spontaneous saccadic activity both in the light
and in the dark. The saccades are characterized
by relatively high peak velocities and large ampli-
tudes, whereas the gaze-holding ability in the
dark is rather poor (Chelazzi et ai., 1989).
Concerning the cerebellar influence on the sac-
cadic performance, we have shown that the lesion
of the vermis does not induce any significant
11. Cerebellar Control of Saccadic Eye Movements
alteration. That saccadic peak velocity and dura-
tion are not affected by this lesion is not unexpec-
ted since it has already been shown in the monkey
that these parameters are unchanged even after
total cerebellectomy (Optican and Robinson,
1980) or cooling of the medial cerebellar nuclei
(Vilis and Hore, 1981). However, a saccadic dys-
metria has been described. When in the monkey
the intracerebellar nuclei are spared, the dysmetria
depends on the saccadic direction (Ritchie, 1976),
whereas when the lesion includes the nuclei, the
monkey shows a saccadic hypermetria (Optican
and Robinson, 1980). The lack of a significant
variation in the average amplitude of the GS in
our experiments may be due to different experi-
mental conditions, since we have analyzed sponta-
neous saccades made to explore the environment,
whereas in the quoted studies target-directed sac-
cades were recorded from trained monkeys. How-
ever, it is also possible that in the rat accurate
saccades are not important and therefore they
are not corrected by the cerebellum. It is known
that the rat has no fovea or any other high acuity
retinal area.
Our results obtained following flocculus-
paraflocculus lesion are in part similar to those
reported in the monkey, where a considerable
reduction of the time constant of the neural inte-
grator has been reported (Zee et aI., 1981). How-
ever, they are different as far as the postsaccadic
drift is concerned. In the monkey, this drift is of
a small amplitude, up to a maximum of 15% of
the GS amplitude (Zee et aI., 1981), whereas in
our experiments it averages around 40% of the
GS. In addition, in the monkey the drift is both
in the onward and in the backward direction with
a predominance of the former one, whereas in our
experiments it is always in the backward direction.
A possible explanation for this discrepancy may
be that in monkey experiments the extent of the
flocculus-paraflocculus lesion was variable in dif-
ferent animals and not complete, whereas in our
experiments in the rats it was possible to ablate
this cerebellar area totally and consistently.
Another major feature of our experiments is that
in the rat the postsaccadic drift in the light is due
in part to the reduced gain of the pulse to step
transformation and in part to the increased leak-
age of the neural integrator, the relative contri-
bution depending on the saccadic eccentricity
223
and on the GS amplitude. It is likely that in the
monkey the leakage contribution is rather modest,
since the time constant of the neural integrator
is relatively longer with respect to the rat, even
after the flocculus-paraflocculus lesion. This fact,
however, does not explain a gain higher than 1
of the postsaccadic position in the monkey.
From our experiments we may draw the con-
clusion that the flocculus-paraflocculus area is
responsible for enhancing the time constant of
the neural integrator and the gain of the pulse to
step transformation. Therefore, at least in the rat,
the flocculus-paraflocculus region is important
to improve the poor gaze-holding capabilities of
the oculomotor system.
Our experiments have also shown that the inte-
grity of the inferior olive is essential for main-
taining the leakiness of the neural integrator
within the physiological range and the gain ofthe
pulse to step transformation at its proper value.
The effects of the inferior olive lesion are also
similar to those of the flocculus-paraflocculus
lesion from a quantitative point of view. There-
fore, it is likely that the inferior olive controls the
ocular motility through the action exerted on the
flocculo-parafloccular Purkinje cells. The simi-
larity between the results of inferior olive and
flocculus-paraflocculus lesions is unexpected and
is worth further discussion.
One simple explanation would be to assume
that following inferior olive lesion, the inhibition
exerted by the Purkinje cells on their target cells
is completely abolished. An almost complete loss
of inhibitory action of these cells has been reported
(Ito et aI., 1979) and has been attributed to a loss
of a trophic factor released by the climbing fibers
(Ito, 1984) or to an irreversible damage of the
Purkinje cells (Rossi et aI., 1987) due to the pro-
longed hyperactivity that follows the inferior
olive lesion for more than 1 month (Benedetti
et aI., 1984; Montarolo et aI., 1982). Such a loss,
however, is only partial (Karachot et aI., 1987;
Montarolo et aI., 1981) and it seems unlikely that
such a partial loss is equivalent to a total Purkinje
cell removal.
A second possibility is that the action of the
inferior olive is not mediated by the olivocere-
bellar fibers, but by their collaterals to the brain
stem. Such collaterals would degenerate as a con-
sequence of the retrograde degeneration of the
224
olivary cells, which follows flocculus-parafloccu-
Ius removal. This hypothesis should be tested by
lesioning the Purkinje cells by means of kainic
acid, which prevents the inferior olive degener-
ation (Ito et aI., 1980) and allows the survival of
the climbing fiber terminal arborizations (Rossi
et aI., 1991).
Previous experiments have demonstrated that
an acute reversible inactivation of this nucleus
leads to increased firing frequency ofthe Purkinje
cells (Montarolo et al., 1982) and decreased firing
of their target neurons in the intracerebellar and
vestibular nuclei (Benedetti et aI., 1983; De'Sperati
et aI., 1985). On the basis of these data, it has been
proposed that the inferior olive is able to change
the gain of any system under cerebellar control
(Strata and Montarolo, 1982). Evidence for this
assumption has been provided (De'Sperati et aI.,
1985; see Strata, 1987; Strata et aI., 1989). One
month after the inferior olive lesion, however,
the firing frequency of the Purkinje cells and of
their target neurons is again at the control level
(Benedetti et aI., 1984; Lopiano and Savio, 1986).
Since in our experiments the ocular movements
have been recorded at least 1 month after the
olivary lesion, the ocular deficits we have de-
scribed are likely not due to such a change of
spontaneous firing. Instead, they may be due to
a direct active involvement of the inferior olive
in controlling the leakiness of the neural inte-
grator and the gain of the pulse to step transfor-
mation.
We suggest that visual information carried to
the flocculus-paraflocculus through the dorsal
cap of the inferior olive plays a specific role in
contributing to the maintenance of proper eye
position after the GS and therefore stabilizes the
gaze. Such a role may be carried out in two ways.
First, the inferior olive may exert an on-line effect
on the gaze-holding ability, and second, it may
be essential to maintain the system in a calibrated
condition. In fact, the flocculus seems essential
fQr the adaptive control of the postsaccadic drift
in primates (Optican et aI., 1986). In addition,
recent experiments have shown that a consist-
ent mismatch appears when vision is suppressed
(Viirre et aI., 1987) or when a visual slip is intro-
duced artificially at the end of a saccade (Kapoula
et aI., 1989; Optican and Miles 1985).
Piergiorgio Strata et al.
Acknowledgments. This work has been supported
by grants from C.N.R. and M.P.1.. We are in-
debted to Mr. M. Orlandini and Ms. G. Milano
for their assistance.
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12
A Possible Connection Between the
Mossy and Climbing Fiber Systems
at Precere bellar Level
Francisco J. Rubia
This chapter addresses the possibility that there is
a precerebellar connection between the two main
inputs to the cerebellum, the mossy (MF) and the
climbing fiber (CF) system. The idea that both
afferent systems could be interconnected before
their entrance into the cerebellum came to us as
we analyzed the results of extracellular recordings
of Purkinje cells (PC), especially the behavior of
their two different spikes, the complex spike (CS)
and the simple spike (SS), and their relationship
in two different preparations: the decerebrate cat
and the awake Rhesus monkey.
Our intent in recording PCs from the decere-
brate cat was to analyze the capability of the CF
system to signal peripheral events. Several authors
have reported that the CF system is able to trans-
mit to the cerebellum information from the pe-
riphery: so did Eccles et al. (1972a-d) and Leicht
et al. (1973), with regard to activation of cuta-
neous, muscle, or mechanoreceptors of the fore-
limb and hindlimb. Similar results were obtained
by Ishikawa et al. (1972a, b) with muscle stretch
and by Maekawa and Simpson (1972) and Simpson
and Alley (1974) with teleceptive stimulation.
Rushmer et al. (1976) also reported that the CF
system was activated with very small displace-
ments of the forelimb. These last authors recorded
the CS responses of the PC and observed that
they appeared to be independent of the quality,
amplitude, and direction ofthe movement. Thus,
they concluded that the CF system acted as an
"event detector," signaling only the touching of
the limb to the ground or its lifting during loco-
motion.
In our own studies, however, we have found
226
that the CF system, as revealed by the recording
of the CS of the PC during passive displacement
of the forelimb, is able to transmit very precise
information about all the parameters of the
movement.
With respect to the question of whether the CF
system is conveying to the cerebellum more tonic
or phasic information, there are also differences
in the literature. Most authors agree that the CF
system is more likely to transmit phasic informa-
tion. However, there are reports of the system's
capability to signal tonic events also. For example,
Ferin et al. (1971) reported a tonic increase of the
CS activity of the PC during or after the end of
caloric and galvanic stimulation of the labyrinth,
and in the frog cerebellum Llimls et al. (1971)
observed tonic increase in the CS responses during
horizontal angular acceleration. Tonic modula-
tion of the CS activity was also observed during
different sleep states by Marchesi and Strata
(1970), Mano (1970), and Hobson and McCarley
(1972). Marini et al. (1976) showed tonic modula-
tion of the CS in the PCs of the nodulus in
response to rotation of the animal around its
longitudinal axis.
Results Obtained
in the Decerebrate Cat
Using the decerebrate cat, we were able to show
that the CF system can be modulated phasically
and tonically in response to passive movements
of the extremity.
The experimental procedure has been described
elsewhere (Rubia and Kolb, 1978). The passive
12. Mossy and Climbing Fiber Systems 227

movement was elicited by a DC motor attached
to the forelimb, so that the foot pad moved around
the wrist joint, the rest of the arm being fixed. The
movement followed in most cases a ramp function
beginning at a starting position and proceeding
to an end position with constant velocity. The
movement was reproduced exactly in order to
average a sufficient number of sweeps and in this
way collect as many CS responses as possible
from the Pc.
The extracellular recording of the PC was per-
formed only within the CI zone of lobule V, the
representation area of the forelimb in the anterior
lobe of the cerebellum. The two spikes of the PC
were reliably separated with an electronic device
and peristimulus time histograms (PSTH) were
constructed.
An example of the capability of the CF system
to transmit peripheral information is shown in
Figure 12.1. In this figure (Fig. 12.1B), the CS of

c
::J J \w=t.oo/s

A
]L..::.: +20
-IS

z -IS
'PH iii -10
"-
o· VI
~
Z
:l
0
u
'PL .10
+ 5

B
i i
o 2.5 55
-20·
] ~w=t.oo/S D
-20. J ~
COUNTS/BIN

~] n=80

i
o 25 55
Figure 12.1. Response of a PC to passive movement
of the cat's forepaw. A: A DC motor-driven device
moves the forepaw around the wrist joint from a low
(L) to a high (H) position and back. 0° is the horizontal
plane. B: response of the PC with the CS is illustrated.
A PSTH shows the CS response to a ramp movement
(from - 20° to + 20°) with constant velocity (40°Is).
o -10 -20
'P /degl
The number of sweeps is n = 80. C: The response of
another PC to displacements of 5° at different starting
positions. D: The mean probability of CS discharge
with 95% confidence limits for upper (H) or lower (L)
positions. (From Kolb and Rubia, 1980, with permis-
sion.)
228 Francisco 1. Rubia

o
-..t
II
C

o
If)
II
'IS o
NIS/SINnO:J
. III
If)
:
O·

_l
~ .0 : • J _ . OJ 00 O.'J o J LI1
II N
C

-"~.::1 -" ..... J-. "oJ ".:.. J ·0-: J

.. o 0

o
 12. Mossy and Climbing Fiber Systems 229

the rec(i)rded PC signals the end position ( + 20°,
0° being the horizontal plane) with a tonic increase
in the firing frequency, the positive acceleration
of the upward movement, the velocity, and the
direction of the movement. The mean frequency
of the tonic discharge increased about five times
when the upper position was changed from + 5°
to + 20°, In Figure 12.1 C we can see that the
response of another PC during the upward move-
ment is different depending on the position at
which the movement has been performed, Al-
though the displacement (5°) and the velocity
(40° Is) are the same, the best responses are ob-
tained at the highest positions, Thus, besides the
signalization of the movement parameters, the
CF system can inform the cerebellum about the
limb position at which the movement has oc-
curred.
This observation can be seen better in Figure
12.2. Here, another PC responded with the CS to
the same movement performed at different limb
positions. At the highest positions, the discharge
frequency is also higher, and at lower positions
the dispersion of the CS responses, as shown in
the dot raster in Figure 12.2A, is greater,
Therefore, the static parameter position can
be conveyed to the cerebellum by the CF system
in two ways: directly by a tonic increase of the
discharge frequency and indirectly in connec-
tion with a dynamic parameter, informing the

A B c
O·f~ __ /~/
______ . w =40 /s
-20. --1/
-40. --' n=45
-----
------- 0

n = 25
'(t: CD
rl.
"l
~~
'.'
IS!
10
5
0·
' 10·
t.
I,,'
oJ
o -
• UP
~;~ ® o DOWN
:~,
" 0· 0,3
',\1, z
,I):':
.~ ',1 ..
......
iii
"'-
Vl
I-
! ·20·
0.2
.
O·T
Z
@

~j
, ::::>

f. ...... +.. .
0
::., ' U 0,1
.
. . '". ..........
"

.......... ·30· o
10 20 30 40
® 6 If> (degl

~
. :~' . ,
.,1.
,'., .
..,
.,' ..
..
.,
'.~
~
, , '0·
0 2,5 55
Figure 12.3. CS discharge of a PC in response to dis- discharge during upward and downward movement
placements of the forepaw of different amplitudes. plotted agaihst the range covered by the movement.
A: Dot raster. B: PSTH. C: Mean probability ofthe CS (From Kolb and Rubia, 1980, with permission.)

~----------------------------------------------------------------------
Figure 12.2. CS discharge of a PC in response to a
displacement of 5° at different paw positions. A: Each
of the dots represents a CS discharge. B: PSTH of the
CS discharge. C: Stack plot of the PSTH. D: Mean
probability of the CS discharge during upward (mu)
and downward (md) movements. The shaded zones
represent the 95% confidence limits (vertical direction)
and movement amplitude (horizontal direction). (From
Kolb and Rubia, 1980, with permission.)
230 Francisco J. Rubia

c;erebellum at which position a certain movement
has been performed.
The amplitude of the movement (displacement)
can also be transmitted to the cerebellum by the
CF system. In Figure 12.3, a PC is responding
with the CS to a movement of constant velocity
(400/s) but different amplitude. We observe that
the shorter the movement, the probability of the
discharge is higher and the response more precise
(Fig. 12.3A, B, specimens 1 and 2). It is interesting
to note that in all cases the movement covered
the displacement in specimen 1 and 2. From this
type of experiment we concluded that the shorter
the displacement, the better the response of the
the amplitude of the movement, the CS ofthe PC
responded changing also the discharge frequency.
This can be observed in Figure 12.4. By increasing
velocity, the mean probability of the unit dis-
charge also increases. The conclusion from these
results is that the higher the velocity, the higher
also the probability of the discharge of the PC
with its CS.
Summarizing the results shown in the last two
figures, we can say that short and rapid move-
ments are the most adequate for transmission
through the CF system. If we observe the PSTH
shown in Figure 12.4B during the movements
with the lowest velocities (12 and 20 /s), there is
0 0

PC with its CS. an increase in the firing rate of the CS at the

By changing the dynamic parameters we also beginning and at the end of the movement. This
obtained consistent changes in the responses of can mean that the cell is responding to both
the PC with its CS. For example, if we changed acceleration signals (positive and negative accel-
the velocity from 12° to 80° Is, by keeping constant eration) of the movement. If this were the case,

A B c

t oO \. "t, •
n: 60 w Ideg/sl
. 'i, :.'1 .
. ....:.:\ " .
'.
,
-:"'.\.;....
~12
' - - --" ''--- --'

.:- P1o.s1 !%1

.. 'oO •

,' , .: r
.) . ,-
~20 'L,- 10'
0.4
'H··
~ ';'i
.' ·r ..
t

...
10
'----' '---' • : mu •
..
o mel
'i: .
· t.:
t,...I; 40
0.2
L.....I L......J

60
'-' L.J
10 20 40 60 80
w Ideg/sl
80

I
o
I I
2.5 5s o 2.5 5s
Figure 12.4. CS discharge of a PC in response to dis- during upward and downward movement plotted against
placements of 20° with different velocities. A: Dot raster. velocity. (From Kolb and Rubia, 1980, with permission.)
B: PSTH. C: Mean probability of the CS discharge
12. Mossy and Climbing Fiber Systems 231

obviously during short and rapid movements
both acceleration signals can be better summed,
producing a higher CS discharge of the Pc.
To test this hypothesis, we used a movement
with a constant amplitude of So and a logarithmic
increase and decrease of the frequency (Fig. 12.S).
By calculating the Pearson correlation coefficient
between the PSTH (Fig. 12.SA) and the move-
ment function, its first, second, and third deri-
vatives and certain sections of the movement
(Fig. 12.SB), we concluded that the best correla-
tion obtained was between the averaged discharge
rate of the CS and the negative acceleration. The
continuous line in the histogram of Figure 12.5A
represents the absolute value ofthe negative accel-
eration. It can be observed that it fits nicely with
the response of the PC with its CS. Thus, the ac-
celeration signal of the movement can be trans-
mitted well to the cerebellum via the CF system.
From these types of experiments we can con-
clude that the CF system is able to transmit to
the cerebellum all the parameters of the move-
ment and especially also at which position the
movement has been performed. The parameter
acceleration is one of the best transmitted by this
system and, therefore, small and rapid movements
produce the best response in the CS of the Pc.
Results Obtained
in the Awake Monkey
It can be argued that these results could be ob-
tained only because the preparation was the de-
cerebrate cat and not the intact animal. However,

A B

IS' ]
fIt) r
10°
1200 c.p (t) 0.31
pos. half wave of c.p(I) 0.32
30
n =40 neg. half wave of 'P(t) 0.25
N
z III
~ (I) - 0,06
CD "-
01
"-
Vl -0
Qj + tP (I) 0,10
t- - tp (t) - 0,20
z
::l
0
u If> It) - 0,58
0 0 + If> (I) - 0,08
,,
"' - If> (I) - 0,80
, , ~, ,, :
" ,, ,, '" ,, if> (t) - 0.D1
, ,, ,, :
, ,
.
•.", , " ,,
+ cp (t )
- if> (I)
0,22
- 0,20
.~ ~ ~

, ,
o 5
Figure 12:5. CS discharge of a PC during a sinusoidal
movement of 5° and logarithmically increasing and
decreasing frequency. A: PSTH and dot raster of the
discharge. The solid line in the PSTH represents the
absolute value of negative acceleration. B: Pearson
correlation coefficients (r) evaluated between the PSTH
in A and different functions (f(t)): qJ (t), sinusoidal move-
ment function; cp (t), velocity; ip (t), acceleration; (jJ (t),
third derivative of (t). The best correlation is found
between the averaged discharge rate and the negative
acceleration-ip (t). (From Kolb and Rubia, 1980, with
permission.)
232
s~milar results have been obtained in the awake
monkey using a similar movement around the
wrist joint. Furthermore, this preparation allowed
us to compare between the movement passively
produced and a similar movement actively in-
duced by the monkey.
The monkeys were trained to move a lever by
flexing and extending the hand in order to pursue
a light signal. The coincidence of a visual target and
the light signal connected to the lever triggered
the reward. Additionally, the lever could also be
connected to a DC motor for the performance of
similar passive flexions and extensions.
The passive movements produced similar res-
ponses in the CS of the PC as those observed in
the decerebrate animal. The acceleration signal
was also best transmitted to the cerebellum via
the CF system. In Figure 12.6 a PC is responding
with its CS to passive movements with different
velocities and accelerations. Figure 12.6B shows
the relationship between the discharge rate ofthe
CS and the acceleration values. Here again, the
higher the acceleration, the higher also the dis-
charge rate of the cell with CS.
A B
n=38
10" ]
0"'5
60"'5 ]
']
z CS
iii
.....
(/)
~
Z
:l
0
U
-300 0 +500ms
Figure 12.6. CS response ofa Rhesus monkey's PC to
passive movements ofthe hand ofthree different mean
accelerations. A: Averaged position curve (P) of 38
similar movements. Velocity curves (V), whereby the
downward slopes provide a measure for the accelera-
tion: 298°/s (left), 661°/s (middle), and 845°/s (right).
PSTH ofthe CS activity. B: Difference between the CS
Francisco J. Rubia
The possibility of comparison between passive
and active movements showed us that in many
cases the active movement was able to suppress
strongly or even cancel the response produced in
the PC by the passive movement. This can be
observed in Figure 12.7, where the CS response of
one PC during passive extensions of the monkey's
hand was completely suppressed during similar
active extensions. The discharge rate seems to be
increased before the movement onset, but this
phenomenon can be observed better in the next
figure.
In Figure 12.8 the response of the CS of one
PC to passive extensions of the monkey's hand
is also suppressed during active extensions, but
the cell started to have a significant increase in
the CS activity as early as 360 ms before the
movement onset.
Thus, during volitional movements the motor
commands seem to have preference upon the
olivary cells with respect to the sensory feedback
produced by the passive movements.
A CS
imp/s
p
6
V
2
0
0 200 'llO 600 800 deg/s2
acceleration
discharge rate during the first lOOms after movement
onset and the 200 ms of the preceding resting period
(in impulsesjs) plotted against mean values of acceiera"
tion of passive movements for the unit in A and three
other PCs. (From Bauswein et aI., 1984, with permis-
sion.)
12. Mossy and Climbing Fiber Systems 233

A B C

PASSIVE FLEX . PASSIVE EXT. ACTIVE EXT.

n = 23 n =20 n = 20

] ] o
,'"

I
.. \
CS
:- '1.
. i
.. I

-
I
~ 10
en
U)
I-
Z CS
~
:::J
0
u 0 ~

- 0.5 0 +0.55 - 0.5 0 +0.55 - 0.5 0 +0.55

Figure 12.7. CS response of a monkey's PC during
passive and active movements. A: Mechanogram of 23
superimposed hand movements, dot raster, and PSTH
of the CS discharge during passive flexion. B, C: The
same during passive and active extension, respectively.
The shaded areas represent the resting period before
the movement lasting 250 (A), 300 (B), and 500 (C) ms,
respectively. Note the responses during passive exten-
sion and its suppression during active extension.
(Reprinted with permission from Bauswein et aI., 1983.)

Relationship Between the Complex
and the Simple Spike of the PC
Since the CS represents only one type of discharge
of the PC, it is interesting to observe what kind
of interaction occurs between this type of spike
and the SS in the same cell.
From the beginning of our recordings we ob-
served that many cells in the decerebrate cat
(approximately 47%) showed what we called an
inverse relationship between the discharges of
both types of spike, that is, an increase in the
firing rate of the PC with the CS was accompanied
by a decrease of the discharge frequency of the
cell with the SS.
Figure 12.9 illustrates two cells showing this
type of relationship. The first cell shows an in-
crease in the firing rate of the CS during upward
movement and end position together with a de-
ctease in the discharge frequency ofthe SS during
the same time (Fig. 12.9B, C). The second cell
shows an increase in the CS discharge frequency
during both dynamic phases of the movement
and a corresponding decrease of its SS during the
same periods (Fig. 12.9D, E).
By changing the parameters of the movement,
this inverse relationship between the two spikes
remains relatively constant. As we can see in
Figure 12.10, the change in the amplitude of the
movement changed also the response of the PC
234 Francisco 1. Rubia

A B

PASSIVE EXT. ACTIVE EXT.

20· ]
J/--~ o
n=60 n=60
z 500 J _ _ _
ro ~r-- J~F

" EMG Figure 12.8. CS and SS

§1~ ~
(f)
discharges of a monkey's
E PC during passive and
_ _ _ _ _-' active movements of the
hand. The minimal resting
, " ~, .'
periods before the
movement onset (200 ms
PC in A and 800 ms in B)
.'
•.1.. ~ " are represented by
shaded zones. In this
figure the electro-
myogram of flexors (F)
CS and extensors (E) are also
z shown. Note an increase
ro in the CS activity before
"z
(f)
I-
the movement during
active extension, which is

§1] ~
significant at - 360 ms.

~~ss
In the dot raster small
dots are for SS and large
dots for CS. (Reprinted
with permission from
I Bauswein et aI., 1983.)
o
i i r, ------~----~I------'I

-0.5 +0.5 -1.0 0 +055

with CS and a corresponding change in the SS
can be observed. The cell in this figure shows the
above-mentioned phenomenon, namely, that the
best response of the PC with the CS was to small
movements and that if the displacement is suf-
ficiently large, both acceleration signals can be
observed separately, each producing an increase
in the discharge of the PC with the CS. The
decrease in the firing rate of the cell with the SS
accompanies the increase every time in the CS,
producing an almost perfect mirror image. This
can also be seen in another PC of the same type
by applying a movement that followed a multiple
ramp function (Fig. 12.10B). Notice that at the
final standing position of + 20° there is a tonic
increase in the firing rate of the PC with the CS
accompanied by a tonic decrease in the SS.
These results can suggest that both afferent
systems could have a kind of connection between
each other. However, it can be argued that the
decrease in the firing rate of the SS could be due
to the effect produced at the PC by the CS. It is
well known that after the occurrence of a CS, a
pause in the firing of the PC with the SS is pro-
duced. Therefore, the decrease in the SS could be
the sum of all the pauses produced by the CS.
However, the inverse relationship between both
types of spike of the PC is not the only possible
relation. A parallel relationship can also occur.
In the decerebrate cat, approximately 15% of the
12. Mossy and Climbing Fiber Systems 235

'PH] 1//,
I II I
111 I
<fl -.J '
i1 : tu :
n m
COUNTS/BIN
CS
B
~ ~ 00 I

~
H
'fll ~ -30 0 n ~ 60

C
SS 100
1 F
15J
I
I

5~r G CS
o ~

]
CS n~100
H SS1~]~
D H
10ms
'PH ~ +10 0

'fll ~ 0 0 n ~100
SS 60
E 30l 0
I I I
0 2.5 5s
Figure 12.9. Relationship between CS and SS of two G and H show an expanded part of the PSTH of B
different PCs (B, C and D, E) during a passive move- and C for better illustration of the time relationship
ment of the eat's forepaw. Note the inverse relationship between CS and SS. (From Rubia and Kolb, 1978, with
between both types of spike in both cells. The dynamic permission.)
sections are represented in the PSTH by shaded areas.

PC recorded showed this last type of relationship,
that is, an increase in the firing rate of the CS was
accompanied by an increase in the SS discharge
ofthe same cell. The rest comprised mixed types.
An example of this parallel behavior of both
types of spike of the PC can be seen in Figure
12.11. Here two different PCs (G, H and I, K)
show during the upward movement of the ramp
function an increase in both types of spike.
In Figure 12.12 we can see two different PCs
showing the two relationships mentioned above:
the first with an inverse relationship between the
two types of spike (Fig. 12.12A) of almost mirror
image character, and the second with a parallel
behavior of both spikes especially during the up-
ward movement of the ramp function (Fig. 12.12B).
It is interesting to note that the increase in the SS
firing occurs with or without the occurrence of
the CS and that when the CS occurs, there is
always a pause in the SS firing, although sometimes
very short.
Fromthese observations we can conclude that
the inverse relationship has not necessarily to be
produced by the effect of the CS on the SS firing
of the Purkinje cell.
In the awake monkey, both inverse and parallel
relationships could be observed. The only dif-
ference with the decerebrate cat was that in the
awake monkey approximately 50% of the cells
showed the parallel behavior compared with 15%
in the decerebrate animal.
236 Francisco 1. Rubia
A B

-J
w=L.Oo/s
20
+ °1
-20·
COUNTS/BIN COUNTS/BIN
n=20
CS ':] CD CS 10]

:~
SS40J
20

i ,
o
i
2.5 5s
CS':]

SS4~ C
20
PIO.51/%)

0.8
..
w=60·/s
' mu

O"y
' i-
CS':]
md
0.6

0.4
"~;;;;: '.:.:~~::!Yo- . '
5540j ..•y/.-: ...•

0.2
20 -30·
0
0 -20 !O .20
'P Ideg)
0 2.5 5s
Figure 12.10. CS and SS discharges of a PC in response a multiple ramp function . C: Mean probability of the
to passive movements of different amplitudes of the CS activity of the cell in B during the dynamic sections
cat's forepaw (A). B: PSTH of another PC of the same of the movement. (From Kolb and Rubia, 1980, with
type (inverse relationship between CS and SS) during permisson.)

In the experiments using the awake monkey
we could also observe that by comparing passive
with active movements, if both types of spike of
the PC recorded had a parallel relationship during
passive movements, during similar active move-
ments there was the previously mentioned shift
of the cell discharge, firing the cell several hundred
milliseconds before the movement onset, but the
parallel relationship remains. This can be ob-
served in Figure 12.13. The cell shown in this
12. Mossy and Climbing Fiber Systems 237

o·
A
-30
I: I : W =l.3°/s
oo~
0
I
I tu
I
I
I
I
I
I
td I
I
F - 30 J W= l.Oo/s

I n m N 1l n m TIL y
COUNTS/BIN
CS CS n=100
30] n =100
70 ]
B 15
G 35

0 0
55 55 180

C
3001 120
H
150
60
of
0
CS 20
n =50 n=100

D 10
CS
30 ]
15

1
0 0
55 100 55
IDO l
E K 50
80
of 0

i i I j
0 2.5 5 0 2.5 5$
Figure 12.11. Relationship between CS and SS dis- of spike can be observed in the cells shown in G, H
charges offour different PCs during passive movements and I, K, respectively. (From Rubia and Kolb, 1978,
of the cat's forepaw. A parallel increase in both types with permission.)

figure had a parallel relationship between its spikes,
and this relationship remains after changing from
passive to active movements.
Spatial Distribution
of the PC Responses
Considering these differences in the behavior of
both types.of PC spikes, it was interesting to find
out if there was a certain spatial distribution of
the different types within the cerebellar cortex.
For this purpose we designed an experiment res-
tricting our recordings to a parasagittal strip of
the CF projection to the anterior lobe, the Cl
zone, and recorded only PC lying in the very
superficial layer of lobules Va, b, and c in the
decerebrate cat.
By using the same passive movement of the
forepaw we recorded PCs in parasagittal or
mediolateral direction (parallel fiber direction),
analyzing also the relationship between their
spikes.
The results show that with respect to the CS
of the PC, the response of all the cells recorded,
no matter where the cells were located, was quali-
tatively very similar. The response consists of
increases in the average discharge rate during the
transitional phases ofthe movement, particularly
238
A B
+200
-2)
cs
Z
III
.......
(/)
] ~.rn","=lc.n°r",,-~.
]
I-
Z
::J
0
SS u
o 5
Figure 12.12. CS and SS discharges of two different
PCs during passive movements of the cat's forepaw.
Note the inverse (A) and parallel (8) relationships
between both types of spike. The dot raster in 8 shows
at the end of the upward movement (negative ac-
celeration). This response remains fairly constant
in the sagittal direction but even moreso in the
mediolateral direction.
This can be seen in Figure 12.14. In this figure
the CS responses of the 157 PCs recorded in para-
sagittal and mediolateral (every 50 !lm) direction
are shown (Fig. 12.14A). The responses are quali-
tatively so similar that by correlating the CS
response of every cell with the average CS res-
ponses of all the recorded 157 cells in all ex-
periments, the correlation was always positive
(Fig. 12.15B).
A completely different picture arises if we ob-
serve the SS activity of the recorded PCs. Some
of them show an inverse relationship between the
CSand SS and others a parallel relationship with
all the intermediate types. Thus, the Pearson cor-
relation between the SS of a PC and the average
CS responses of all the recorded PCs can be posi-
tive or negative, respectively. Surprisingly, even
at a distance of only 50!lm two PCs show an
Francisco J. Rubia
[
]
100
n=1.0
.;
" .
I I
o 5s
that there is an increase in SS discharge in spite of the
CS occurrence. (Reprinted with permission from
Marini et aI., 1982.)
inverse and a parallel behavior, as shown in
Figure 12.15C. Nevertheless, as a rule, by record-
ing along the mediolateral direction, every 250
to 300!lm the same relationship can be again
recorded (Fig. 12.16).
It is difficult to interpret these results in a
physiological sense, but if we consider the output
of the PC it seems obvious to assume that in the
case of a parallel relationship between both types
of spike, a strong inhibition should result on the
target cells, whereas in the case of an inverse
relationship one can assume that the inhibition
exerted on the target cells by one type of spike
can be counterbalanced by the decrease of inhi-
bition through the other type. If so, PCs showing
an inverse relationship between their spikes
should not be effective in inhibiting their target
cells. Since they are the majority of the cells re-
corded, at least in the decerebrate cat, we can
suppose that the cerebellum is receiving more
information signals than those leaving the cere-
bellar cortex. The comparison between the number
12. Mossy and Climbing Fiber Systems 239

A B C D
PASSIVE EXT ACTIVE EXT. PASSIVE FLEX . ACTIVE FLEX .

E~

PC

~,----~,r_--_" ,.-----,.---_., ~,----~jr----., r,----~Ir_--_.i

-0.5 0 .0.5 -0.5 0 .0.5 -0.5 0 .0.5 -0.5 0 .0.55

Figure 12.13. CS and SS activity of a PC of an awake (B and 0), as shown by shaded areas. Note the shift of
monkey during passive and active extensions and cell discharge in o. (Reprinted with permission from
flexions of the hand. The minimal resting periods before Bauswein et aI., 1983.)
movement onset were 250 ms (A and C) and 500 ms

of cells with a parallel behavior in the decerebrate Motor Cortex Effect

cat and in the awake monkey can be interpreted
in the sense that in the awake animal the cere-
bellum is more active than in the decerebrate
preparation.
The different types of relationship observed
between the two types of spike of the PC let us
presume that somewhere a functional link between
both afferent systems may exist. It is difficult to
assume that this link can be established at the
level of the PC itself and, therefore, we surmise
that this link should exist at a precerebellar
level.
The results obtained with electrical stimulation
of the motor cortex point also to this conclusion.
on the Relationship Between
the PC's Spikes
In a series of experiments we stimulated the motor
cortex electrically in order to observe its effect on
the sensory feedback signals impinging on the PC
during passive movements of the forepaw. The
electrical stimulation was applied to the repre-
sentation area of the forepaw in the motor cortex
of the cat, trying to produce the same type of
movements as those passively induced by the
DC motor. As soon as the desired movement was
produced, the stimulus strength was reduced until
the movement was no longer visible. Then, the
 240 Francisco J. Rubia

B
1 2

z 200
~
z 50,.
ro "
(,/) ./
"~
(,/)

100
I-
Z
5 100
:::> u
o
u

o 5,12 s o 5,12 s

200
z
ro
"
(,/)
I-
Z 100
A :::>
o
u

o 5,12 s

z
ro
"Z
I-
(,/)

:::>
o
u
o 5,12 s

Figure 12.14. CS responses of several PCs recorded in
three different cerebellar folia to passive movements of
the cat's forepaw. A: Diagram of the cerebellar anterior
lobe showing the c1 zone (shaded) and the different
penetrations of the microelectrode. The distance be-
tween two penetrations in the mediolateral direction was
50 f.lm . B: Stack plots of the different PSTH. Binwidth:
640 ms. Note the qualitative similarity of the responses.
(From Rubia and Tandler, 1981, with permission.)
 !'-'
-
s::o
'<
'"'"
$>0
::s
0-
o
S·
0'
S·
(JQ

cs "l"j
ss 5'
o
"1
+1
CIl
'<
z ;!;.
Q t t o
:]1 ~ t--
«
fTff tf!1fT Uff! H a
..J '"
W
1000 cr f 11
z 15 0
to
"- l 1
III
t--
z
:::> 50 11m
! t
0
u f
j
50 11m
A 0
Jt"~L~57_~ 5.~2 s B
ij -1 C

Figure 12.15. Pearson correlations between CS and SS of PCs recorded within a mediolateralline and the average CS response
in an experiments. A: Average CS response of an 157 recorded Pc. Binwidth: 5 ms. B: Values of the correlation between CS of 21 PCs
within a representative recording line and the average response of A. The presence of a blood vessel prevented the recording of
three PCs on the right side of the correlation diagram. The decreasing positive values on the right are due to decreasing responsiveness
of PCs at the border of the cl zone. The bars at each value represent the 99% confidence interval of the correlations. C: Values
of the correlation between SS of eight different PCs and the average CS response. (From Rubia and Tandler, 1981, with permission.)

~
-
242
A B
CS/5S
+1
z CS-A
0
I-
«
f CD
-l CD
W 55
a: Q)
a:
0 0
u
z
0
'"«a: 1 o
w
a..
CD
-1 50J.lm
Figure 12.16. A: Pearson correlations between SS of
11 different PCs and the average CS response of
Fig. 12.15A. B: PSTH of CS and SS of four different
cells whose correlation values are shown in A. The
motor cortex was stimulated during different
phases of the passive movement, recording both
types of PC spikes.
From the observations of the results obtained,
two phenomena should be emphasized. Electrical
stimulation during the dynamic phases of the
passive movement can result in increasing or de-
creasing the sensory feedback signals recorded at
the PC as CS and SS discharges, but without
changing the relationship between the two types
of cell spikes. This can be seen in Figure 12.17. In
this figure, two different PCs are shown. Figure
12.17 A illustrates the result of the electrical sti-
mulation of the motor cortex during the upward
and downward movements of the forelimb of the
ketamine anesthetized cat. In both cases we ob-
tained in this PC of the inverse type (increase in
the CS response and decrease in the SS response)
a reduction of the response, the relationship re-
maining approximately constant.
In the second PC shown in Figure 12.17B, we
have a cell of the parallel type (increase in both
CS and SS responses). Stimulation of the motor
cortex during the upward and downward move-
ments results in an increase in the responses of
the cell with the CS and SS in both cases. Here,
also, the relationship between the two different
spikes remains constant.
However, in other cells the electrical stimula-
Francisco J. Rubia
~-
. -
5,125 o 5.125
correlation was calculated only for upward movement
and upper position (shaded zone). Binwidth: 160 ms.
(From Rubia and Tandler, 1981, with permission.)
tion of the motor cortex was able to disrupt the
relationship between the spikes, as can be ob-
served in Figure 12.18. In the PC shown in this
figure, the relationship between the spikes was of
the inverse type and the electrical stimulation of
the motor cortex during the upward movement
produced a decrease in the CS response, but the
corresponding suppression of the SS was greater
instead of smaller. The same holds true for the
downward movement.
The conclusions we have inferred from these
types of experiments is that a functional link
should exist at the precerebellar level and that the
motor cortex can influence this link in two ways:
either affecting the sensory feedback signals at a
previous level to the link and, therefore, keeping
the functional relationship constant (Fig. 12.17),
or impinging on the link, disrupting in this way
the functional relationship between the two
afferent systems (Fig. 12.18). Although this
hypothesis appears rather speculative, it seems
to us interesting enough to be analyzed further.
With respect to the forepaw, important infor-
mation from this part of the body comes as MFs
to the cerebellum through the cuneo cerebellar
pathway. On the other hand, it is well known that
there are connections between the main cuneate
nucleus (MCN) neurons and the inferior olive
(10). Thus, one of the candidates for the site at
12. Mossy and Climbing Fiber Systems
A B
.20°]
:j
-20°
n-20
CS
I I
110 j
55
C5
5S
C5
55
j
o 5
Figure 12.17. CS and SS discharges of two different
PCs to passive movements of the cat's forepaw with
and without electrical stimulation of the motor cortex.
The electrical stimulation of the cortex is marked by
horizontal bars. A: PSTH of a PC showing an inverse
relationship between CS and SS. B: PSTH of another
which the presumed functional link can be is the
cuneate nucleus. For this reason we questioned
whether there were MCN neurons projecting
simultaneously to both the cerebellum and the
10
N euroanatomical Experiments
To approach this issue we examined the cuneo-
cerebellar and cuneo olivary projections in the
cat using two tracer combinations: Fast Blue
(FB) and Diamidino Yellow Dihydrochloride
(DY.2HCI) or horseradish peroxidase conjugated
243
.20 0
J w-40i's
~o
n-20
:~
110j
]
l~j
2]0 ----..
] 301JA
~
15
o 55
PC showing a parallel type of relationship. Note in A
the reduction ofthe response affecting both spikes and
in B an increase in the response of the cell with both
spikes. (Reprinted with permission from Marini et a!.,
1982.)
to wheat germ agglutinin (WGA- HRP) and Dia-
midino Yellow Dihydrochloride.
Injection ofWGA- HRP into the 10 results in
retrogradely labeled neurons in the caudal, middle,
and rostral subdivisions of the contralateral MCN
(Fig. 12.19). In the caudal and middle MCN the
neurons were concentrated ventrally. They were
predominantly multipolar-, triangular-, and fusi-
form-shaped.
After WGA-HRP injection into the cerebellar
anterior lobe, especially lobules Vb and Vc, the
retrogradely labeled neurons were found ipsil-
aterally and predominantly concentrated in the
244
A B
+200[ ~
-200
[ ~s [~
CS ; :; JOt
ID
U)
0 t
SS \< 501 7sf IJti
§ 25 ~ 50
" ............ 30IJA
I I
0 5s
Figure 12.18. CS and SS response of a PC to passive
movements ofthe eat's forepaw with and without elec-
trical stimulation of the motor cortex (horizontal bars).
rostral subdivision of the MeN, bordering the
external cuneate nucleus (Fig. 12.20).
Although in the rostral MeN the neurons pro-
jecting to the 10 were most numerous medially
and ventrally and those projecting to the cere-
bellum were located laterally, there was consider-
able overlapping ofthe two neuronal populations,
especially in the ventral region.
A quantitative distribution of the labeled neu-
rons after injection of WGA-HRP into the 10
and the anterior lobe of the cerebellum is shown
in Figure 12.21. It can be observed that the majority
of the MeN cells labeled after 10 injection are
located caudally to the obex (Fig. 12.21A, B),
whereas after injection into the cerebellum the
labeled neurons are concentrated rostrally to the
obex (Fig. 21,21 C). However, there is a region
around the obex rostral to it, where both neuronal
populations overlap.
By using double-labeling techniques in the com-
binations mentioned above, the results indicated
that MeN neurons projecting to the cerebellum
represent a different population of neurons from
those projecting to the 10. Thus, the hypothesis
that the same MeN neuron can project simul-
taneously to the cerebellum and to the 10 was
discarded (Fig. 12.22).
However, the possibility remains of a connection
between the neurons of origin of the cuneocere-
bellar and cuneoolivary paths within the MeN
either directly or through interneurons. To analyze
Francisco J. Rubia
C
n: 40
t
75J
50
'C..
1~ ~30uA
i I
Note the different behavior of both spikes as compared
to the cell shown in Figure 12.17 A. (Reprinted with
permission from Marini et aI., 1982.)
this hypothesis, WGA- HRP injections into the
subdivisions of the MeN were performed.
The injections of WGA-HRP into the sub-
divisions of the MeN without affecting other
nuclei showed retrogradely labeled neurons locat-
ed within the MeN, as can be observed in Figure
12.23A. The neurons are grouped in the ventral
zone of the nucleus, although some of them were
also found in the dorsal rim of the MeN.
The quantification of the distribution of the
labeled neurons indicates, however, that the
number of neurons projecting in an ascending
direction within the MeN is greater than that of
a descending direction (Fig. 12.23B). These dif-
ferences could be appreciated in all the cases
studied, from which a summary can be observed
in Figure 12.23C.
These results hint at the possibility that MeN
neurons have projections within the nucleus. The
localization of these neurons in the ventral zone
of the MeN is similar to that of the cuneoolivary
neurons. However, using the double-labeling tech-
nique, by injecting Diamidino Yellow into the 10
and WGA-HRP into the rostral subdivision of
the MeN, we were able to show that the neurons
with a presumed intrinsic projection and the cuneo-
olivary projection neurons are two different popu-
lations. The proximity of the two neuronal groups
is remarkable, although within the ventral zone
the first group was located more dorsally than
the group neurons projecting to the fO (Fig. 12.24).
12. Mossy and Climbing Fiber Systems
Figure 12.19. Drawings of transverse sections through
the MCN showing the distribution of retrogradely
labeled neurons after WGA-HRP injections into the
10. Each dot represents a labeled neuron. Numbers at
the lower left of each section indicate the distance in
245
mm from the obex. ECN, external cuneate nucleus;
GR, gracile nucleus; c, d, v, vi, vm, central, dorsal,
ventral, ventrolateral, and ventromedial parts of the
MCN, respectively. (Reprinted with permission from
Alonso et aI., 1986.)
246 Francisco J. Rubia

Figure 12.20. The same as in Fig. 12.19 but after injec- (Reprinted with permission from Alonso et aI., 1986.)
tion of WGA~HRP into the cerebellar anterior lobe.
12. Mossy and Climbing Fiber Systems 247

A 80
- contralateral
c:: 70 ••.• ipsi lateral
..;:::;0 60
&l 50
-...
en
.!!!
Qj 40
u
-c 30
~
Qj 20
.0
.!!! 10

+1 -1 -2 -3 -4 -5 -6 -7
OBEX
B 80
c:: 70
..;:::;0 60
&len 50
'Ui
Qj 40
u
-c 30
~
Qj 20
.0
.!!! 10

+2 +1 -1 -2 -3-4 -5 -6 -7 -8mm
OBEX
ROSTRAL I MEDIAL CAUDAL

C 40
c::
..;:::;0 30
u
-...5l
.!!!
Qj 20
u
-c
~
Qj 10
.,
.0
.!!!
... A ...

+2 +i -1 -2 -3 -4 -5 -6 -7 -8 mm
OBEX
Figure 12.21. Distribution of retrogradely labeled different animals) and the cerebellar anterior lobe (C).
neurons in the contralateral and ipsilateral MCN after o in the abscissa represents the obex. (Reprinted with
WGA-HRP injections into the 10 (A and B, two permission from Alonso et ai., 1986.)
248 Francisco J. Rubia

Figure 12.22. Fluorescent photomicrography of the inferior olive) but not double-labeled neurons. The
rostral MCN showing labeled cells either with Fast location of one DY. 2HCliabeled cell is indicated by an
Blue (injected into the cerebellar anterior lobe) or arrow. (Reprinted with permission from Alonso et aI.,
Diamidino Yellow Dihydrochloride (injected into the 1986.)

In those experiments in which the lectin PH A- interpretation of the results and make the hy-
Land WGA- HRP were used as anterograde pothesis of the intrinsic connectivity within the
tracers, we could also observe labeled terminals MeN more plausible. In any case, the evidence
within the MeN even in those cases where the of an intrinsic connection within the nucleus can
injections were restricted to a small group of be obtained by means of intracellular injections.
neurons (Fig. 12.25). The results obtained in ant- It remains also to be elucidated if the presumed
erograde direction with both tracers were similar. connection is exclusively intrinsic or not.
The labeled terminals within the rostral subdivision The results obtained so far indicate that in the
of the MeN were preferentially localized in a MeN they are several anatomically independent
transitional zone between the MeN and the ex- populations of neurons since each one projects
ternal cuneate nucleus. In some cases in which to a different target. Several authors have found
the cuneo cerebellar neurons were labeled with similar results by trying to identify neurons pro-
Diamidino Yellow, injections ofWGA-HRP into jecting simultaneously to the thalamus and tectum
the medial and caudal subdivisions of the MeN (Berkley et aI., 1980; Bull and Berkley, 1984;
showed labeled terminals around the cuneocere- Wiberg and Blomqvist, 1984) or to the spinal
bellar neurons. A precise localization of these cord and thalamus (Bromberg et aI., 1981).
terminals can probably be obtained only with the However, electrophysiological studies show that
electron microscope. in some cases the same MeN neuron has a func-
Nevertheless, the technique required for the tional relationship with two different structures
use of the PHA-L, namely, the iontophoretic in- (Haring et aI., 1984; Johnson et aI., 1968; Gordon
jection, as well as some of the characteristics of and Seed, 1961). Therefore, considering the anato-
this tracer, for example the uptake by the dendrites mical independency ofthe various populations of
exclusively and the anterograde transport, reduce neurons within the MeN, the hypothesis of the
considerably the probability of errors in the existence of interneurons connecting functionally
12. Mossy and Climbing Fiber Systems 249

B 90 100
c: 90
.g 80 c:
80

I
0
~ 70 70
60
";;r
~ 50
60
u a; 50
al 40 u
=
1l
30
"0 40
.!1 30
!!
20 :8 20
10 ~
10

c DIIIECTlOtt 0' THI! INTIIINSIC CONNECTIONS

Figure 12.23. Intrinsic connections within the MCN.
A: Drawings of transverse sections through the MCN
showing retrogradely labeled neurons after injections
ofWGA- HRP into the rostral and caudal parts of the
MCN. B: Quantification ofthe labeled neurons showed
in A. The abscissa represents the distance from the
obex in mm. C: Diagram showing the direction of the
....
intrinsic connections based on the results obtained
after injections into the rostral, medial, and caudal
parts of the MCN. The size of each arrow is related to
the mean number of neurons obtained from different
injections in every part of the nucleus. (From
M.J. Blanco, Doctoral dissertation, 1988, with per-
mission.)
250
Figure 12.24. Drawings of transverse sections through
the MeN showing retrogradely labeled neurons (dots)
after injection into the rostral part of the nucleus. The
the different neuronal populations appears more
attractive.
Rustioni et aL (1984) have described small
gamma-aminobutyric acid (GABA)-ergic inter-
neurons in the medial subdivision of the MCN,
probably involved in local circuitries. The work
Francisco J . Rubia
stars represent the cells of origin of the cuneoolivary
projection. (From MJ. Blanco, Doctoral dissertation,
1988, with permission.)
of Cooke et al. (1971a, b) also shows that the
information from the periphery to the cerebellum
is transmitted polysynaptically, suggesting the
existence of interneurons within the MCN. Al-
though this has not yet been established, our
results with retrogradely labeling techniques could
12. Mossy and Climbing Fiber Systems
Figure 12.25. Fluorescent photomicrography of the
caudal part of the MCN showing WGA-HRP (asteriks)
labeled cells after injection into the rostral part of the
be interpreted in the sense that they are the effect
of interrupting interneuronal pathways from the
MeN to the cerebellum. This would explain why
after injections of WGA-HRP into rostral parts
of the nucleus we found labeled neurons at any
level caudally to the injection but definitely not
the presence of labeled neurons rostrally to the
injections into the caudal parts of the nucleus.
The experiments with double-labeling tech-
niques indicate that the neuronal population pre-
251
nucleus and DY.2HCllabeled neurons after injection
into the 10 (arrows). (From M.l Blanco, Doctoral dis-
sertation, 1988, with permission.)
sumably projecting within the MeN is also an
independent population. The functional meaning
of these neurons could be the establishment of a
connection between different neuronal populations
ofthe MeN, which could put together the results
obtained with anatomical and electrophysiological
experiments.
Summarizing these results, we can conclude
that the existence of an anatomical connection
between the cuneocerebellar and cuneo olivary
252 Francisco 1. Rubia

Figure 12.26. Anterogradely lab-

A eled terminals in the rostral part
of the MeN (A) after injection of
PHA-L into the caudal part of the
nucleus (8). (From c.L. Paino,
Doctoral dissertation, 1988, with
permission.)

:.\-.h. r{
,
BI
I

..
" -.

\ \
1
r
' ,
,.
~

neurons is possible. This hypothesis is supported
by the following findings: the close vicinity between
the group of cuneo olivary neurons and that of
presumed intrinsic connection, the greater number
of ascending projections, and the visualization of
WGA-HRP or PHA-L labeled terminals around
the cuneo cerebellar neurons. All these facts speak
in favor of a possible connection between the
cuneoolivary neurons and those neurons project-
ing to the cerebellum.
12. Mossy and Climbing Fiber Systems
Finally, it is interesting to note that the cerebral
cortex projects preferentially to the ventral zone
of the MeN, making possible a cortical influence
on this hypothetical anatomical link between the
two afferent systems to the cerebellum.
References
Alonso, A., Blanco, M.J., Paino, C.L., and Rubia, F.I
(1986): Distribution of neurons in the main cuneate
nucleus projecting to the inferior olive in the cat.
Evidence that they differ from those directly project-
ing to the cerebellum. Neuroscience, 18, 671-683.
Bauswein, E., Kolb, F.P., and Rubia, FJ. (1984): Cere-
bellar feedback signals of a passive hand movement
in the awake monkey. PflUgers Arch., 402, 292-299.
Bauswein, E., Kolb, F.P., Leimbeck, B., and Rubia, F.J.
(1983): Simple and complex spike activity of cere-
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Berkley, K.J., Blomqvist, A., Pelt, A., and Flink, R.
(1980): Differences in the collateralization of neuronal
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Blanco, MJ. (1988): Proyeccion cuneo-cerebelosa y
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Bromberg, M.B., Burnham, J.A., and Towe, A.L. (1981):
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Bull, M.S., and Berkley, KJ. (1984): Differences in the
neurons that project from the dorsal column nuclei
to the diencephalon, pretectum and tectum in the
cat. Somatosen. Res., 1, 282-300.
Cooke, J.D., Larson, B., Oscarsson, 0., and Sjolund,
B. (1971a): Origin and termination of cuneocerebellar
tract. Exp. Brain Res., 13, 339-358.
Cooke, J.D., Larson, B., Oscarsson, 0., and Sjolund, B.
(1971b): Organization of afferent connections to
cuneocerebellar tract. Exp. Brain Res., 13, 359-377.
Eccles, J.e., Sabah, N.H., Schmidt, RF., and Taborikova,
H. (1972a): Cutaneous mechanoreceptors influencing
impulse discharges in cerebellar cortex. I. In mossy
fibers. Exp. Brain Res., 15, 245-260.
Eccles, J.e., Sabah, N.H., Schmidt, RF., and Taborikova,
H. (1972b): Cutaneous mechanoreceptors influencing
impulse discharges in cerebellar cortex. II. In Purkinje
cells by mossy fiber input. Exp. Brain Res., 15, 261-
277.
253
Eccles, Ie., Sabah, N.H., Schmidt, R.F. and
Taborikova, H. (1972c): Cutaneous mechanorecep-
tors influencing impulse discharges in cerebellar
cortex. III. In Purkinje cells by climbing fiber input.
Exp. Brain Res., 15,484-497.
Eccles, Ie., Sabah, N.H., Schmidt, RF., and Taborikova,
H. (1972d): Integration by Purkinje cells of mossy
and climbing fiber inputs from cutaneous mechano-
receptors. Exp. Brain Res., 15,498-520.
Gordon, G., and Seed, W.A. (1961): An investigation
of the nucleus gracilis of the cat by antidromic
stimulation. J. Physiol., 155, 589-60l.
Haring, J.H., Rowinski, MJ., and Pubols, B.H. (1984):
Electrophysiology of raccoon cuneocerebellar neu-
rons Somatosens. Res., 1,247-264.
Hobson, J.A., and McCarley, RW. (1972): Spontaneous
discharge rates of cat cerebellar Purkinje cells in
sleep and waking. Electroenceph. CUn. Neurophysiol.,
33,457-469.
Ishikawa, K., Kawaguchi, S., and Rowe, M.J. (1972a):
Actions of afferent impulses from muscle receptors
on cerebellar Purkinje cells. I. Response to muscle
vibrations. Exp. Brain Res., 15, 177-193.
Ishikawa, K., Kawaguchi, S., and Rowe, M.J. (1972b):
Actions of afferent impulses from muscle receptors
on cerebellar Purkinje cells. II. Responses to muscle
contraction: Effects mediated via the climbing fiber
pathway. Exp. Brain Res., 16, 104-114.
Johnson, J.I., Welker, W.I., and Pubols, B.H. (1968):
Somatotopic organization of raccoon dorsal column
nuclei. J. Comp. Neurol., 132,1-44.
Kolb, F.P., and Rubia, FJ. (1980): Information about
peripheral events conveyed to the cerebellum via the
climbing fiber system in the decerebrate cat. Exp.
Brain Res., 38, 363-373.
Leicht, R., Rowe, M.J., and Schmidt, R.F. (1973):
Cutaneous convergence onto the climbing fiber input
to cerebellar Purkinje cells. J. Physiol., 228, 610-618.
Llinas, R, Precht, W., and Clarke, M. (1971): Cerebellar
Purkinje cells responses to physiological stimula-
tion of vestibular system in the frog. Exp. Brain Res.,
13,408-43l.
Maekawa, K., and Simpson, 1.1. (1972): Climbing fiber
activation of Purkinje cells in the flocculus by im-
pulses transferred through the visual pathway. Brain
Res., 39, 245-25l.
Mano, N. (1970): Changes of simple and complex spike
activity of cerebellar Purkinje cells with sleep and
waking. Science, 170, 1325-1327.
Marchesi, G.F., and Strata, P. (1970): Climbing fibers of
cat cerebellum: Modulation of activity during sleep.
Brain Res., 17, 145-148.
Marini, G., Provini, L., and Rosina, A. (1976): Gravity
responses of Purkinje cells in the nodulus. Exp. Brain
Res., 24, 311-323.
254
Marini, R., Rubia, FJ., Kolb, F.P., and Bauswein, E.
(1982): Cortical influence upon cerebellar Purkinje
cells responding to natural, peripheral stimulation in
the cat. Neuroscience Letters, 33,55-59.
Paino, c.L. (1988): Conexiones efferentes del nucleus
cuneatus en e1 gato. Doctoral dissertation, Uni-
versidad Complutense, Madrid.
Rubia, F.J., and Kolb, F.P. (1978): Responses of cerebel-
lar units to a passive movement in the decerebrate
cat. Exp. Brain Res., 31, 387-401.
Rubia, F.J., and Tandler, R. (1981): Spatial distribution
of afferent information to the anterior lobe ofthe eat's
cerebellum. Exp. Brain Res., 42, 249-259.
Rushmer, D.S., Roberts, WJ., and Augter, G.K. (1976):
Climbing fiber responses of cerebellar Purkinje cells
FraIfcisco J. Rubia
to passive movements of the cat forepaw. Brain Res.,
106,1-20.
Rustioni, A., Schmeche~ D.E., Cheema, S., and Fitzpatrik,
D. (1984): Glutamic acid decarboxylase containing
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Somatosen. Res., 1, 329-357.
Simpson, J.I., and Alley, E. (1974): Visual climbing
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13
Eye Movements and the Zonal Structure
of the Rabbit Flocculus
John I. Simpson, Johannes Van der Steen, and Joep Tan
The flocculus is part of the compensatory eye
movement control system that can stabilize gaze
during head rotations about any axis in space.
Therefore, understanding signal processing in the
flocculus requires knowledge of how the multi-
dimensionality of these eye movements is repre-
sented. Studies of the messages carried by the
visual climbing fibers (CFs) as well as studies of
the eye movements and eye muscle activity elicited
by electrical stimulation suggest that the basis of
this representation is the zonal structure of the
flocculus.
The zonation of the flocculus, like that of the
rest of the cerebellum, is rooted in the anatomical
organization of the CF inputs and the Purkinje
cell outputs. The inferior olive can be anato-
mically subdivided such that each subdivision's
terminal field in the cerebellar cortex has the
form of a zone or strip oriented approximately
orthogonal to the long axis of the folia (e.g.,
Brodal and Kawamura, 1980; Gerrits and Voogd,
1982; Groenewegen and Voogd, 1977; Voogd,
1969; Voogd and Bigare, 1980). A similar zonal
arrangement is also apparent in the pattern ofCF
activation of Purkinje cells produced by electri-
cal stimulation of peripheral nerves (e.g., Oscars-
son, 1969; Andersson and Oscarsson 1978a, b,
Oscarsson and SjOlund, 1977). These CF termi-
nation zones necessarily define corresponding
Purkinje cell zones because each Purkinje cell is
contacted by only one CF. Anatomical and phys-
iological studies have shown that Purkinje cells
of a given zone terminate in the same cerebellar
or vestibular nucleus (Brodal and Kawamura,
1980; Oscarsson and Andersson, 1978a, b; Voogd,
1969). Oscarsson (1969,1979) has suggested that
each zone constitutes a functional computing
unit receiving information related to a different
motor control mechanism, but the nature of this
computation is still unknown. Even so, elabora-
tion of Oscarsson's proposal in a specific, be-
haviorally tractable context has been possible
through studies of CF signals and eye move-
ments in relation to the zonal structure of the
flocculus.
In both the anesthetized and alert rabbit, floc-
cular CF activity is modulated in relation to the
speed and direction of movement of large, tex-
tured visual patterns (Leonard, 1986; Simpson
and Alley, 1974). Recordings from the dorsal
cap of the inferior olive and from floccular
Purkinje cells (Graf et aI., 1988; Leonard et aI.,
1988; Simpson et aI., 1981) have shown that the
visual CF modulation can be divided into three
classes according to the axis about which rota-
tion of the visual world evokes the greatest modu-
lation. For one class of CFs the preferred axis is
vertical; for the other two classes the preferred
axes lie close to the horizontal plane at about 45°
or 135° to the midsagittal plane. The CFs domi-
nated by the ipsilateral eye have preferred axes
that are either vertical or close to 135° aximuth;
the CFs dominated by the contralateral eye have
preferred axes close to 45° azimuth. These findings
support the idea that retinal image movement is
represented by visual CF activity in a reference
frame whose axes have spatial orientations quite
like those of the best-response axes of the ves-
tibular semicircular canals (Simpson and Hess
1977; Simpson et aI., 1979). Furthermore, the
255
256 John I. Simpson, Johannes Van der Steen, and Joep Tan
similarity of the orientation of both of these sets
of axes to the rotation axes of the extraocular
muscles (Graf and Simpson, 1981; Simpson and
Graf, 1981) suggests a relatively simple relation-
ship between the preferred axis of each class of CFs
and the axis of eye rotation produced by activa-
tion of the Purkinje cells upon which that class
of CFs synapses. These geometrical considera-
tions point to the zonal structure of the flocculus
as the structural correlate of a reference frame
representing eye movements.
A zonation of the flocculus has also been pro-
posed on the basis of the topographical distri-
bution of the sites where microstimulation elicited
different classes of eye movements or influenced
different vestibular ocular reflex (VOR) pathways
(Dufosse et aI., 1977; Ito et aI., 1982; Nagao
et aI., 1985; Yamamoto, 1979). However, the re-
ported localization and extent of these zones is
not consistent in these studies. Presumably, the
difference arose in part because no independent,
anatomical delineation of the zones was concomi-
tantly available for comparison with the physio-
logy and in part because two different measures
of the influence of floccular stimulation on eye
muscles were used, namely, eye movements and
electromyography.
In previous experiments, three classes of eye
movements (horizontal, rotatory, and vertical)
were evoked by electrical stimulation in the
rabbit's flocculus (Dufosse et aI., 1977; Nagao
et aI., 1985). The question naturally arises as to
whether a one-to-one correspondence exists
between the three preferred axes of the visual
CFs and these three classes of eye movements.
Whereas the eye rotation axes of the horizontal
and rotatory classes do correspond to the pre-
ferred axes of the visual CFs, the vertical eye
movement class comprises rotations about the
roll (nasal-occipital) axis, which is not a preferred
axis of the visual CFs. Furthermore, the vertical
eye movement class is not predicted by combining
the results of electromyographic measures of the
effects of electrical stimulation on the various
VOR pathways (Ito et aI., 1973, 1977, 1982) with
measurements of the kinematic action of indivi-
dual eye muscles in the rabbit (Graf and Simpson,
1981; Simpson and Graf, 1981, 1985). This incon-
sistency is made apparent when the finding that
the flocculus does not directly influence the post-
erior canal VOR pathways is coupled with the
fact that an eye movement about the roll axis can-
not be produced by inhibition of only the path-
ways from the ipsilateral anterior canal. Clearly,
the relation between eye movements and the
zonal structure of the flocculus needs to be
addressed further.
Recently, a zonation of the cerebellum was also
demonstrated using acetylcholinesterase (AChE)
histochemistry (Hess and Voogd, 1986; Voogd
et aI., 1987). With this technique, five compart-
ments separated by raphes and running oliquely
from caudomedial to rostrolateral can be identi-
fied within the white matter ofthe rabbit flocculus
(Tan et aI., 1989; Van der Steen et aI., 1989). The
most lateral compartment is part of the white
matter of zone C2 (of Voogd); the four other
compartments are labeled 1 through 4, counting
from lateral to medial. The majority of the white
matter is made up of the serially adjacent com-
partments 1, 2, and 3. The C2 compartment is
small and present in only the most caudoventral
part of the flocculus, and compartment 4, which
is quite thin, does not reach the cortex until the
most rostral part of the flocculus. AChE staining
of the rabbit flocculus reveals a possible anatom-
ical substrate for different classes of eye move-
ments since it delineates white matter compart-
ments that presumably have projections to
different parts of the vestibular nucleus complex.
The ability of the AChE method to provide an
independent delineation of floccular zones invited
experiments in which physiological and anatomi-
cal techniques are combined in the same animal
to determine the patterns of eye movement evoked
by stimulation of each compartment.
Methods
The eye movements evoked by electrical micro-
stimulation in the flocculus were recorded in 26
awake, pigmented rabbits. During the experiment
the animals were comfortably restrained and the
head held with the nasal bone at 57° to the hori-
zontal. The movements of both eyes were meas-
ured simultaneously in three dimensions using
two orthogonal coils attached to each eye (Van
der Steen and Collewijn, 1984). At least 5 days
before the experiment, two search coils, one hori-
zontal on top of the superior rectus muscle and
13. Eye Movements
Figure 13.1. The orthogonal
coordinate system used to
measure the angular dis-
placements of the eyes. See
text for explanation.
1350 CCW
'~ ......
. .'.--.*---
.............
,,- "
/
/
one vertical following the limbus, were implanted
under the conjunctiva of each eye with the rabbit
under general anesthesia. The orthogonal co-
ordinate system used to measure the angular dis-
placements consisted of a vertical axis and two
horizontal axes oriented at azimuths of 45° and
135° (Fig. 13.1). The zero reference was rostral in
the midsagittal plane and the azimuthal coordi-
nate was taken to increase to each side of the
reference direction. The components of the evoked
eye movements were named after their respective
axis (vertical axis, 45° axis and 135° axis) referenced
to the ipsilateral (left) or the contralateral (right)
eye. The sense of the rotation components about
the horizontal axes was defined as clockwise (CW)
or counter-clockwise (CCW) according to how
they would be seen by an observer looking along
each axis towards the rabbit's eye. With these
conventions, the 135° axis of one eye is parallel
to the 45° ,axis ofthe other eye and a CW rotation
of one eye is conjugate to a CCW rotation of the
other eye. The sense of the rotation components
about the vertical axis was described for each eye
as leftward or rightward. The vertical axis (hori-
zontal) component of rotation was measured with
257
VA
~L
............
...
the vertical coil using the phase-angle detection
technique (Collewijn, 1977), whereas the rotation
components about the 45° and 135° axes were
measured with the horizontal coil using the
amplitude detection technique (Robinson, 1963;
Van der Steen and Collewijn, 1984). The vertical
coils had absolute calibration, whereas the hori-
zontal coils were calibrated before implantation.
The three eye position components for each eye
were charted on a pen recorder and stored on
magnetic tape. In the off-line computer analysis,
each position component was digitized at 125 Hz
with a resolution of 10 s of arc.
One to 3 days before the experiment, the rabbit
was reanesthetized, the bone above the para-
median lobe of the cerebellum on the left side
was removed, and the opening was surrounded
by a chamber made of dental acrylic. The dura
was covered with an antibiotic gel and a thin
sheet of silicone rubber and then the chamber
was filled with bone wax and paraffin. On the day
of the experiment, the dura was opened under
local anesthesia. The flocculus was identified by
recording the modulation of Purkinje cell CF
activity in response to movement of handheld
258 John I. Simpson, Johannes Van der Steen, and Joep Tan

visual patterns. With the awake rabbit in dark-
ness, the glass or metal recording microelectrode
was then used to deliver mono polar electrical
stimuli at 200-jlm intervals during withdrawal
through the flocculus. The standard stimulus was
a I-s train ofO.2-msec pulses at 200Hz and -20
jlA. Some ofthe more effective locations for evok-
ing eye movements were marked by electrolytic
lesions. After termination of the experiment, the
animals were deeply anesthetized and perfused.
In serial sections reacted for AChE the lesion
sites and electrode tracks were located with re-
spect to the anatomically distinguishable com-
partments.
Results
Classification of the Evoked
Eye Movements
An effective site of electrical stimulation was de-
fined as a site at which the standard stimulus (a
I-s train ofO.2-msec pulses at 200 Hz and - 20 jlA)
evoked an eye movement of at least 0.5° about
at least one of the six recorded axes. The amplitude
of these slow eye movements was typically 1° to
5°, but occasionally amplitudes of up to 20° were
observed. Eye movements were more readily
produced by stimulation in the deep granular
layer and white matter than from the Purkinje
cell or molecular layers. The onset of the major
components of the eye movements usually oc-
curred between 8 and 48 ms after the stimulus
onset. The peak speed (maximum 20 °/sec) oc-
curred 100 to 200 msec after movement onset. In
most instances the eye returned to the initial posi-
tion within 4 to lOs after the end of the stimulus.
The eye movements were classified according to
the largest recorded component of the response.
Of the 12 possible classes (two eyes x three axes
x two senses of rotation), two of them alone
accounted for more than 75% of the responses.
For 59% (82/140) of the effective stimulation
sites, the largest component was a CCW rotation
of the ipsilateral (left) eye about its 135° axis
(Fig. 13.2). In about half of these cases the re-
sponse was conjugate in that the largest com-
ponent of the contralateral (right) eye was a CW

Figure 13.2. Example ofthe class of evoked

-
Stimulus
- - eye movements for which the largest com-
ponent was a CCW rotation of the ipsi-
Right (Contra) Eye lateral (left) eye about its 135° axis. The
largest component of the contralateral
VA]: (right) eye movement was in this case a
conjugate rotation about its 45 0 axis. In

~
this and the subsequent figures the three
jCCW
45 traces for each eye show the angular dis-
CW placement about the indicated axes (L, left-
T N ward; R, rightward; CW, clockwise; CCW,
135~CCW counterclockwise). The periods of electrical
CW stimulation of the left flocculus are indic-
ated at the top. The schematic drawings on
Left (Ipsi) Eye the righ~ show how the eye movements
would appear if a cross had been placed

VA]: on the cornea. The dashed cross indicates

the initial position and the solid cross

<i@
shows the position at the end ofthe stimu-
lation (T, temporal; N, nasal). Note that
135JCW neither eye movement was "vertical." The
CCW movement of the ipsilateral eye is due:;
N T largely to the actions of the vertical recti,
45JCW whereas the movement of the contralateral
CCW ,--, eye is due largely to the actions of the
4 sec obliques.
13. Eye Movements
rotation about its 45° axis. However, in some
instances the component of conjugacy of the
contralateral eye was essentially absent. The azi-
muthal orientation of the average rotation axis,
which lay close to the horizontal plane, was esti-
mated for each eye by "vector" summation of the
45° and 135° components. Although angular
displacements are not vector components, com-
pounding the small displacements involved pro-
vides a reasonable indication of the orientation
of the rotation axis. For the ipsilateral (left) eye
the axis of rotation, projected into the horizontal
plane, was located at 141.0° azimuth. For those
cases in which the component of conjugacy was
the largest component of the accompanying move-
ment ofthe contralateral (right) eye, the similarly
computed rotation axis was located at 32.0° azi-
muth. On some occasions when the largest re-
sponse component was a CCW rotation about
the ipsilateral 135° axis, disjunctive rotations of
both eyes about the vertical axis were also evoked.
In those cases the latency of the adducting com-
ponent of the ipsilateral eye was about lOOms
VA]:
135~
Figure 13.3. Example ofthe class of evok-
~
ed eye movements for which the largest
component was abduction of the ipsi- 45
lateral (left) eye. The format is the same as
that of Figure. 13.2.
259
longer than the latency of the CCW component
about the ipsilateral 135° axis.
For 19% (26/140) of the effective stimulation
sites, the largest response component was abduc-
tion of the ipsilateral eye; that is, a leftward rota-
tion of the left eye about its vertical axis (Fig. 13.3).
The vertical axis component of the accompanying
movement of the right eye was nearly always
conjugate; that is, leftward (adducting). The ex-
ample shown in Figure 13.3 had nearly only verti-
cal axis components, but not infrequently a modest
CCW component about the ipsilateral (left) 135°
axis was also present.
Although the above two classes accounted for
most of the responses, several additional types of
responses were recorded. Of these responses, one
that was often found was a CCW rotation about
the contralateral (right) 135° axis that was larger
than or comparable to an accompanying CW
rotation about the contralateral (right) 45° axis.
In the latter case, the net result of the two compo-
nents about the 45° and 135° axes of the contra-
lateral eye was essentially an upward rotation
Stimulus
• • •
Right (Contra) Eye
Left (Ipsi) Eye
<0
CW
CCW
CW N T
CCW
~o
260 John I. Simpson, Johannes Van der Steen, and Joep Tan
alJout the roll (nasal- occipital) axis (shown in
Fig. 13.5 as nearly equal, but opposite rotations
of the contralateral eye about its 45° and 135°
axes). However, the component about the 135°
axis typically had a longer latency than the com-
ponent about the 45° axis. Even when the re-
sponse of the contralateral eye was "vertically"
upward, the response of the ipsilateral eye was
not a conjugate "vertically" downward move-
ment, but instead it was essentially a CCW rota-
tion about the 135° axis.
Eye Movement Classes in Relation
to the Anatomical Compartments
When the middle of the three largest compart-
ments (zone 2) was stimulated, the largest re-
sponse component was abduction of the ipsi-
lateral eye about the vertical axis (Fig. 13.4). For
B Stimulus
VAJ:
A
45JCCW
CW
135JCCW-
CW
VAr~
135JCW
CCW
45J CW ------------------
CCW
' 4 sec '
Figure 13.4. Eye movements evoked by stimulation of
the middle compartment (zone 2). The location of the
electrolytic marking lesion is indicated on a frontal
section ·through the left flocculus by the arrow in A.
From lateral to medial, compartments 1, 2, and 3 are
the accompanying contralateral eye movement,
the largest component was a smaller, varying
amount of adduction about the vertical axis. When
either of the two neighboring compartments (zones
1 and 3) was stimulated, the largest component
usually was a CCW rotation of the ipsilateral
(left) eye about its 135° axis (Figs. 13.5,13.6). In
about half of these cases, the largest component
of the contralateral (right) eye movement was a
CW rotation about its 45° axis. In addition,
stimulation of these two compartments some-
times resulted in a smaller, varying amount of
horizontal convergence resulting from adduction
of the ipsilateral and contralateral eyes (Fig. 13.5).
Because the most lateral compartment (C2) and
the most medial compartment (zone 4) are com-
paratively small, the eye movements,. if any,
evoked by stimulating only the fibers within each
of these two zones have not been determined.
Right (Contral Eye
@
T N
-e
Left (lpsi) Eye
N T
indicated by large dots, medium dots, and fine dots,
respectively. Compartment 4 is indicated by the clear
area between compartment 3 and the brachium pontis.
The abducting response of the ipsilateral eye is shown
in B. Stimulus strength 10 /lA.
13. Eye Movements
B Stimulus
VAJ:
~--------------
A
451ccw____________
135
JCw
Ccw____--____
JCW
VAJ:~
135JCW \------.
CCW - ~
4S]CW ____-------------
CCW
'4
Figure 13.5. Eye movements evoked by stimulation of
the medial compartment (zone 3). The location of the
marking lesion is indicated by the arrow in A. Note in
The predominant components of the evoked
eye movements closely reflect the action of indi-
vidual pairs of eye muscles (Graf and Simpson,
1981; Graf et aI., 1988; Simpson and Graf, 1981).
The movement of the ipsilateral eye evoked by
stimulation of the middle compartment (zone 2)
was due predominantly to the horizontal recti
(lateral rectus contracting). The movement of the
ipsilateral eye evoked by stimulation of the two
bordering compartments (zones 1 and 3) was due
predominantly to the vertical recti (inferior rectus
contracting); in about half the cases the accom-
panying contralateral eye movement was pro-
duced largely by the obliques (superior oblique
contracting).
Relation between CF Afferents
and the Anatomical Compartments
The association between different classes of eye
movements and the anatomical compartments
261
Righi (Contra) Eye
--~r----
~
T N
Left Opsi) Eye
<@
N T
r,
sec ' LSo
B that the largest response component was a CCW
rotation of the ipsilateral (left) eye about its 135° axis.
Stimulus strength 2 f1A.
raises the question of whether axons originating
from physiologically different parts of the dorsal
cap of Kooy and its ventrolateral outgrowth re-
spect the boundaries of the AChE compartments.
This point has been investigated by Tan, Voogd,
and Gerrits (personal communication) using
anterograde horseradish peroxidase (HRP) tech-
niques. Injections into the rostral part of the
dorsal cap and the adjacent ventrolateral out-
growth, whose cells are maximally sensitive to
visual world rotation about axes close to 135°
contralateral azimuth or 45° ipsilateral azimuth
(Leonard et aI., 1988; Simpson et aI., 1981), re-
sulted in labeled fibers in the contralateral floc-
culus in white matter areas corresponding to the
anatomically identified compartments 1 and 3.
HRP injections into the caudal part of the dorsal
cap, whose cells are maximally sensitive to rota-
tion of the visual world about the vertical axis,
resulted in labeled fibers in that part of the floc-
cular white matter corresponding to the anatomi-
cally defined compartment 2.
262 John I. Simpson, Johannes Van der Steen, and Joep Tan

B Stimulus

Right (Contra) Eye

VAJ:~
~
A
"lCCW______________
Jcw T N

13J ccw____________----__----
Jcw
Left (ipsi) Eye

4 5] CW ~
CCW [50
'4 sec '
Figure 13.6. Eye movements evoked by stimulation of B that the largest response component was a CCW
the lateral compartment (zone 1). The location of the rotation of the ipsilateral (left) eye about its 135° axis.
marking lesion is indicated by the arrow in A. Note in Stimulus strength 5/lA.

Discussion three are excitatory. Two of them influence the

Classes of Evoked Eye Movements-
Past and Present
The present and previous findings (Dufosse et aI.,
1977; Nagao et aI., 1985) of the eye movements
produced by electrical stimulation in the rabbit
flocculus should be considered in conjunction
with the effects of flocculus stimulation on the
various VOR pathways. Purkinje cells ofthe floc-
culus monosynaptically inhibit some of the vesti-
bular nuclei neurons in the three-neuron arc of
the VOR (Baker et aI., 1972; Fukuda et aI., 1972;
Highstein, 1973; Ito et aI., 1970; Kawaguchi, 1985).
The selectivity of this inhibition for particular
VOR pathways was shown in the rabbit by record-
ing the influence of flocculus stimulation on eye
muscle activity produced by vestibular nerve stim-
ulation (Ito et aI., 1973, 1977, 1982). Of the six
pathways influenced, three are inhibitory and
pathways from the horizontal canal to the ipsi-
lateral medial and lateral recti. Increased activity
of the related Purkinje cells results in increased
activity ofthe lateral rectus muscle and decreased
activity of the medial rectus muscle. The other
four pathways under direct inhibitory control are
the anterior canal pathways, which excite the ipsi-
lateral superior rectus and contralateral inferior
oblique muscles and inhibit their antagonists, the
ipsilateral inferior rectus and contralateral supe-
rior oblique muscles.
The major, short latency components of the
eye movements evoked in the present study are
in good agreement with those predicted by com-
bining the effects of flocculus stimulation on the
various VOR pathways (Ito et aI., 1973, 1977,
1982) with measurements ofthe pulling directions
of the individual extraocular muscles (Ezure and
Graf, 1984; Graf and Simpson, 1981; Simpson,
13. Eye Movements
1983; Simpson and Graf, 1981, 1985). A conse-
quence of this result is that the classes of eye
movements found in the present study differ in
some respects from those found earlier. For one
of the two predominant classes of eye movements
found in the present study, abduction of the ipsi-
lateral eye was the largest component. This class,
which corresponds to the horizontal class found
before, can be explained by the known influence
ofthe flocculus on the horizontal canal pathways
to the lateral and medial recti. For the most
common class of eye movements encountered in
the present study, the largest component was a
CCW rotation ofthe ipsilateral eye about its 135°
axis. The largest component of the accompanying
movement of the contralateral (right) eye was
often a CW rotation about its 45° axis. This class
of eye movement is similar to the rotatory class
reported before, although there are some notable
differences with respect to the frequency with
which this class was encountered and with respect
to the relative amplitudes of the ipsilateral and
contralateral movements. In contrast to the pre-
sent findings, the rotatory class described before
was relatively rarely found and the movement
of the contralateral eye was larger than that of
the ipsilateral eye. The rotatory movements of the
contralateral eye were considered to be due to the
inhibitory influence of the flocculus on the path-
ways from the ipsilateral anterior canal to the
contralateral oblique muscles, but an equally
satisfactory explanation was not given for the
rotatory movements of the ipsilateral eye. In the
present study, the axis of rotation ofthe ipsilateral
eye movements in the rotatory class was located
at 141 ° azimuth, which is very close to the location
(148° azimuth) of the rotation axis of the paired
vertical recti (Graf et aI., 1988). This similarity
indicates that the ipsilateral "rotatory" eye move-
ments are essentially the result of floccular inhibi-
tion of the pathways from the ipsilateral anterior
canal to the ipsilateral vertical recti.
The vertical eye movement class composed of
rotations of both eyes about the roll axis (Dufosse
et al., 1977; Nagao et aI., 1985) was not found in
the present study. Whereas the relatively small
contralateral "vertical" eye movements found by
us are apparently the same as the contralateral
eye movements in the vertical class of Dufosse
et al. (1977) and Nagao et aI. (1985), the accom-
263
panying ipsilateral eye movement was not "verti-
cal," but rather it was predominantly a CCW
rotation about the 135° axis. In the previous
studies, the ipsilateral "vertical" eye movements
are considered to be due to the influence of the
flocculus on the pathways from the ipsilateral
anterior canal to the ipsilateral vertical recti.
However, this conclusion is at variance with ana-
tomical and physiological measurements of the
actions of these muscles (Ezure and Graf, 1984;
GrafandSimpson, 1981; Simpson, 1983; Simpson
and Graf, 1981). From those measurements it can
be concluded that the "rotatory" movements of
the ipsilateral eye are due to the action of the
vertical recti and that a combined action of the
obliques and vertical recti is required to produce
"vertical" movements. Therefore, the presence of
"vertical" eye movements indicates that the floc-
culus stimulation influenced both anterior and
posterior canal pathways. In our study, the laten-
cies of the contralateral 135° axis component
were longer than those of the contralateral 45°
axis component. This difference implies that the
effect on the posterior canal pathways was less
direct and possibly not the result of stimulation
of Purkinje cells.
The eye rotation axes of the horizontal and
rotatory classes, as defined in both the present
and previous experiments, are consistent with the
preferred axes of the visual CFs referenced to the
dominant eye (Graf et aI., 1988; Simpson et aI.,
1981). That is, the abduction component of the
response of the ipsilateral eye to stimulation of
zone 2 is a rotation about the preferred axis of
the vertical axis CFs, whereas the predominant
ipsilateral and contralateral components evoked
by stimulation in zones 1 and 3 are spatially
closely associated with the ipsilateral 135° axis
CFs and the contralateral 45° axis CFs, respec-
tively. For all three relations the evoked eye move-
ment would have resulted in excitation of the
respective CFs if the eye had been viewing a
stationary visual world. It should be noted that
the absence of a vertical class of eye movements
for the ipsilateral eye substantially improves the
correspondence between the classes of visual CFs
and the classes of evoked eye movements since
the roll (nasal-occipital) axis is not a preferred
axis of the CFs.
264 John I. Simpson, Johannes Van der Steen, and Joep Tan
Topographical Organization ofthe Flocculus
The organization of the floccular white matter
into compartments revealed by AChE histochem-
istry can be directly related to particular classes
of eye movements and, thus, to particular VOR
pathways. Of the three largest compartments, the
middle one (zone 2) is related to the horizontal
canal pathways since the major component of the
eye movements evoked by stimulation in this
compartment is abduction of the ipsilateral eye.
The two bordering compartments (zones 1 and
3) are both related to the anterior canal pathways
since the major components of the evoked eye
movements are the consequence of decreasing the
activity of the ipsilateral superior rectus and the
contralateral inferior oblique muscles ·and/or in-
creasing the activity of the ipsilateral inferior
rectus and the contralateral superior oblique
muscles.
In this study we were unable to distinguish
zone 1 from zone 3 on the basis of differences in
the patterns of evoked eye movements. Stimula-
tion of both of these zones typically resulted in
eye movements whose largest component was a
CCW rotation of the ipsilateral (left) eye about
its 135° axis. Furthermore, the range of amplitudes
ofthe accompanying conjugate component about
the contralateral 45° axis was also similar for
stimulation of zone 1 and zone 3. Although, these
findings suggest that both zones receive ipsilateral
135° axis CFs and contralateral 45° axis CFs, they
do not preclude the possibility of a differential
distribution within each zone of these two CF
classes, which arise from different parts of the
rostral dorsal cap and the adjacent ventrolateral
outgrowth. From an autoradiographic study of
the CF projections to the cat flocculus, Gerrits
and Voogd (1982) concluded that the rostral dor-
sal cap and the ventrolateral outgrowth each
project differentially to both zones 1 and 3. Since
there is no anatomically precise border between
the rostral dorsal cap and the ventrolateral out-
growth, a first approximation association may
be made between the rostral dorsal cap and the
45° axis CFs and between the ventrolateral out-
growth and the 135° axis CFs (Leonard et aI.,
1988; Simpson et al., 1981). The extent to which
these two physiologically distinguishable classes
of CFs project to separable areas in the rabbit
flocculus remains to be determined. Nevertheless,
the shared organization of the preferred axes of
the visual CFs and the rotation axes of the pre-
dominant classes of eye movements evoked by
stimulation in anatomically distinguishable floc-
cular compartments provides a framework-in
a literal sense-for investigating the meaning of
the geometry inherent in the CF zones and the
orthogonally directed parallel fibers.
Acknowledgments. Supported by National Insti-
tutes of Health Grant NS-13742, Medigon Grant
900-550-092, and the Netherlands Organization
for Scientific Research (NWO).
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to lateral vestibular nucleus from cerebellar c1im bing
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Andersson, G., and Oscarsson, O. (1978b): Climbing
fiber microzones in cerebellar vermis and their pro-
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Baker, R.G., Precht, W., Llinas, R. (1972): Cerebellar
modulatory action on the vestibulo-trochlear path-
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Brodal, A., and Kawamura, K. (1980): Olivocerebellar
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Ezure, K., and Graf, W. (1984): A quantitative analysis
of the spatial organization of the vestibulo-ocular
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Grar, W., and Simpson, J.1. (1981): The relations between
the semicircular canals, the optic axis, and the extra-
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mals. In: Progress in Oculomotor Research Dev. N euro- cerebellar anterior lobe as revealed by the projection
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14
The Dynamic Selection Hypothesis:
A Proposed Function for
Cerebellar Sagittal Zones
James R. Bloedel and Thomas M. Kelly
This chapter reviews our recent studies of the
climbing fiber's heterosynaptic action on individ-
ual Purkinje cells and presents a new hypothesis,
the "dynamic selection" hypothesis, regarding the
functional basis for the sagittal zones of the cere-
bellum. This hypothesis is developed through a
discussion of our findings as well as those from
other laboratories in the context of two theories of
climbing fiber function: one proposing that these
afferents perform a major role in establishing en-
grams in the cerebellar cortex required for motor
learning and the other arguing that climbing fibers
are most critical for real-time operations per-
formed by the cerebellum during motor execution.
Our experiments support the latter view by dem-
onstrating a specific short-term action of climb-
ing fibers on simple spike responses in passive
paradigms as well as in multiple, sagittally organ-
ized Purkinje cells during perturbed locomotion.
Other studies examining the effect of cerebellar
ablation on the classically conditioned nictitating
membrane/eyeblink response in rabbits substan-
tiate our previously held contention that the cere-
bellum is neither sufficient nor necessary for this
type of conditioned behavior currently being used
as models for motor learning.
The sagittal organization ofthe cerebellum has
been well recognized since its original description
by Jansen and Brodal (1940). Since that time, this
principle has been progressively refined with il-
lustrations of its organizational details, its specific
fiber systems, and its modifications occurring
throughout phylogeny. The original description
of the sagittal zones focused on the organization
ofthe cotticonuclear projection, the projection of
Purkinje cells to nuclear neurons. This projection
was divided into three large sagittal zones, each
corresponding to a region of the cerebellar cortex
and a nonoverlapping region of termination
within the deep cerebellar nuclei.
The historic observations of Voogd (1969)
based on the characteristics of fiber systems found
within the white matter underlying the cerebellar
cortex revealed that the cerebellum may be divis-
ible into more than the three sagittal zones des-
cribed by Brodal and Jansen (1940). Subsequent
studies clearly showed that the multiple sagittal
bands reported by Voogd also describe the sagittal
distribution of Purkinje cells projecting to discrete
nonoverlapping regions in the cerebellar nuclei
(Courville et aI., 1973; Courville and Diakiw, 1976;
Haines et aI., 1982). The careful anatomical experi-
ments of Dietrichs and colleagues (Dietrichs, 1981;
Dietrichs and Walberg, 1980) further characterized
the discrete, nonoverlapping projections of cortico-
nuclear fibers to regions of the cerebellar nuclei by
illustrating the wormlike progression ofPurkinje
cell terminals from progressively more medially
or laterally located sagittal bands of Purkinje
cells.
Parallel anatomical experiments in other labo-
ratories revealed that the termination of climbing
fibers in the cerebellar cortex also was localized
to sagittal bands (Courville, 1975). Several studies
in many species have now substantiated that
cells located in specific regions of the inferior
olive terminate in the contralateral cerebellar
cortex in a specific distribution of sagittal bands
(Brodal and Kawamura, 1980). Intriguingly, the
dimensions of these sagittal bands are similar to
267
268
those describing the organization of the cor-
ticonuclear projection (Voogd and Bigr, 1980;
Groenewegen et al., 1979; Groenewegen and
Voogd, 1977). The correspondence between the
sagittal bands describing the termination patterns
of olivocerebellar fibers and those characterizing
the origin of corticonuclear fibers is critical to the
hypothesis developed in the discussion below.
In addition to the olivocerebellar and cortico-
nuclear fibers, major components of the nucleo-
cortical projection, the recurrent collaterals of
cerebellar nuclear neurons coursing to the granu-
lar layer of the cerebellar cortex as mossy fibers
(Tolbert et ai., 1976), also appear to be organized
sagittally (Tolbert et ai., 1978). Although excep-
tions exist, nucleocortical fibers originating from
specific sites within a cerebellar nucleus project
to relatively nonoverlapping, sagittally distrib-
uted regions of the cerebellar cortex (Dietrichs,
1981; Dietrichs and Walberg, 1980).
The electrophysiological experiments of
Robertson and. Laxer (1981) provide evidence
that the sagittal organization of the climbing fiber
system also characterizes its somatotopy. Based
on their findings, complex spike responses of
Purkinje cells evoked by discrete stimuli applied
to specific regions of the body surface occur in
neurons organized sagittally within the cerebellar
cortex. This electrophysiological observation fits
well with the combined morphological and elec-
trophysiological studies of Gellman et ai., (1983,
1985), illustrating that neurons at least within
the dorsal accessory subnucleus of the inferior
olive are somatotopically organized. Consequently,
olivary neurons at anyone site not only would
respond to stimulation of specific regions of the
body surface but also would project to a specific
sagittally distributed termination site. This pattern
of termination also characterizes the sagittal dis-
tribution of collaterals from individual olivary
neurons (Rosina and Provini, 1983).
Last, the elegant anatomical studies of Houk
and Gibson (1987) illustrate that specific compo-
nents of the corticonuclear and olivocerebellar
projections comprise subcircuits of cerebellar
afferent and efferent pathways that relate specific
olivary and cerebellar cortical regions with a
specific population of cerebellar nuclear neurons.
These studies show that olivocerebellar fibers
project to discrete groups of Purkinje cells that
James R. Bloedel and Thomas M. Kelly
in tum impinge on cerebellar nuclear neurons,
providing an input to the olivary site from which
the climbing fiber projection originates.
Also important to the hypothesis developed
in this chapter is the observation that the soma-
totopic organization of mossy fiber inputs to the
cerebellum does not appear to be organized in
sagittal strips similar to those characterizing the
olivocerebellar and corticonuclear projections.
The studies of Shambes et ai., (1978) and Bower
and Woolston (1983) provide strong evidence
that stimuli applied to specific regions of the
body surface activate mossy fiber projections to
widespread regions of the cerebellar cortex in
a distribution that has been termed a "patchy
mosaic." Each body region is represented in
multiple patches, and the patches are organized
in a complex map of noncontiguous cutaneous
regions. Consequently, mossy fiber inputs acti-
vated from any specific site on the body surface
actually are distributed to multiple sagittal zones.
As a result, the afferent projection conveying most
of the information pertaining to time- and ampli-
tude-dependent modulation of peripheral inputs
and activity in central pathways does not appear
to obey the most pervasive anatomical organiza-
tional feature of the cerebellum: the sagittal zones.
Operation Performed by the
Climbing Fibers
in the Cerebellar Cortex
Despite the fact that the electrophysiological
events evoked by the climbing fibers on the
dendritic tree of Purkinje cells is now reasonably
well understood due largely to the studies of
Eccles et ai., (1966) and the subsequent work of
Llinas and colleagues (Llinas, 1985; Llinas and
Nicholson, 1976; Llinas and Sugimori, 1982;
Pellionisz and Llinas, 1977; Tank et ai., 1988), the
aspect of motor function to which this unique
afferent system contributes remains a subject of
extensive experimentation and discussion. Al-
though there are currently several specific hypo-
theses regarding the precise function of this system,
most proposals fall into two categories. One view
argues that the climbing fiber system plays a
critical role in establishing engrams important
for motor learning within the cerebellar cortex.
14. Dynamic Selection Hypothesis
This argument has been supported principally
by two avenues of experimentation. The first ex-
amined the effectiveness of conjointly stimulating
climbing and mossy fiber inputs to Purkinje cells
in producing a long-term modification in Purkinje
cell responsiveness (Ekerot and Kano, 1985; Ito
et aI., 1982; Ito and Kano, 1982; Kano and Kato,
1987). A second group of experiments has focused
on providing evidence that sites within the cere-
bellum are both sufficient and essential for the
establishment of certain types of motor learning.
Most of these experiments have employed the
classical conditioning of the nictitating mem-
brane eyeblink reflex in the rabbit as a model
(McCormick and Thompson, 1984a, b; Thompson,
1986; Yeo et al., 1984, 1985a, b). These studies
have used both electrophysiological recording
within cerebellar nuclei as well as ablation experi-
ments to support this view. The data indicate that
lesions in a specific region ofthe interposed nuclei
(McCormick and Thompson, 1984a, b) or lobule
HVI of the cerebellar cortex (Yeo et aI., 1984,
1985a, b) eliminate the capacity of a conditioned
animal to execute the conditioned reflex but do
not produce significant effects on the performance
of the unconditioned reflex. Furthermore, the
climbing fiber system has been strongly implicated
in the process responsible for establishing the
proposed memory traces within the cerebellum
(McCormick et aI., 1985; Thompson, 1986).
Alternate views of climbing fiber function have
focused on the operational role this system may
exercise during ongoing motor behavior (Bloedel
and Ebner, 1985; Llimis, 1985). An extended series
of experiments in our laboratory supports this
view (for review see Bloedel and Ebner, 1985;
Bloedel and Zou, 1987; Bloedel and Zuo, 1989)
and was was the basis for proposing the gain
change hypothesis (Bloedel et aI., 1983; Bloedel
and Ebner, 1985). A general overview of the initial
findings leading to the formulation of the gain
change hypothesis was presented previously
(Bloedel and Ebner, 1985; Bloedel and Lou, 1987)
and therefore will be reiterated in only enough
detail to provide the background necessary for
the argument to be presented below.
The initial series of experiments done together
with Ebner (Bloedel et aI., 1983; Ebner and Bloedel,
1981, 1984; Ebner et aI., 1983) employed passive
paradigms in decerebrate, unanesthetized animals.
269
In general, the experiments were designed specifi-
cally to eliminate the need for electrically sti-
mulating the climbing fiber input to a recorded
Purkinje cell, consequently minimizing any effects
due to abnormal recruitment patterns or activa-
tion rates of the climbing fiber inputs. Two types
of paradigms were developed. One used spon-
taneously occurring complex spikes to trigger
peripheral stimuli activating mossy fiber inputs
at specified times after the occurrence of the cell's
climbing fiber input (Ebner and Bloedel, 1981,
1984). The second approach used a trial-sorting
technique comparing the simple spike respon-
siveness of Purkinje cells in trials in which the
climbing fiber input was activated with that in
trials in which complex spikes were not evoked by
the peripheral stimulus (Ebner et al., 1983).
In studies examining the effects of sponta-
neously occurring climbing fiber inputs on the
responsiveness of Purkinje cells to their mossy
fiber-granule cell-parallel fiber input, the complex
spikes were electronically discriminated and used
to trigger the application of either natural peri-
pheral stimuli (Ebner and Bloedel, 1981) or
electrical stimuli applied to the surface of the
cerebellar cortex to activate a parallel fiber beam
(Ebner and Bloedel, 1984). With these paradigms
it was possible to determine any changes in the
simple spike responsiveness of Purkinje cells at
various intervals after their climbing fiber input.
Furthermore, possible "conditioning" effects of
repeatedly coupling the activation of climbing
fiber and parallel fiber inputs to the same Purkinje
cell could also be tested.
In brief, these experiments demonstrated that
the climbing fiber input to a Purkinje cell modified
its responsiveness to the activation of mossy
fibers. However, in contrast to the studies em-
ploying the conjunction stimulation paradigm
(Ekerot and Kano, 1985; Ito and Kano, 1982; Ito
et aI., 1982), the responsiveness of Purkinje cells
increased rather than decreased. This increase in
responsiveness was reflected by changes in ampli-
tude of both excitatory as well as inhibitory re-
sponse components. Furthermore, the effect of
climbing fiber inputs on the responsiveness of
Purkinje cells was short lasting, persisting for
only 200 msec. Successively pairing climbing fiber
and parallel fiber inputs to the same Purkinje cell
did not produce a prolonged modification in its
270 James R. Bloedel and Thomas M. Kelly

A responsiveness. Consequently, when a peripherally

activated mossy fiber input or a directly activated
Non-CF Trial
parallel fiber input was no longer coupled with
SS-PSTH the cell's climbing fiber input, the Purkinje cell's
responsiveness returned to control levels within
an extremely short period of time.
An example of the data obtained with the
sorting paradigm is shown in Figure 14.1. With
CF Trial SS-PSTH
this technique, two histograms were constructed,
one a nonclimbing fiber trial simple spike histo-
gram and the other a climbing fiber trial simple
spike histogram. The nonclimbing fiber trials
were those in which a tap to the forepaw of the
All Trial CF-PSTH acutely decerebrate cat did not evoke a complex
spike within the response window indicated by
the arrows below the abscissa of each histogram .

• o n = 200
In contrast, the climbing fiber trials were those
in which the Purkinje cell responded to the tap
with a complex spike within the same response
All Trial SS-PSTH window. The amplitudes of the histograms were
then normalized to the number of trials (n). The
standard all-trial histogram combining the non-
,... climbing fiber and climbing fiber trials is shown
~ . 20 n = 46 in Figure 14.lD.
>-1-
CF-Trial Aligned ~ :E Critical to the hypothesis to be developed from
~~ these data is the fact that the simple spike re-
SS-PSTH ~: sponsiveness of Purkinje cells was greater in the
Q:~
~
climbing fiber trials (B). Note that the amplitudes
msec 600 of both the excitatory (E) as well as the inhibitory
Tap (I) response components were increased. These
2.5 mm
modifications in response amplitude were quanti-
Figure 14.1. Increase in the amplitude of both an exci- fied by calculating the gain change ratio for each
tatory as well as an inhibitory component in a Purkinje component. In this example, the excitatory re-
cell's response to a tap applied to the dorsum of the
sponse component increased by a factor of 3.63
forepaw. The all-trial histogram (D) illustrates the re-
(RE = 363%), and the inhibitory component in-
sponse of this Purkinje cell to 200 successive stimuli
applied with the time course shown by the tap trace creased in amplitude by a factor of 1.86 (R) =
below the histograms. The arrows indicate the response 186%). These data typify the statistically verified
window in which the occurrence of complex spikes was finding in these experiments: the occurrence of a
considered a response evoked by the peripheral stimu- climbing fiber input resulted in an increased
lus and consequently was used as the basis for sorting amplitude of both excitatory as well as inhibitory
the 200 trials into two groups. One group, consisting responses evoked by the mossy fiber- granule cell-
of 154 trials, was used to construct the non climbing parallel fiber input to the same cell.
fiber trial SS-PSTH (A); the other group of 46 was used These data, together with a multiunit study in
to construct the climbing fiber trial SS-PSTH (B). A which the responses of up to three Purkinje cells
histogram from these trials aligned on the evoked
were recorded simultaneously (Bloedel et aI., 1983),
complex responses is shown in E. The excitatory and
inhibitory response components are demarcated in
were used as a basis for proposing the gain change
both A and B. A comparison of the climbing fiber trial ~----------------------------------
(B) and nonclimbing fiber trial (A) simple pike histo- the gain change ratios resulting from this calculation is
grams was done by calculating the gain change ratios shown in B. (Reprinted with permission from Ebner
for each of the two response components. The value of et aI., 1983.)
14. Dynamic Selection Hypothesis 271

hypoth~sis. This operational view contends that ing fiber activation during the conditioning phase
the action of the climbing fiber demonstrated in should persist after acquisition, even though the
the experiments just reviewed reflects a mech- climbing fiber inputs may diminish or cease. In
anism by which these afferents produce a short- other words, if the climbing fiber inputs were
lasting increase in a Purkinje cell's responsiveness acting as a "teacher," the modification in Purkinje
that is critical to the processing of information cell responsiveness should persist even after these
conveyed by the mossy fiber inputs to the cerebel- afferents are no longer activated. In contrast, the
lar cortex. It should be emphasized that the find- data demonstrated that the modulation of simple
ings on which this hypothesis is based are very and complex spike activity occurred in parallel:
different in two respects from those resulting from increases or decreases in climbing fiber activation
the studies employing the conjunction paradigm. were statistically associated with parallel changes
First, the data from our experiments indicate that (i.e., increases or decreases, respectively, in the
the responsiveness of Purkinje cells after climbing depth of modulation of simple spike activity
fiber inputs increases rather than decreases due throughout the period of observation). No data
to the heterosynaptic action of climbing fibers. indicated that a persistent effect in the simple
Second, the effect observed in our studies is spike responses of Purkinje cells was induced by
shortlasting. A persistent change in Purkinje cell the action of the climbing fiber system. Conse-
responsiveness could not be produced by re- quently, the findings in this study are appreciably
peatedly coupling a cell's spontaneous climbing more consistent with the gain change hypothesis
fiber input with the electrically or naturally evoked than the learning hypothesis.
mossy fiber-granule cell-parallel fiber input to Also inconsistent with the learning theory were
its dendritic tree. the results of studies examining the effects of
Despite the findings supporting the existence chronically removing the entire ipsilateral cere-
of a mechanism by which climbing fibers can bellar cortex and nuclei on the capacity of decere-
exert a functionally important short-term effect brate ferrets to acquire this task (Bloedel et aI.,
on Purkinje cell responses, discussions continue 1987; Bloedel and Zuo, 1989). Although a motor
regarding an alternate or even an additional role deficit was apparent in both the unperturbed as
for climbing fibers in motor learning (Kano and well as the perturbed step cycles of these animals,
Kato, 1987; McCormick et aI., 1985; Thompson, they were capable of learning to step over the
1986). Our laboratory has approached this gen- bar despite the fact that massive regions of the
eral issue using two different preparations. The cerebellum had been removed. Clearly, the cere-
first employed a perturbed locomotion paradigm bellum was not necessary or essential for the
in which an acutely decerebrate locomoting cat manifestation of this type of conditioned behavior.
or ferret encountered a bar placed in the trajectory To clarify whether or not the classical condi-
of one of its forelimbs during swing phase on each tioning of the nictitating membrane response of
successive trial (Lou and Bloedel, 1986). After an the rabbit occurs in the absence of the cerebellum,
amazingly few (5-30) trials, these animals were experiments were performed in acutely decerebrate
conditioned to step over the bar (Lou and Bloedel, rabbits with functionally total cerebellar abla-
1987a). In one of the experiments examining the tion. In these preparations all the cerebellum was
role of the cerebellum in this process (Lou and removed, including the nuclei, except a small
Bloedel, 1987b), up to five Purkinje cells were portion of the lateral-most tips of the hemi-
simultaneously recorded in regions of the cere- spheres. Decerebrate rabbits were employed
bellar anterior lobe receiving climbing fiber inputs because of our interest in recording from single
activated in response to this type of perturbation. neurons throughout the entire time course of the
The relationship between simple and complex acquisition and performance of the conditioned
spikes was assessed throughout the acquisition, nictitating membrane/eye blink reflex in subse-
performance, and extinction of this conditioned quent experiments. These preparations made it
behavior. It was hypothesized that if the learning possible to apply enough stimuli successively to
theory were correct, any change in simple spike condition the reflex while recording from a single
activity associated with the Purkinje cell's climb- neuron.
272 James R. Bloedel and Thomas M. Kelly

:\/,
A CR Failure
C Tone Only

\/
t
+T +8
t
+T
B D Tone Only-Extinction
CR-UCR

t
+T
~.
+8
•
--
-.-
>-
.::1
lIIe

a:: t
0
+T
,
msec
~ ,
600

Figure 14.2. Individual trials obtained from an experi-
ment in which the eyeblink reflex was classically condi-
tioned in an acute decerebrate-decerebellate rabbit,
The times at which either a tone stimulus (T) or a
periorbital electrical stimulus were applied are shown
below the time axis of each record. The trials in A and
B were obtained during the conditioning of the eyeblink
reflex, A showing a trial in which there was a failure of
the conditioned response and B a trial in which a
conditioned response was evoked. After conditioning
a conditioned response could be evoked in trials in
which only the tone stimulus was applied (C) until
extinction occurred (D).

The recordings in Figure 14.2 illustrate single the decerebrate animal is very similar to that
trials obtained from a series of experimental ob- observed in intact rabbits (Gormezano et aI.,
servations in one rabbit. In these experiments a 1983). Our studies have also demonstrated that
500-Hz tone was used as a conditioned stimulus, the conditioned response in decerebrate rabbits
which was terminated coincidentally with the is temporally related to the interstimulus interval.
unconditioned stimulus, a single periorbital Changing the interval between the conditioned
electrical pulse. The nictitating membranejeye- stimulus and the unconditioned stimulus also
blink reflex was monitored using an emitter- modifies the interval between the conditioned
transducer system that measured changes in the stimulus and the conditioned response. Conse-
reflection of an infrared beam of light off the quently, the temporal relationship between the
conjunctiva of the eye. In Figure 14.2A the con- onset of the conditioned response and the un-
ditioned and unconditioned stimuli were both conditioned stimulus remained relatively fixed,
applied but no conditioned response was evoked, as expected in this type of associative pheno-
a so-called failure. Figure 14.2B illustrates a trial menon (Gormezano et aI., 1983).
in which both a conditioned and an unconditioned Although there always remains some debate
response were evoked. Once the conditioning as to whether the mechanisms of associative
was complete, the reflex was activated by the ap- conditioning in this task are comparable in de-
plication of only the tone stimulus (Figure 14.2C). cerebrate and intact animals, the data clearly
After extinction, no response was evoked by the reveal that the cerebellum is not essential or
tone (Fig. 14.2D). Similar data in five animals necessary for this type of conditioned behavior
have been completely analyzed systematically, to occur. These findings also imply that the climb-
and controls for sensitization have been perform- ing fiber system is not establishing engrams within
ed (see Kelly et aI., 1990). Plots of the acquisition the cerebellar cortex critical for this type of motor
and extinction of this conditioned behavior illus- learning, as previously proposed (Thompson,
trate that the time course of the conditioning in 1986).
14. Dynamic Selection Hypothesis 273

In our: view, there are two feasible explanationsanterior lobe. Similar to the motor learning
for the difference between the findings in our experiments, the forelimb of cats and ferrets was
experiments and those of Thompson's laboratory perturbed by a bar interposed into the swing
(McCormick et aI., 1984a, b; Thompson, 1986) phase during locomotion. In the experiments
and Yeo and colleagues (Yeo et aI., 1984, 1985a, b).reported in this section, the perturbation was
First, cerebellar ablation can have potent, long- applied intermittently, on the average of every 7
lasting effects on the excitability of brain stem to 9 step cycles.
nuclei (see Gilman et aI., 1981, for review). In a For the purpose of examining the relationship
recent preliminary report on adaptation of the between the activation of the cells' climbing fiber
VOR in goldfish (Baker et aI., 1988; Weiser et aI., inputs and their simple spike responsiveness to
1988), the prolonged tonic effects produced by the perturbation, a new multiunit data processing
cerebellar ablation were found to last for as long algorithm was developed. This method, referred
as 1 month. These data not only reiterate the to as the RTPR (real time postsynaptic response)
importance of the cerebellum's tonic action on (Lou and Bloedel, 1986), quantitatively assesses
extracerebellar nuclei, but they also demonstrate the amplitude of the population's response to a
these effects in the context of a specific type of peripheral event on a trial-by-trial basis. This
motor learning. analysis has been described extensively elsewhere
Second, cerebellar ablation can produce a defi- (Lou and Bloedel, 1986). In brief, the algorithm
cit in motor performance that may be responsible is based on the assumption that neighboring,
in part for the negative findings in some ablation sagittally oriented Purkinje cells localized to a
studies. A recent report by Welsh and Harvey surface folium have components oftheir terminal
(1989) showed that cerebellar ablation decreases fields that contact the same nuclear neuron. This
the force associated with the movement of the assumption has substantial anatomical support.
nictitating membrane over the conjunctiva. Conse- Sagittally oriented Purkinje cells have been shown
quently, a system requiring a specific force to to project to the same discrete region within the
move a lever arm attached to a low-friction cerebellar nuclei (Bishop et aI., 1979; Dietrichs,
potentiometer in order to measure the amplitude 1981; Dietrichs and Walberg, 1980; Houk and
of the nictitating membrane reflex may have too Gibson, 1987). Second, anatomical studies de-
much inertia to detect adequately the reflex after monstrated that the termination field of indivi-
cerebellar ablation. The infrared-emitting diode dual Purkinje cell axons within a region of the
detec.tor used in our paradigm measured the re- cerebellar nucleus is fairly extensive, likely con-
traction of the eyeball associated with the reflex tacting dendrites of several neighboring nuclear
rather than the nictitating membrane's movement neurons (Bishop et al., 1979; see also Palkovits
across the eyeball. Consequently, a reflex weak- et aI., 1977). For the purposes of the analysis, it
ened by cerebellar ablation may be detectable was assumed that each of the recorded neurons
with the infrared detector system while being impinges on a simple lumped-circuit linear model
immeasurable with a transducer system requiring of a nuclear neuron and that the unitary post-
the displacement of a mechanical load. synaptic potential (PSP) evoked by each of the
Purkinje cells is comparable, an argument that
also has some support in the literature (Fanardjian
and Sarkissian, 1980).
Response Properties of Sagittal Analytically, each simple spike generated by
Strips of Purkinje Cells during each Purkinje cell is converted to an analog signal
Perturbed Locomotion with a time course comparable to that of the
inhibitory PSP (IPSP) evoked in nuclear neurons
The perturbed locomotion paradigm introduced by the action of Purkinje cells (Ito et aI., 1964,
above has been actively employed in our labora- 1970). The simulated PSPs are then summed
tory to investigate the response properties of linearly in each trial using the lumped-circuit
sagittally oriented strips of 3 to 6 Purkinje cells model of the nuclear neuron. This method pro-
localized within various sagittal zones within the vides an analog response characterizing the
274 James R. Bloedel and Thomas M. Kelly

,activity of all the recorded neurons. Further-
more, the amplitude ofthis analog representation
of the population's output can be integrated bet-
ween windows demarcating the beginning and
end of the response period.
The data shown in Figure 14.3 characterize the
initial findings from this set of experiments. This
figure shows data from two selected trials obtained
while recording responses from a set of five sagit-
tally oriented Purkinje cells in the paravermal
region of the anterior lobe of an acute decerebrate
ferret. Records obtained from a trial in which the
locomotor cycle was not perturbed are shown in
A-I, and records from a trial in which the forelimb
ipsilateral to the recording site was perturbed
during swing phase are shown in J-R. For each
trial the digitized spike trains of each cell's simple
spike activity are shown in A-E and J-N, re-
spectively. The time of occurrence of complex
spikes in each cell is indicated by symbols located
beneath the time axis of each digitized spike train.
Different symbols are used to represent the
climbing fiber activity of each cell. The time of
occcurrence of all the cell's complex spikes during
the entire 1600 ms of recording is shown in F and
O. The RTPR calculated from the spike train of
each neuron is shown in G and P for the unper-
turbed and perturbed trials, respectively. The time
course of the protraction and retraction of the
ipsilateral forelimb is shown in Hand Q. These
records represent the movement of a low-friction
potentiometer ·arm attached by a string to the
forelimb of the ambulating anima,l. The time
course of the perturbation bar's movement is

Unperturbed Perturbed

Cell 1 Cell 1

A L I I
i!i
"
I 1111 I Complex Spikes
,
J l'l;;,,__.uIl1llIlIUII---lLJ..JIIlJ..I__J Complex Spikes

Cell 2 F J " " Cell 2 o J~

Q ~+

B L I " Ig II lIP I RTPR

j 'if' "," ,,,,,,,,.,
GI¥ "1' 'H' I '
Ktl I I III II 1111 II I
'" '"
Cell 3 Cell 3

C lU!HL-~~__~I I~I~I I~1 Right Leg L 141____ , ~I!I~I LI- -l . - '

Cell 4
~ Cell 4

t
H I I

o I I 1111 I " I 11111 Perturbation ML II II III 1111 I II

+
III
Perturbation

CeliS
~;:::::=:; R~
E Lilli III III
I .1:=1

11111111
x
'-----'-__-'-__-'---', co
II II III ..
o
,
1600

9430,DEO

Figure 14.3. Analysis of the responses of five simul-
taneously recorded Purkinje cells during an unper-
turbed (A-I) and perturbed (J-R) locomotor cycle.
A- E and J - N: Digitized simple spike activity recorded
from each Purkinje cell during the unperturbed and
perturbed trials. The time of occurrence of each cell's
complex spike activity is shown below each trace with
different symbols. The time of occurrence of these
complex spikes is shown in the composite diagrams in
F and 0, with each cell's climbing fiber responses
shown on a separate line. G and P: The RTPR cal-
culated from the unperturbed and perturbed trials. H
and Q: The protraction and retraction of the ipsilateral
forelimb. I and R: Time course ofthe movement ofthe
perturbation bar. SS, simple spikes; CS, complex spikes;
RTPR, real-time postsynaptic response. (Reprinted
with permission from Bloedel and Lou, 1987.)
14. Dynamic Selection Hypothesis
shown i:Q. R. The arrow in Q illustrates the time
the ferret's forelimb contacted the perturbation
bar. The arrowheads above the axis in P and G
indicate the visually determined response window
used to calculate the response amplitude and the
synchrony of the climbing fiber inputs to the
recorded neurons.
Two marked differences between the responses
observed in the unperturbed and perturbed trials
are clearly evident in this figure. First, the pertur-
bation evoked synchronous climbing fiber inputs
to the simultaneously recorded Purkinje cells
(compare F and 0). Second, this synchronous
activation of climbing fiber inputs was also as-
sociated with a much more dramatic modulation
ofthecombined simple spike activity ofthe recor-
ded neurons (compare G and P).
These two response characteristics, the syn-
chronous activation of climbing fiber inputs and
the amplitude of the RTPR, were analyzed syste-
matically to determine quantitatively the differences
between the observations in the perturbed and
unperturbed trials. These analyses revealed (see
Lou and Bloedel, 1986, for further details) that
the occurrence of the perturbation resulted in a
significant increase in the synchronous activation
of climbing fiber inputs during the phase of loco-
motion demarcated by the response windows
across all sets of neurons analyzed. Furthermore,
there was a statistically significant correlation
between the extent of the synchrony and the
amplitude of the RTPR. The gain change ratio
was also determined. This quantity depicts the
percent increase in response amplitude of the
RTPR when the climbing fiber inputs to at least
50% of the recorded neurons were activated.
These data, together with the results from the
other analyses, strongly imply that there is a
relationship between the synchronous activation
of the climbing fiber inputs to the simultaneously
recorded neurons and their simple spike modula-
tion in response to the perturbation.
In addition to showing that climbing fiber
inputs to sagittally distributed Purkinje cells are
synchronously activated under appropriate be-
havioral conditions, these findings also strongly
support the gain change hypothesis reviewed
above. The correlation between the response
amplitude and the synchrony index as well as the
increased gain change ratio implies that this
275
synchronous volley of climbing fiber inputs
induces an increase in the responsiveness of the
Purkinje neurons to inputs from the mossy fiber-
granular cell-parallel fiber input activated by the
same behavioral conditions.
The Dynamic Selection Hypothesis:
An Initial Proposal Regarding
the Functional Importance
of Cerebellar Sagittal Zones
Based on these initial findings and some of the
distinctive morphological features of the organ-
ization of both climbing and mossy fiber inputs
to the cerebellar cortex reviewed above, we are
formulating a hypothesis regarding a mechanism
by which the sagittal zonation of the cerebellar
cortex could meaningfully contribute to the in-
formation processing required for the cerebellum's
role in regulating coordinated motor behavior.
The hypothesis focuses on providing a mechanism
that functionally implements the refined topo-
graphy characterizing the corticonuclear and
olivocerebellar systems as well as the output pro-
jections from the deep cerebellar nuclei (see Houk
and Gibson, 1987), together with the unique short-
lasting heterosynaptic action of climbing fibers
described above.
As emphasized earlier, functionally there are
interesting dichotomies in the topographic and
somatotopic organization of cerebellar afferent
and efferent systems. The relatively comparable
distribution of zones describing the olivocere-
bellar and corticonuclear projections provides an
elegant example of a precise spatial relationship
between an afferent projection and the population
of output neurons on which they impinge. Intri-
guingly, the input system in this circuit does not
contain the afferents carrying critically important
quantitative information regarding ongoing
changes in the periphery or activity in descending
pathways. This information is conveyed by the
mossy fiber system, an afferent system consisting
of many different projections activated by a spec-
trum of peripheral receptor systems, spinal path-
ways, and descending projections (Bloedel and
Courville, 1981).
Furthermore, the mossy fiber system is a much
more distributed afferent system than the climbing
276
Perturbed Passive
Locomotion Tap
Figure 14.4. Distribution of mossy and climbing fiber
inputs associated with three different conditions, each
resulting in a perturbation applied to the dorsum of
the animal's forepaw. The distribution of mossy fiber
inputs (stippled patches) is the same in each of the three
fiber projection (see Bloedel and Courville, 1981,
for review). Although the mossy fiber input pro-
jects to the cerebellar cortex in a well-defined
organizational pattern characterized as a patchy
mosaic (Llinas, 1982; Bower and Woolston, 1983;
Shambes, et aI., 1978), these mosaic patterns con-
sist of multiple representations of noncontiguous
body regions extending over rather substantial
regions of the cerebellar surface. As a consequence,
punctate stimuli are likely to activate Purkinje
cells in several sagittal zones (Bloedel and Burton,
1970; Yu et aI., 1985). This dispersion ofmodula-
ted Purkinje and nuclear cell activity presumably
would minimize the contribution of the topogra-
phy characterizing the corticonuclear and nuclear
efferent projections to the spatial distribution of
inputs to extracerebellar sites.
Based on the premise that a mechanism must
exist to produce a spatially focused modulation
of Purkinje and nuclear cell activity appropriate
to a set of existing physiological conditions, we
are proposing the "dynamic selection hypothesis."
According to this view, the distribution of climb-
ing fiber inputs activated by specific patterns of
convergence in the inferior olive determines those
James R. Bloedel and Thomas M. Kelly
Perturbed
Volitional
Movement
conditions, hypothetically resulting from the activation
of afferents projecting from the forepaw (see text for
qualifications). The distribution of climbing fiber inputs
is shown as crosshatched strips.
populations of Purkinje cells that will be most
highly modulated by mossy fibers activated under
the same behavioral conditions. Functionally
relevant patterns of afferent convergence in the
inferior olive would selectively activate climbing
fibers projecting to a specific distribution of sagit-
tally distributed bands of Purkinje cells within
the cerebellar cortex (Robertson and Laxer, 1981).
These termination patterns would specify compo-
nents of the corticonuclear system that in turn
project to discrete populations of cerebellar nu-
clear neurons. Critical to this hypothesis is that
these specified populations of Purkinje cells would
be much more dramatically modulated by ac-
tivated mossy fiber inputs than Purkinje cells in
other sagittal zones due to the heterosynaptic
action of climbing fibers on the simple spike re-
sponsiveness of these neurons.
To provide a better qualitative understanding
of this view, three conditions are compared in
Figure 14.4. This example assumes that a stimulus
applied to the forepaw activates the same mossy
fiber input under three different behavioral condi-
tions: a passive tap of the forepaw dorsum, a tap
of the forepaw resulting from a perturbation of an
14. Dynamic Selection Hypothesis
"automlltic" movement such as locomotion, and
a tap of the forepaw dorsum resulting from a
perturbation applied during a volitional goal-
directed movement. The distribution of this input
is shown as stippled patches in each of the three
panels. It is acknowledged that this is a simplifi-
cation. Certainly, integration occurring in pre-
cerebellar nuclei could modify the distribution of
mossy fiber inputs responding to the peripheral
stimulus. However, this possibility is not pertinent
to the inferences to be drawn from the example.
It is also acknowledged that mossy fiber inputs
in addition to those responding to the forepaw
would be activated in each ofthe three conditions.
However, this example focuses only on the mossy
fiber input activated as a consequence of the tap
on the dorsum ofthe forepaw. Consequently, the
climbing fiber and parallel fiber inputs both con-
verge on different populations of Purkinje cells in
each circumstance. Based on the gain change hy-
pothesis, it is assumed that these patterns of con-
vergence "select" or specify the components of
the corticonuclear system to be most highly modu-
lated by the mossy fiber-granule cell-parallel
fiber inputs under each behavioral condition.
Because of the topography of the corticonuclear
projection, this interaction will also specify the
cerebellar output neurons to be most affected by
cerebellar cortical interactions.
Preliminary Experimental
Evaluation of the Dynamic
Selection Hypothesis
Our laboratory is currently engaged in a series of
experiments designed to test aspects of the dy-
namic selection hypothesis. These experiments are
being performed in locomoting, acutely decere-
brate cats in which two sagittal strips of Purkinje
cells are recorded simultaneously using two arrays
of five electrodes each. One set of electrodes is
placed in a more medial sagittal zone, usually
within the A or B zone, and the more lateral array
is localized on the same folium in either the C 1 ,
C z, or C 3. zone. The precise zone in which the
recording array is located is assessed based on its
distance from midline (Yu et aI., 1985) as well as
the criterion of Oscarsson (1979) regarding the
somatotopic organization of climbing fiber inputs
to each of the sagittal zones.
277
The objective of the experiment is to record
simultaneously from two zones receiving mossy
fiber inputs evoked by the perturbation of the
forelimb, with only one zone receiving climbing
fiber inputs activated by the same stimulus. For
the dynamic selection hypothesis to be tenable,
there should be a substantially greater modula-
tion of simple spike activity in the zone receiving
the synchronously activated climbing fiber input.
Figure 14.5 illustrates the data from one of the
sets of neurons recorded in the course of this
study. The format of this figure is similar to that
of Figure 14.3. The cells were recorded with both
a medial (array 2, K-Q) and a lateral (array 1,
A-J) array during a single trial in which the step
cycle of the cat was perturbed with the bar inter-
posed into the trajectory during swing phase. The
digitized simple spike activity of the six cells
recorded with the lateral array and the three cells
recorded with the medial array is shown in B-G
and L-N, respectively. The time of occurrence of
each cell's complex spike is shown beneath each
record using a different symbol. A and K show
the temporal relationship of the complex spikes
evoked in all of the neurons on both arrays, with
the complex spike activity of each Purkinje cell
shown on separate lines. The protraction and
retraction of the perturbed forelimb are shown in
identical records in I and P; and the contact of
the bar by the limb is shown in J and Q, also in
identical records. Last, the RTPR calculated from
the responses of each subgroup of neurons is
shown in Hand O.
Notice that four of the six Purkinje cells record-
ed with the lateral-most array received synchro-
nously-activated climbing fiber inputs in response
to the perturbation (A). A comparable synchrony
was clearly absent in the more medial zone (K).
Similarly the simple spike activity of the neurons
in the lateral zone was much more highly modu-
lated, as revealed by a comparison of the RTPRs
in Hand O. The responses recorded from this
particular set of neurons have been analyzed for
both the synchrony index as well as the RTPR
amplitude and gain change ratio. The synchrony
index for the lateral and medial groups of Purkinje
cells was 00405 and 0.114, respectively. The gain
change ratio for the lateral cells was 204.65%.
The value of this quantity for the medial group
could not be calculated because of the low syn-
278 James R. Bloedel and Thomas M. Kelly

ARRAY 1 ARRAY 2 chrony index for these neurons.

(C1 Zone) (A Zone) Clearly, in this experiment there is a dramatic
difference between the responses of the cells re-
.. ..
CS

A ·• K
. .. corded with the two arrays. Consistent with the
dynamic selection hypothesis, the simple spike
modulation was much more dramatic in the strip
of cells also receiving a synchronous volley of
Cell 1 climbing fiber inputs. More studies are required
.·
B I II I
Cell 2
II II
Cell 7
in order to evaluate this question further. How-
ever, these initial findings provide strong en-
C I I I111 I 111111 L .
1111111 III III 1111 I couragement for continuing to test the idea.
Cell 3 Cell 8
D 111111111 111111 III M ""~II",,illll~~---::-....,....,~ Conclusion and Comment
Cell 4 Cell 9
E JI N 111111110111 The data employing the perturbed locomotion
IllillUlllIlIllllW II
paradigm support the notion that at least a part
CeliS
F I11II1 111111 W H ofthe action of the climbing fiber system is related
to its capacity to produce a short-lasting enhance-
Cell 6 ment of the Purkinje cell's simple spike respon-
G 1111
• siveness to the mossy fiber-granule cell-parallel
fiber input. There continues to be little evidence
in any of our experiments for the view that the
o~ climbing fibers produce a long-term change in
the responsiveness of Purkinje cells. Not only
p ,-j_/'~~---!l have we failed to find any long-lasting changes,
but the changes we have observed consist of an
Pb
enhancement rather than a depression of the cell's
J IlIl responsiveness as required by several of the learn-
o msec 1600
ing theories. Furthermore, behavioral data from
other laboratories (Kim et aI., 1987; Mano et aI.,
Figure 14.5. Analysis of responses recorded simulta1 1986) have provided additional evidence that the
neously from electrodes in two different arrays, one
climbing fiber system is activated in relationship
located more medially in the A zone (K-Q) and one
to volitional arm movements in a manner con-
located laterally in the C, zone (A-J). Cells 1 to 6 were
recorded with array 1, whereas cells 7 to 9 were recorded
sistent with an operational role of the climbing
with array 2. All records were obtained during the fiber system in regulating the output of the cere-
same perturbation trial in an ambulating, acutely bellar cortex (Andersson and Armstrong, 1987;
decerebrate cat. B-G and L-N: Digitized simple spike Armstrong and Edgley, 1984; Bloedel and Ebner,
activity of each recorded Purkinje cell. The time of 1985; Llimls, 1985; Pellionisz, 1985) rather than
occurrence of each cell's complex spikes is indicated a role in establishing engrams in the cerebellum
beneath each record. These complex spikes are shown required for motor learning (Thompson, 1986).
in a composite diagram for each electrode array in A Furthermore, the lesion studies in our laboratory,
and K, with the complex spikes from each cell being (Bloedel et aI., 1987; Bloedel and Zuo, 1989; Kelly
shown on a separate line. Hand Q: RTPR calculated
et aI., 1990) continue to cast considerable doubt
from the cells' responses recorded with the lateral and
on the premise that the cerebellum is essential
medial electrode arrays, respectively. I and P: The
protraction and retraction of the forelimb recorded and sufficient for at least two types of conditioned
with a potentiometer attached by string to the ipsilat- reflex behaviour: the nictitating membrane/
eral forelimb. J and Q: The sensor output illustrating eyeblink reflex in the rabbit and the avoidance
the time of contact of the forelimb with the perturbation conditioning of the forelimb movement in the
bar. ferret.
14. Dynamic Selection Hypothesis
The dynamic selection hypothesis is intended
to provide an initial proposal regarding the
functional role of the cerebellar sagittal zones.
Although at present this view is highly qualitative
and very speculative, the initial studies designed
to test the suppositions on which it is based have
been supportive. One of its most appealing
features is its consistency with most ofthe organi-
zational and physiological features of the olivo-
cerebellar system. For example, this view can easily
incorporate the electrotonic coupling of inferior
olivary neurons (Llinas et at, 1974, Llinas and
Yarom, 1981) as well as the data from the multi-
unit recording studies of Llinas and colleagues
illustrating the inherent rhythmicity in the olivo-
cerebellar system and the expression of this
rhythmicity in the activation of sagittal strips of
Purkinje cells (Bower and Llinas, 1983; Sasaki
et at, 1989). In addition, the new electrophysio-
logical findings regarding the dynamic properties
of the dendritic tree of the Purkinje cells presented
in this symposium (see presentations by Llinas
and Sugimori in this volume as well as Llinas and
Sugimori, 1980a; Tank et at, 1988) provide direct
evidence for the existence of active modifications
in dendritic characteristics that could underlie
the change in responsiveness of Purkinje cells.
In closing, it should be emphasized that the
hypothesis presented here addresses only one
feature of the operation performed by the climbing
fiber input to the cerebellum. Specifically, this
view addresses its action on the responsiveness
of Purkinje cells and the spatial integration that
may result due to the overlapping relationship of
the olivocerebellar and corticonuclear projections.
The precise contribution of the climbing fiber
system to motor execution and the way in which
this system is integrated into the control systems
responsible for regulating ongoing movements
are not addressed. However, the consistency of
these data with other electro physiological and
anatomical findings from several laboratories is
contributing useful insights into the functional
characteristics of a neurobiological system whose
precise function has been an elusive subject for
investigators. It is appropriate that these increased
insights into this system's operational character-
istics are occurring at a symposium held to honor
the contributions of Ramon y Cajal, who so
279
elegantly systematized the fundamental charac-
teristics of the cerebellar circuitry.
Acknowledgements. We wish to thank Mr. Joel
McAlduff for his help in the experiments and
Ms. Deirdre Janus for her help in preparing the
manuscript. This research was supported by NIH
grant NS 21958 and NIH grant NS 07309.
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15
Cerebellar Output: Multiple Maps and
Modes of Control in Movement Coordination
W. Thomas Thach, S.A. Kane, J.W. Mink, and H.P. Goodkin
For the scientist, one of the chief goals in cerebellar
research has been to find physiological meaning
in the function of the anatomical circuitry. Ramon
y Cajal, in providing many of the anatomical
details of the circuitry, did not hesitate to specu-
late on its function. Since then, elucidation of.the
synaptic actions of the different neurons forming
those circuits has brought a functional explana-
tion closer to hand (Eccles et aI., 1967; Llinas,
1981). Yet there has remained the technical pro-
blem of relating the actions of the neural cir-
cuitry to the sphere of activity that it undoubtedly
principally controls, bodily posture and move-
ment.
For the neurologist, a chief goal has been to
recognize, from some abnormality in behavior,
when the cerebellum is injured, and whether dif-
ferent parts of the cerebellum give different mani-
festations of injury. From the classic ablation
studies of Fluorens and Luciani in animals through
to the elegant clinical studies of Babinski and
especially Holmes in man, it has been clear that
the behavioral deficits are principally if not ex-
clusively of posture and movement. Yet there has
been disagreement on whether or not function is
localized and if so how, and what the one over-
riding function of the circuitry really is.
The following studies attempt to answer some
of these questions through use of two relatively
new techqiques, single-unit recording and transient
multicell inactivation in awake trained animals
(Evarts, 1966; Hikosaka and Wurtz, 1983; Hubel,
1960; Ricci et aI., 1957). Two types of behavior
were examined. One battery of tasks consisted of
five overtrained instrumented movements con-
fined to a single joint, the wrist. The second battery
of tasks consisted of fiye untrained "natural"
movements that involved multiple joints.
These studies test two hypotheses. One is the
hypothesis, developed from prior work (Asanuma
et al., 1983; Thach, 1970, 1978; Thach et aI., 1982),
that within the cerebellum there are multiple
maps of the body and multiple modes of motor
control. Each deep cerebellar nucleus is proposed
to contain a separate somatotopic representation
of the body (Fig. 15.1), and to have a unique
control function over the body: the fastigius is
proposed to assist stance and gait; interpositus,
to speed initiation of movements triggered by
sensory cues that guide the response, to stabilize
holds, and possibly both to damp and to promote
oscillation; and dentate to speed initiation of
movements triggered by mental or arbitrary sen-
sory cues, and to control multijointed movements
more than single-jointed movements. The second
hypothesis is that there is a common function
across maps and modes of movement that is con-
sistent with the physiological experiments and the
anatomical circuitry. That function, as originally
proposed by Fluorens in 1824, is coordination of
movement.
Methods
Two Complementary Methods
to Define Control Signals
from Cerebellar Nuclei
Two methods were used to determine the nature
of the control signal that is generated by the
283
 tv
00
.!>-
Motor Cortex
<J VL Thalamus 0

Interneurons (Cord), Inferior Olive, OUTPUT

Other brain stem nuclei <J Red Nucleus 0

Motor ond Inter - neurons(Cord + Brain Stem) <J Vestibular Nuc o

~~
~~

INPUT
<J "Association" Cerebral Cortex
Pontine Nuclei

<J------ Somatosensory Receptors(Limbs), Motor Cortex

Mossy Fibers (Input) Spinocerebella rand
~
Nuclear Cell Flbers(Output) ....,
Pontine Nuclei p-
Excitatory o C> <J~---------------- Somatosensory Receptors(Trunk), Aud.- Vis. Info. o
Spinocerebellar Nuclei (Aud. and S
I>'
Vis. pathways unknown)
Purkinje Cell Fibers Labyrinths '"....,
o p-
Inhibitory • Vestibular Nuclei I>'
(')
p-
Figure 15.1. Diagram of the body representation with each of the deep cerebellar nuclei. (Adapted with permission from Asanuma et aI., 1983. Copyright by 2
Elsevier Science Publishers.) ~
15. Cerebellar Output
cerebellar nuclei. One method was the extracel-
lular recording with metal microelectrodes of the
discharge of single cerebellar nuclear cells during
motor performance. Since this discharge is in fact
the cerebellar output signal, modulation of neural
discharge in relation to time of onset of motor
performance and to some aspects and not others
provides one of the strongest clues we have as to
what aspects of behavior the cerebellar signals
are capable of controlling. A second method is
the temporary or permanent inactivation of pools
of neurons with muscimol (Hikosaka and Wurtz,
1983) or kainic acid (McGeer and McGeer, 1978),
respectively. A Hamilton syringe with a 26-gauge
needle was inserted into the microdrive system
and 1 JLI of muscimol or kainic acid injected
stereotaxically where task-related unit activity
had been recorded. Judging from the type of
deficit produced as a function of the site of the
injection, and of the volume of necrosis after
kainic acid injections, the sphere of inactivated
neurons was about 1 mm in diameter. More than
30 muscimol injections were made to map the
nuclei without permanent injury, the effects typi-
cally wearing off in 7 to 10 hours. This removes
the cerebellar output signal from the downstream
circuits that more directly control the behavior.
The type of deficit that this causes in behavior
should be complementary to the type of correla-
tion that the neural discharge has with behavior.
Theoretically, the neural discharge would be the
positive image of the negative deficit in behavior
caused by cerebellar ablation. It is important to
emphasize the complementary nature of the two
methods of evaluation, since the restricted use of
just the one or the other can lead to logical errors
of circuit analysis. These errors are different and
particular to the method employed. Seeing that
a recorded neural signal correlates with the be-
havior does not necessarily mean that the signal
caused the behavior or that the behavior caused
the signal. This is the error of false or indirect
correlation. Seeing a deficit in behavior produced
by ablation may suggest that a particular neural
control signal has been removed, but is not suffi-
cient to prove that the signal really ever existed.
This is the error of deduction from insufficient
evidence. But the recording of a neural signal that
is the complement of the neurological deficit caused
by its removal are together necessary and suffi-
285
cient for proposing that the signal controls the
behavior in question.
Battery of Five Instrument-Trained
Multijoint Movements
Figure 15.2 shows the monkey seated in a modified
Forenger primate chair with its head, trunk, and
elbow restrained. The wrist (and eyes) alone are
allowed to move freely. The hand is inserted,
fingers extended, into a glove-shaped manipulan-
dum that is moved in a horizontal plane by flexing
or extending the wrist. This involves chiefly the
long flexors and extensors of the fingers (Schieber
and Thach, 1985). Electromyography (EMG) is
recorded periodically with electrodes pasted onto
the skin over the muscles of many parts of the
body in addition to the operant arm. A poten-
tiometer monitors angular wrist position; a tacho-
meter, angular velocity; velocity is differentiated
to give acceleration. A visual target consists of a
vertical band or "window" on an oscilloscope
screen in front of the animal. The target moves
right and left under the control of a Rockwell
AIM-65 microcomputer. The manipulandum is
coupled to a cursor, which appears as a vertical
line on the oscilloscope. The job of the animal is
to line up the cursor within the target window,
and to maintain the alignment despite movements
of the window.
The Tests. Animals perfomed five tasks designed
to dissociate the functions of the longitudinal cere-
bellar zones (Eager, 1966; Jansen and Brodal, 1940)
by flexing and extending the wrist (Fig. 15.2). To
test the dentate nucleus, subjects performed a) jerk
(no stop on target): a prompt visual-triggered
move (Fig. 15.2, top). The subject aligned his cur-
sor on the band by holding the wrist 35° in flexion
or extension. At an interval randomly varied be-
tween 2 and 4 s, the band shifted instantaneously
70° in the opposite direction to a new position.
The subject was required to begin to move within
the allowed reaction time and to extend (or flex)
the wrist the full 70° through and past the target.
This tests the hypothetical function of the dentate
nucleus. Neural discharge should change before
EMG change and movement onset. A functional
deficit would show itself as a delayed onset time;
b) jump (stop on target): a prompt visual-trig-
gered move identical to that above, except that
 286 W. Thomas Thach et al.

FLEX TIME
I
EXTEND ·
5 I~RWD
,
I 70.
: : load: Torque
-----,----', :
I

Jerk
~ RT 'MT

Jump
---~~ RT :MT'
____~-;;Rwii--
--------~I ~'----------
Pert : : Load: Torqve
-- - - - - ______1 I

Torque RT ~WD

5
/
Ramp

RAM

Figure 15.2. Two batteries of behavioral tasks. Trained jointed movements are on the right. RT, reaction time;
wrist movements are on the left. Nontrained multi- MT, movement time; RWD, reward.

the subject was required to move and stop within
the visual target and hold there for at least 0.9 s
(Fig. 15.2, second from top). This tests the hypo-
thetical functions of the dentate and interpositus.
Dentate signals should change before onset of
EMG and movement. A deficit in dentate function
would show itself as a prolonged reaction time.
Interpositus signals should change before termi-
nation of movement and movement terminating
EMG changes, and correlate with terminal tremor.
15. Cerebellar Output
A deficit in interpositus function would show it-
self as a delay in ending the movement, and a
pronounced endpoint tremor in attempting to
hold the final position; to test the interposed
nucleus further, subjects performed c) pert: return
to central hold position after perturbation from
it by torque step (Fig. 15.2, third from top). For
this task, the target band was centered in the
middle of the oscilloscope screen. As for the above
tasks, the subject aligned his cursor on the band,
in this task by holding the wrist in a "central"
position of 10° of flexion. At an interval randomly
varied between 2 and 4 s, the torque load main-
tained against flexion (or extension) suddenly
switched to oppose extension (or flexion), per-
turbing the hold and displacing the hand from
the central position. The subject then had to react
within the allowed reaction time to return the
hand to the central position. This tests the hypo-
thetical function of the interposed nucleus. A
deficit in interpositus function would show itself
as a delay in the return ofthe hand after perturba-
tion ofthe central hold position, and an increased
terminal oscillation around that position; d) rainp:
ramp tracking of visual target against viscous
and negative viscous loads (Fig. 15.2, fourth from
top). As for the above tasks, the subject aligned
his cursor within the target band on the oscillo-
scope by holding the wrist 35° in flexion or exten-
sion. At an interval randomly varied between 2
and 4 s, the target band began to move at a
velocity of 28° per s. The subject had to begin to
move before the target band left his cursor behind
to track the target band remaining within it~
boundaries for an excursion of 70° until it stopped,
and to maintain the hold for an additional 0.9 s.
This tests the hypothetical function of the inter-
posed nucleus and its cerebellar cortical input.
Interposed neural signals should change in rela-
tion to physiological tremor. A deficit in inter-
positus function would show itself as an increase
of the amplitude of physiological tremor from
less than a degree to as much as 10 to 15° of wrist
arc, and a slowing of tremor frequency from the
normal 8 to 12/s to 3 to 6/s; e) RAM: self-paced
rapidly alternating movements (Fig. 15.2, fifth
from top). The subject was instructed to flex and
extend as rapidly as possible. This tests the func-
tion of the interposed nucleus and its cerebellar
cortical input. Interposed neural signals should
287
modulate in relation to RAM; discharge from
other nuclei should not. A deficit in interpositus
function would show itself as a slowing of the
frequency of the alternating movements and a
loss of regularity of period.
Battery of Five Natural Nontrained
Postures and Movements
A second stereotyped battery of natural move-
ments was also studied before and after nuclear
inactivation (Fig. 15.2, right column). This group
of movements consisted of standing, sitting,
walking, reaching for pieces of fruit from the
examiner's hand, and using thumb and forefinger
to pick pieces of fruit from deep, narrow food
wells. Stance, sitting, and walking were evaluated
immediately after the animal was released from
the primate chair. Reaching involved coordinated
movement of shoulder and elbow; reaching was
performed while sitting and standing on the floor
and while sitting in the primate chair. The chair
reduced the requirements for active postural ad-
justments of trunk and other limbs and permitted
easier assessment of the relative contributions
of shoulder and elbow. The precision pinch task
required coordinated movements of the hand and
fingers, including the pinch per se of thumb and
forefinger, and "tea-cup" posturings of the other
digits to keep them out of the way ofthe edges of
the food well. These movements were movie-filmed
or video-taped from front, back, and side views,
and analyzed frame by frame. Drawings were
made of individual frames or evenly spaced frame
sequences. Distances were measured for these
natural movements by comparison with known
lengths of the examiner's hand, the monkey's
body, and the grid pattern of floor tiles. Eye move-
ments were also visually examined after drug
injections.
Penetration tracks, injection tracks, and lesion
sites were verified using standard histologic pro-
cedures.
Results
Unit Recording
Single-unit activity was recorded across tasks
performed in standard order and across dent-
ate, interposed, and fastigial nuclei. Change in
288
discharge frequency was systematically compared
with a number of different movement variables.
These variables included the times of onset and
termination of movement and the direction, velo-
city, acceleration, and force of movement. During
the steady-state hold position before and after
movement, unit discharge was compared to the
hold position per se, the pattern of muscle activity
used to hold the position (systematically varied
by varying the torque load), the intended direction
of next movement, the oscillatory error in moving
and holding, if any (tremor), and the type of
stimulus situation eliciting the movement (per-
turbation of the visual target or somatosensory
perturbation of the limb to be moved, or self-
paced).
Figure 15.3 shows some typical units, one in
each of the three cerebellar nuclei, as they fire
across the trained wrist movement tasks. Dis-
charge displays are in the raster format, where
each dot represents an action potential, and
each row of dots, the discharge during a trial. The
trials are aligned on the onset of movement, re-
presented by the vertical line. Within each hori-
zontal row, the stimulus onset and the movement
stop are indicated by short vertical bars. The first
trial is at the bottom of each raster, and each
subsequent trial is the next row up. A moving
average of the discharge frequency across trials
is given above the raster rows. Units on the
abscissa are ms before (left) and after (right) the
onset of movement; on the ordinate, discharge
frequency is in impulses/so
The unit located in the fastigius nucleus (Fig.
15.3F, left column of raster displays), although
located in a region presumed to be concerned with
forelimb movement, did not change its discharge
frequency in relation to any of the trained wrist
movements-jerk, jump, pert, ramp, or RAM.
This was typical ofthe units in this area. No part
of fastigius was found to contain units related to
these movements.
The unit located in interpositus (Fig. 15.31,
middle column of raster displays) did change
markedly, and in relation to all of the movements.
For both jerk and jump, the unit commenced to
fire about 40 ms before the onset of movement,
which is about 10ms before the onset of first
EMG change (not shown; cf. Thach, 1970, 1978).
The discharge pattern was similar for these two
w. Thomas Thach et al.
movements, although the movements were dif-
ferent: jump required a termination on target;
jerk, none. The discharge time-aligns better
on the start of movement than on the stop. For
pert, there is a prompt response of the unit at
about 14ms, which is at about the same time or
a ms earlier than the earliest EMG change (not
shown; cf. Thach, 1978). This persists through
and after the stop. Oscillations in unit discharge
at 12 Hz correlate with oscillations in the wrist as
it settles into the final hold position. For ramp,
the discharge increases before the onset of move-
ment and persists throughout the movement.
Small oscillations in the unit discharge relate to
and follow tremor of the wrist at around 6 Hz
(Elble et ai., 1984). For RAM, unit discharge is
time-aligned on the extreme offlexion and is seen
to modulate as greatly as in the above tasks with
flexion (peak) and extension (trough).
The unit in dentate (Fig. 15.3D, right column
of raster displays) also changed its discharge
frequency, but only in relation to some of the
tasks-especially jump and jerk. For jerk, the
discharge increased about lOOms before move-
ment (cf. also Thach, 1970, 1975). For jump, the
discharge increased even earlier-about 130 ms
before onset of movement (cf. also Thach, 1978).
For pert, the increase in discharge was not as
great or consistent trial-to-trial as for interpositus,
and began about 50 ms after the perturbation.
Neither were there any oscillations in unit dis-
charge that correlate with the oscillations at the
wrist as it settled into position. For ramps, there
was hardly any change in unit discharge, there
being a slight decrease. For RAM, there was no
change in discharge frequency of this unit, al-
though there was for others.
Inactivation
Inactivation deficits were evaluated by comparing
the pre- and postinjection movement traces and
by movie film and video analysis. Several variables
were examined, including onset and termination
times, movement time, velocity, amplitude, and
EMG patterns.
For fastigius (Fig. 15.4F, left column), inactiva-
tion with muscimol or kainic acid caused no
detectable abnormality in the performance of any
of the five trained wrist movements. The animal
15. Cerebellar Output 289

I D

RAM

"
'"

, , \
. ', I .
, "

"
• I _ \

"
Figure 15.3. Discharge of single fastigius (F), interposed
(I), and dentate (D) neurons across the five trained wrist
movements. For jerk,jump, and ramp, rasters of single-
unit discharge are aligned on movement onset (center
vertical line). Tic marks in the raster before the center
vertical line represent the onset of the visual stimulus;
tic marks after represent the onset of reward. For pert,
rasters are aligned on the perturbation onset (center
vertical line), The first tic mark represents the onset of
return from displacement; the second tic mark repre-
sents onset of reward. For RAM, rasters are aligned on
peak flexion (or extension), and the first and second tic
marks represent peak extension (or flexion), Above the
rasters is the average frequency summed across trials.
290
Bt:fore
fo
Time
Figure 15.4. Major deficits produced by microinjection
ofmuscimol and kainic across the tasks. F, top, repre-
sents stance after each of two fastigial muscimol injec-
tions, an anterior one causing adduction of hindlimb
(left), and a posterior one causing adduction offorelimb
(right) . The figures below represent sequential video
frames in the course of falling that was caused by an
ipsilateral muscimol injection. I , top, represents wrist
position during successive trials of extension before
did not appear to be any different in its behavior
than when performing during a training session.
Jerks, jumps, and perts were performed with the
same reaction times and accuracy. Ramps were
performed without tremor, unless the injection
was made near the fastigial border with inter-
positus (see below). RAMs were performed at
the same frequency. We at first suspected that
the injection had missed the nucleus and was
in the white matter. Nevertheless, immediately
upon getting down from its primate chair, the
monkey showed severe difficulties with sitting,
w. Thomas Thach et al.
D
ft r
rt
r+
r+ rtr+r1lr+ r-
= Before
I
~ - fter
I'
~ .... ~
(left) and after (right) interpositus injection of muscimol.
Under each graph is the power frequency spectrum
of the tremor. Below is a figure using three successive
video frames to show tremor. D, top, bar graph show-
ing reaction times before (white) and after (hatched)
dentate injection of muscimol for each of four direction/
load combinations (extension/extensor, extension/
flexor, flexion/extensor, and flexion/flexor) and the
totals. Bottom, deficits in reaching and pinching.
stance, and gait. In the extreme case, it imme-
diately fell to the ground (to the side of the injec-
tion), and could not rise to a standing position.
There was no apparent weakness of the limbs
distally or proximally, and the animal made fre-
quent vigorous efforts to attain the upright stance.
After 5 or so minutes of trying, the animal
could often stand upright, only to fall again when
it attempted to walk. An example of one of these
falls is shown in Fig. 15.4F. Video frames at fixed
intervals are shown as the animal falls to the right
(side ofthe muscimol injection). The cause for the
15. Cerebellar Output
falls was not apparent from any abnormality in
trunk or limb strength or position. When standing
and walking, there was a tendency to place the
limbs on the affected side in a more adducted
position. This by itself did not appear sufficient
to cause a fall. But during a fall, the efforts that
the animal made to prevent itself from falling
were not sufficiently abducted to succeed.
Sitting was similarly affected, with falls back-
ward and to the side of the lesion. The animal
would approach the examiner and lean against
his leg for support, only to fall when the examiner
moved away. With some injections the tendency
was not so severe and the animal would sit-quietly,
only to fall when it attempted to reach for a food
bit.
There was a tendency to somatotopic locali-
zation. Most injections into the fastigius caused
falling that appeared due to defective control of
arm and leg. Two anterior injections caused pref-
erential adduction of the hind limb sparing the
forelimb, with falls precipitated from the hind-
quarters. One posterior injection caused just the
reverse, with preferential adduction of the fore-
limb, and falls precipitated from the forequarter.
The eye movements appeared normal; there
was no nystagmus. The animal appeared to have
no vertigo, and not to be ill at ease. There was no
retching or vomiting. It reached for raisins or
apple with either hand, without tremor, and fed
itself avidly, while lying on the floor, unable to
stand. The animals could pick food bits out of a
deep food well using a precision pinch without
apparent deficit.
For interpositus (Fig. 15.41, middle column),
deficits were apparent in both the trained move-
ments at the wrist and in the "natural" multijoint
movements. The most prominent feature was a
large amplitude, low frequency (3 Hz) tremor. This
appeared as a terminal tremor of the wrist in the
trained single-joint movements at the end of
jumps and perts and during ramps, and at the
shoulder and elbow in the natural multijoint
movements during reaching. With several injec-
tions the hind limb was involved, manifesting as
a tremor while sitting.
There was occasionally a slight delay in the
reaction time on perts (1O-20ms) and a loss of
regularity and rapidity of rapid alternating move-
ments. Otherwise, movements appeared unim-
291
paired. Jerks and jumps were performed with
normal reaction times. Sitting, standing, and
walking were normal, except for the very occa-
sional manifestation of tremor in certain posi-
tions, and the tendency to drag the dorsum of the
foot of the affected limb. Picking food bits out of
the foodwell was normal, except as affected by
tremor at shoulder, elbow, and wrist in certain
positions.
For dentate (Fig. 15.4D, right column), the
principle deficits were noted in reaching and in
picking food bits out of food wells. In reaching,
the animal would overshoot the target by as much
as 6 cm, bumping its hand against that of the
examiner's hand that held the food bit. This oc-
curred in the absence of any tremor. The over-
shoot resulted from a combined overextension of
the elbow and overflexion of the shoulder by as
much as 30°. This joint angle error was not seen
in the single-joint movements of the wrist in jumps
and perts, which landed on target with little or no
position error. Although interpositus injections
resulted in tremor that sometimes affected reach
at the endpoint, the position errors were never as
great or as sustained as those after dentate injec-
tions.
In the food well task, a variety of errors were
seen that also pointed to a coordinative problem
across joints. After dentate injections, the monkey
did not employ the precision pinch with the
accuracy and frequency as it did previously.
Instead, it resorted to "single finger" strategies.
These consisted of poking the raisin at the bottom
of the food well and either impaling it on the
forefinger'S nail or raking it up the side of the well
into the palm or against the side of the thumb.
Frequently the adjacent digits 3, 4, and 5 would
get caught over the side of the food well, instead
of being held up and out of the way or folded
together so as to allow the whole hand into the
food well. The relative inability to coordinate the
adjacent digits and to use digits 1 and 2 in a
precision pinch appeared out of proportion to
the fairly good use of the single index finger.
Not all dentate injections produced a deficit,
and when one did, the deficit could either be one
of reaching or pincning or both. The worst reach-
ing deficit was produced by an injection into the
most lateral part of dentate, and the worst
pinching deficit by an injection into the medial.
292
For these two injections, the reach deficit was
accompanied by a normal pinch, and the pinch
deficit by a normal reach. Injections between the
two gave deficits in both reaching and pinching.
This fits with the body mapping scheme depicted
it Fig. 15.1, with the forelimb mapped onto the
coronal dimension of the nuclei, with proximal
limb lateral and distal limb medial.
Discussion
Coordination of the movement of the many
muscles and joints ofthe body is one ofthe oldest
ideas on the fundamental function of the cerebel-
lum (Fluorens, 1824). This idea is based on the
impression that, after cerebellar damage, move-
ments at a single joint are less impaired than are
movements involving multiple joints. The idea
implies that within the cerebellar architecture,
there are circuit functions that are specialized to
span and coordinate the activities of multiple
joints. This would in turn imply a precise relation
ofthat architecture to the many joints and muscles
of the body.
Yet even at the present time there are appar-
ently conflicting views as to how the body and its
various parts are functionally represented within
the cerebellum. The question has been studied
repeatedly, using ablation, electrical stimulation,
and single-neuron activity during movement
within different regions of the cerebellum. These
studies have resulted in the following different
schemata: a) no localization (Holmes, 1917; Manni
et aI., 1964) b) medial cerebellum controlling
trunkal musculature and lateral cerebellum con-
trolling distal musculature (Adrian, 1943; Brown,
1949; Goldberger and Growden, 1973; Hampson,
1949; Hampson et aI., 1952; Sasaki et aI., 1976;
Snider and Eldred, 1952; Snider and Stowell, 1944;
Thach, 1967; Victor et aI., 1959), c) lateral cere-
bellum controlling proximal musculature to pro-
vide a postural base upon which fine, distal move-
ments are performed under intermediate cerebellar
control (Massi on, 1979), and d) medial cerebellum
controlling whole body postural mechanisms and
lateral cerebellum controlling whole body voli-
tional movements (Botterell and Fulton, 1938a, b;
Chambers and Sprague, 1955a, b; Dow and
Moruzzi, 1958; Sprague and Chambers, 1953).
It may well be that these different schemata for
w. Thomas Thach et al.
body representations are not at all contradictory,
but instead represent real differences in the topo-
graphic organization as it exists at the levels of
sensory input, cerebellar cortex, and cerebellar
nuclear output, and as it is revealed by different
methods of study. Recent anatomical studies have
given a rather clear and relatively simple view
of how the body is represented within the cere-
bellar nuclei (Allen et aI., 1977; Allen et al., 1978);
Asanuma et al., 1983; Kalil, 1982; Stanton, 1980).
These studies support a pattern of multiple repre-
sentation of the body within the nuclei: in the last
ofthese, there is a complete representation within
each nucleus, with the tail anterior and the
head posterior, limbs medial and trunk lateral
(Asanuma et aI., 1983).
The present nuclear mapping study using the
two methods of single-unit recording and repeated
muscimol injections across the two batteries of
tasks provides physiological support for the cere-
bellar nuclear output scheme first described by
anatomical studies above.
Fastigius
Previous studies of the deficits following ablation
of the fastigial nuclei have reported abnormalities
of upright stance and gait (Botterell and Fulton,
1938a; Sprague and Chambers, 1953). Antziferova
et aI. (1980) found unit activity in fastigius that
was well related to fictive scratching and walking,
whereas Arshavsky et aI. (1980) found little or no
such activity in inpterposed and dentate.
One study (Brown, 1949) and most clinical
neurologists have interpreted the stance deficit
of midline cerebellar lesions as being due to an
abnormality of the function of proximal trunk
muscles. This may in part be the result of Snider's
sensory input maps in cerebellar cortex (Snider
and Eldred, 1952; Snider and Stowell, 1944), which
show trunk in the midline and limbs laterally. But
iffastigial inactivation affects only the trunk and
the hip and shoulder girdle musculature, then the
animal should a) have abnormalities in reaching
with arm or leg, which require these muscles, yet
b) still be able to support itself upright by exten-
sion at elbow and knee. In our study, neither con-
dition was observed. When the monkey's trunk
was supported in a sitting position in the primate
chair, the animals had no difficulty in reaching,
15. Cerebellar Output
and they performed this task as if fully normal.
By contrast, stance and gait were often so severely
affected that the animal could not prevent itself
from falling, even though elbow and knee exten-
sion appeared of normal force and coordination
in reaching while seated in the chair. In sum, the
abnormality appeared not one of weakness of
anyone muscle group, but rather of the inability
to place and hold the limb properly to serve as a
support while standing.
A probable clinical correlation is the syndrome
of hypertensive intracerebellar hemorrhage (Fisher
et aI., 1965). The presenting complaint is an in-
ability to stand and walk. This usually occurs
initially in the absence of incoordination of willed
movements, or signs of brain stem damage. The
hemorrhage is just to one side of the midline deep
within the cerebellum. From the similarity of the
clinical presentation and the findings of this study,
the syndrome would appear to be caused purely
by damage ofthe fastigial nuclei.
I nterpositus
Electroanatomical studies have shown that inter-
positus receives receptor-specific information at
short latency directly and indirectly (via cortex)
from the large fiber, rapidly conducting spino-
cerebellar pathways (Allen et aI., 1977; Burton
and Onoda, 1977, 1978; MacKay and Murphy,
1974; Soechting et aI., 1978; Thach, 1978). Single
interpositus neurons of awake moving monkeys
have been shown to carry a signal proportionate
to physiological tremor that is similar to the
tremor signal also carried by Ia spindle afferents
(Elble et aI., 1984; Schieber and Thach, 1985).
There has so far been no such sharp distinction
between the motor deficits that follow interposed
and dentate ablation; the ablation of either is
supposed to include limb ataxia and action tremor
(Botterell and Fulton, 1938b; Goldberger and
Growden, 1973). Nor has cooling of the inter-
posed and dentate shown striking differences
(Brooks et aI., 1973; Uno et aI., 1973). Indeed, the
above cited ablation and cooling experiments
and the cooling experiments of Cooke and
Thomas (1976) and Vilis and Hore (1977, 1980)
attribute cerebellar tremor specifically to involve-
ment of the dentate. We believe that the distinc-
tion that we make between interposed and dentate
293
nuclear ablation deficits is allowed by the finer
spatial resolution of our two methods (unit re-
cording and chemical inactivation), neither of
which involves fibers of passage.
Our results confirm that there are movement
task-specific differences between interposed and
dentate nuclei. This is true for both methods of
study: the correlation of single-neuronal dis-
charge with behavior, and the behavioral deficits
after inactivation. Moreover, the neuron-behavior
correlations correspond to the ablation-behavior
deficits. Thus, interpositus neurons signal wrist
oscillation, and interpositus inactivation increases
the amplitude and slows the frequency of the
oscillation. Interpositus neurons fire at short
latency to wrist perturbation, and interpositus
inactivation delays the response time to this
stimulus. Interpositus neurons fire in relation to
self-paced rapid wrist alternation, and interposi-
tus inactivation decreases the regularity of these
movements.
What is the function of interpositus? If inter-
positus reponds at critically short latencies to
perturbation from a holding position, and if in-
activation of interpositus delays the movement
response to return to the hold position, then it
follows that interpositus may playa role in that
response. This is the conclusion of some previous
unit-recording studies (Strick, 1983; Thach, 1978),
which point out the similarity to the segmental
and especially the "long loop" stretch reflex. The
question remains as to the specificity of the re-
sult. This question is raised by the inconstancy
and rather meager magnitude of the delay. If the
"functional stretch reflex" per se were fully routed
through interpositus, then one might have ex-
pected a more robust impairment of its perfor-
mance by interpositus inactivation. This result
not having been obtained, one wonders if the
more important and global function is something
bigger but yet beneath the surface.
Perhaps a more important clue is the relatively
dramatic correlation of neural discharge and of
inactivation deficit with oscillation. We have
elsewhere entertained the idea (Elble et aI., 1984;
Thach et aI., 1986), as have others (Glaser and
Higgins, 1966; Henatsch, 1967; Wiener, 1948), that,
since oscillation is a problem inherent in the mech-
anical reflex design of the motor system, the cere-
bellum may have been included to damp the
294 w. Thomas Thach et al.

oscillation. Mechanical systems have their dash- However, previous studies of dentate ablation
pots and electronic systems have their resistors, had not shown a conspicuous difficulty in initiat-
and it is possible that a similar solution is also ing voluntary movement (Botterell and Fulton,
taken by the nervous system. If the cerebellum 1938; Goldberger and Growden, 1973; Luciani,
were to damp oscillation actively, then the ques- 1911; Chambers and Sprague, 1955b). The reac-
tion becomes whether this is at fhe level of seg- tion time delay of some 200 ms after human cere-
mental stretch reflexes or of long loops, or both. bellar injury, first reported by Holmes (1917), has
This question also has been discussed in the above been confirmed by subsequent ablations of den-
and other references. tate (vide supra; Thach, 1975; Trouche and
Does the cerebellum also promote oscillation? Beaubaton, 1980), but the delay has shrunken to
It is of interest that two of the most reliable one as small as 25 to 50ms. The large and phy-
empirical tests for cerebellar injury include both logenetically increasing size of the dentate would
the presence of unwanted oscillation and the in- appear to argue for some more important func-
ability to produce oscillation. The observation tional contribution.
that interpositus neurons may best signal, and It can be argued that small reaction time dif-
interpositus inactivation most impair self-paced ferences may be very critical in certain rapid and
rapid alternating movements fits with the idea of skilled motor performances. But another inter-
Walshe (1977) that the most rapid of oscillations pretation is that the timing delay may be a clue
may be produced by undamping stretch reflex to some larger and more important function
mechanisms. lurking just beyond our present knowledge. In
The phenomenon of the interpositus link to this vein, one piece of work in human cerebellar
oscillation is in keeping with the work of Gilman patients has suggested that dentate functions may
and colleagues (Gilman, 1969; Gilman et al., 1976) be perceptual or mental. The finding was that
and of Smith and colleagues (Frysinger et aI., patients with lateral cerebellar lesions are unable
1984; Smith and Bourbonnais, 1981; Wetts et aI., to distinguish small duration differences in tone
1985) on agonist-antagonist muscle control by pips presented serially (Ivry and Keele, 1988).
the cerebellum. The idea would be that this part Other speculative articles have suggested even
of the cerebellum may adjust and coordinate in more abstract functions, such as the coordination
time and across muscles the bias and/or gain of of thought and mental skills (Leiner et aI., 1986;
stretch reflexes of agonist and antagonist muscles. Leiner et aI., 1987).
This could be for a variety of purposes other than Our experiments of recording and inactivating
the prevention of oscillation. Oscillation could neural discharge within the dentate nucleus con-
simply be the by-product of maladjustment. firmed and extended previous results on initiation
of volitional movement. Dentate neurons changed
Dentate firing well before movement onset, especially the
The parallel phylogenetic development of the visually triggered jerks and jumps, and it was
cerebral neocortex and lateral cerebellum, to- these movements that were delayed by dentate
gether with the connections between them, have inactivation. On perts, dentate discharge lagged
long suggested that the former must work along that of interposit us, and inactivation had no effect
with the latter. In trained monkeys, dentate neural on the reaction time. Dentate neurons carried no
discharge was reported to lead the discharge of tremor signal, and dentate inactivation had no
motor cortex neurons and movement (Lamarre effect on tremor. In some animals, inactivatiQn of
et aI., 1983; Thach, 1975, 1978), whereas dentate dentate causes tremor of hand tracking during
inactivation delayed both the discharge of motor the ramp task only. This movement is usually a
cortex neurons and movement (Meyer-Lohman compound one of eyes and wrist though some
et al., 1977; Spidalieri et aI., 1983). This supported animals learn to complete the ramp-track without
the idea that the dentate was involved in initiating looking apparently from memory. This may ex-
volitional movement from motor cortex (Allen plain the inconstancy of the result across animals
and Tsukahara, 1974; Brooks and Thach, 1981; and the results of Vilis and Hore (1977, 1980).
Eccles, 1967; Evarts and Thach, 1969). Dentate neurons (like interpositus neurons) fre-
15. Cerebellar Output
quently discharged in relation to RAMs, but den-
tate inactivation did not impair the alternations
beyond a slight slowing.
These results are consistent with some previous
interpretations as to the differences between den-
tate and interpositus (Allen and Tsukahara, 1974;
Botterell and Fulton, 1938a, b; Brooks and Thach,
1981; Chambers and Sprague, 1955a, b; Eccles,
1967; Evarts and Thach, 1969). Dentate discharges
earliest in relation to stimuli that give no informa-
tion as to the nature of the movement that is to
follow. These stimuli do not ordinarily cause a
movement, and the monkey has to make the
association through operant conditioning. Once
learned, the monkey will not respond to the
movement unless it is motivated to do so. These
criteria can be used to create an operational defi-
nition of "volitional movement." It seems appro-
priate that it is the part of the cerebellum that is
best connected to the cerebral association cortex
that is most critically concerned with these tasks.
Tasks in which the stimulus is delivered to the
part to be moved, in which the stimulus direction
determines the movement direction (as in the
Figure 15.5. Model of
granule cell parallel fiber
control of muscular co-
ordination.
--i6-4-.....I(H-----tc:::J.-1.--kl-+---KH---....-I- -
-.J-~+---tc::H--<K}-+--t<J-+-t:J_-._-----
--+--I--~-+---f-+--+--tr-+__+--,--
295
stretch reflex), appear to be more critically con-
trolled by interpositus than dentate. Do these
observations then reveal the full identity of dentate
control?
The "room behavior" as casually observed after
dentate inactivation did not prepare us for what
we observed under systematic testing (Kane et aI.,
1989). To be sure, there was a "clumsiness and
awkwardness" of the hand in picking up items or
warding off threats, and a tendency to lift the
affected foot a little higher than its fellows in
walking, both of which were described by Botterell
and Fulton (1938b) after large hemispheric abla-
tions. But when the animal reached into deep
narrow food wells in which raisins, bits of apple,
or M&M candies were placed, the effects of den-
tate inactivation became dramatic. The animals
suffered an impaired ability to pinch in a precision
grip. This normally involves the coordinated
opposition of thumb and index. When specifically
looked for, the animals had also lost this ability
in retrieving food bits from the examiner's hand:
the deficit did not depend on the food well alone.
The animals retained the ability to move one digit
- -
- - - - - -
296
at a time, and made use of this to get food out of
the food well. This resulted in "poking," "stabbing"
with the fingernail, and "raking" up the side to
get the food bit out of the well. The other fingers
would move independently, lacking the previous
synchrony with each other and with the thumb
and index.
The deficits were not confined to the hand
alone, but also affected shoulder and elbow. This
was best seen in standing and reaching for profer-
red food bits, and especially while seated in the
primate chair. The principle visible error was an
overshoot ofthe hand past the target. This resulted
from an overflexion at the shoulder and overex-
tension at the elbow, compounding to give the
endpoint error. In a single joint, one might have
attributed the overshoot purely to a delay in
stopping the movement. But the time delay as
measured at the wrist was small, and the position
overshoot scarcely detectable (the animal easily
landed within the 10-15° target window). For the
two joints, the error had to have resulted more
from excessive angle than from late timing, and,
judging from the negligible position error at the
wrist, to be uniquely a deficit of multijointed
movements.
Mackel (1987) lesioned portions of the deep
nuclei with kainic acid and studied a variety of
movements, especially movements of the digits.
His lesions involved both interpositus and dentate,
and he did not dissociate tremor from the other
deficits. He did remark upon a striking difficulty
of the monkeys in making independent digit move-
ments, which he likened to the corticospinal deficits
described by Lawrence and Kuypers (1968). These
observations, along with the earlier ones of
Botterell and Fulton (1938b), support our obser-
vations of abnormalities of finger movements after
dentate lesions. Nevertheless, it seems to us that
they missed an essential point. That is, that move-
ments of individual joints, including independent
finger movements, are relatively normal. It is the
movement of two or more joints, as well exem-
plified by the thumb and index in precision pinch,
and the shoulder and elbow in reaching, that are
qualitatively more severely affected by dentate
lesions. This is in keeping with the rather mild
deficits seen in single-joint movement after dentate
ablations.
w. Thomas Thach et al.
Parallel Fibers, Purkinje Celis,
M yotomes in the Coronal Dimension,
and Coordination
Does cerebellar circuitry provide a mechanism
for multijoint coordination? These results are
interpreted in context of the prior anatomical
(Asanuma et aI., 1983; Kalil, 1982; Stanton, 1980)
and physiological (Thach et al., 1982) findings of
somatotopic representation of the body within
each of the three cerebellar nuclei. In each repre-
sentation, the mapping is of the caudorostral
dimension of the body onto the sagittal dimension
of the nucleus. Thus, the hindlimbs are represented
anteriorly, the forelimbs more toward the middle,
and the head posteriorly in the nuclei. This orient-
ation suggests that the myotomes, running ortho-
gonal to the long axis of the body, run in the
coronal dimension and parallel to the trajectory
of the parallel fibers. Since the parallel fibers are
connected to the nuclear cells by Purkinje cells, a
coronal "beam" of parallel fibers would control
though inhibitory modulation the nuclear cells
influencing the synergistic muscles in the myotome.
The parallel fiber in this way would be a single
neural element spanning and coordinating the
activities of multiple synergic muscles and joints.
Fig. 15.5 shows a coronal beam of parallel
fibers running parallel to the myotomes in the
forelimb representation within the deep nuclei.
At one level of organization, the parallel fiber
spans the synergic muscles within the myotome,
and the strength of the contact on the Purkinje
cell determines the strength of activity of the
muscle within the limb. It is clear that, as drawn,
the action of one parallel fiber will have an effect
very different from that of another. The top
parallel fiber, acting through the Purkinje cell and
the nuclear cell, will be seen to inhibit distal
musculature relative to proximal, whereas the re-
verse is true for the second parallel fiber from the
top. The assumptions are that a parallel fiber may
have different effects on the successive Purkinje
cells it passes. At a second level of organization,
the parallel fibers that span nuclei will perforce
have a coordinating effect on the outputs of the
different nuclei (e.g., of stance, of stretch reflex
gains, and of voluntary movements). It is clear that
motor activities are coordinated at both these
levels, that of synergist muscles, and that of different
15. Cerebellar Output
modes of control. The question remains, "What
is the coordinating circuitry?" Exactly like the
modern circuit designer's labeled logic line, the
parallel fiber obviously makes contact with both
elements of organization. This anatomical fact
suggests that it can perform the function we tenta-
tively ascribe to it. Future work will be needed to
prove its sufficiency in this role.
It is just as clear that the cerebellum is not the
only mechanism that coordinates multijointed
movement. Coordination is built into antagonist
muscles and alternating stepping in the spinal
cord, and into antigravity stance and head-limb
synergies in the brain stem. It may be that move-
ment of single digits in combinations and se-
quences is achieved at the level of motor or pre-
motor cortex, just as visual cortex may build up
complex receptive fields from simple fields. These
synergies, relatively simple and hardwired, may
need modification if variety and change of combi-
nations and sequences ofthe moving components
is required.
We have pointed out the favorability of the
parallel fiber and its connections with the Purkinje
cells through to coronal beams of nuclear cells
and thence ultimately to synergic muscles and
modes of control as a logical candidate for the
coordinating link. Given such a link, the amount
of variation that the mechanism permits should
go up with the number of such coordinating links.
Adjustment and adaptation would also appear
to be desirable attributes of the coordinating
principle, and at the level of the contact of the
linking fiber its linked cells. The cerebellar cortical
circuitry appears to have all these desirable
features.
Verification of this mechanism will require
a) showing that, after cerebellar cortical injury,
multijointed movements are sufficiently more
impaired than single-jointed movements such that
the sum of the abnormalities at the single joints
cannot account for the magnitude of the abnor-
malities in the compound movement, b) recording
from Purkinje cells to show that they are differen-
tially cQntrolled in single versus multijointed
movement, and c) that the parallel fiber is the
sole exclusive agent of this control.
297
Summary
The deep cerebellar nuclei were mapped in awake
Rhesus monkeys using a) the behavioral corre-
lation of recorded single-unit discharge and b) the
behavioral deficits produced by muscimol and
kainic acid microinjection across two task bat-
teries employing five trained wrist movements and
five nontrained multijointed movements. The
following conclusions were made.
1. There is a separate somatotopic representation
within each nucleus, with caudal parts anterior,
rostral parts posterior, trunk lateral, and limbs
medial. Each nucleus controls a different type
or "mode" of movement.
2. The medial fastigial nucleus controls muscles
only in the modes of sitting, standing, and
walking: its neurons did not fire in relation to
the trained wrist movements, and inactivation
produced no deficit in these movements. In-
activation did produce severe deficits in sitting,
standing, and walking, with inability to main-
tain an upright position, or frequent falls to
the ipsilateral side. There was a tendency for
somatotopic organization, with hind limb ante-
rior and forelimb posterior in the nucleus.
3. The intermediate interposed nucleus controls
stretch and other somatosensory reflexes and
may help damp the inherent mechanical reflex
oscillation of body parts: it fires in relation to
mechanical reflex tremor and to prompt move-
ment initiated by perturbation of the part to
be moved (the so-called long loop reflex); its
inactivation increases the amplitude and slows
the frequency ofthe tremor, and may prolong
the reaction time of an attempted long loop
reflex. Interpositus may also preferentially con-
trol self-paced rapid alternating movements: it
fires in relation to them, and its inactivation
slows their frequency and impairs their regu-
larity.
4. The lateral dentate nucleus helps initiate (via
thalamus and motor cortex) those movements
that are triggered by mental or arbitrary sen-
sory cues and therefore called "volitional": it
fires before such movements; its inactivation
delays onset ofthese movements. Inactivation
affected multijointed movements much more
than single-jointed movements. The dissoci-
ation was sufficiently extreme to warrant re-
298
consideration of the old idea that a cardinal
purpose of cerebellar circuitry is the coordi-
nation of multijointed movements. A model
whereby this might be achieved is presented.
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16
The Role of the Cerebellum in
Voluntary and Reflexive Movements:
History and Current Status
John P. Welsh and John A. Harvey

Because important scientific questions are usually ter. Whereas the occurrence of such voluntary
examined by a number of research groups that acts can be increased in frequency by association
may differ from each other in both methodology with environmental events, the relationship be-
and theoretical orientation, it is not unusual to tween stimulus and response remains probabilistic.
have opposing interpretations of a research out- It has been concluded that the consequences of a
come. Moreover, the interpretation of any single voluntary act must in some manner determine its
research group may be powerfully influenced and frequency of occurrence. For this reason, such
rewarded by the culture, politics, and historical behavior is also referred to as instrumental in that
precedents to which it has been exposed. There- it is assumed to be instrumental in obtaining
fore, the validity of a scientific interpretation some reward. Those who prefer to eliminate any
and its recognition as "truth" by the scientific teleological inferences prefer to use the term
community is greatly enhanced when that truth "operant" to indicate simply that the behavior
is arrived at by independent research groups operates on the environment.
differing in their intellectual backgrounds and The second group, located predominantly in
coming from altogether different historical frame- the USSR, derived its interest in cerebellar function
works. Such has been the case with the study of from the perspective of reflexology as propounded
cerebellar function. Thus, one purpose of this by Sechenov and later Pavlov. Students of Pavlov
chapter is to review how similar conceptions of began to examine the role of the cerebellum in
cerebellar function were arrived at by two different the regulation of both unlearned (i.e., uncondi-
and to a large extent noninteractive groups of tioned) reflexes and in learned (i.e., conditioned)
scientists: the first located in the "West," by which reflexes. The focus was on behavior that occurs
is meant western Europe and the western hemi- in a relatively stereotyped manner in response to
sphere, and the second in the USSR. a identifiable antecedent events. In contrast to in-
Scientists in the West focused their attention strumental conditioning, which requires that the
on the role of the cerebellum in voluntary be- experimenter wait for an animal to make a re-
havior-behavior that occurs without any perceiv- sponse before providing a reward, Pavlovian con-
able external cause and is therefore considered to ditioning requires the experimenter to elicit the
be self-generated. Causality for voluntary acts desired response. It is not difficult to see the
can only be inferred by what the act accomplishes, relationship between the historical and political
thereby giving such behavior a purposive charac- context within which these two groups operated
and their views concerning the important deter-
minants of behavior.
a Unless otherwise noted, citations to historical works
were derived from the following references: Holmes Instrumental behavior and conditioned reflexes
1917; Luciani, 1915; Pollock and Davis, 1927; Popov, have in common the fact that they represent learn-
1929a; Rawson, 1932. ed responses. Indeed, Pavlov, along with many

301
302
learning theorists in the West, believed that condi-
tioned reflexes formed the basis of instrumental
behaviors. As we shall see, the early consensus of
scientists in the West and USSR was that the
cerebral cortex was involved in the acquisition of
instrumental behaviors and conditioned reflexes
whereas the cerebellum was necessary for their
optimal performance. However, the vulnera-
bility ofthese two learned behaviors to cerebellar
damage has led to frequent speculations that the
cerebellum was somehow involved not only in
the expression of learned behavior, but with the
learning process itself(see Rawson, 1932). Recently,
a strong claim has been made that the cerebellum
is not only essential but actually the center for
learning and memory of some reflexive motor
acts. In this view, the plastic events that underlie
motor learning are located within the cerebellum.
Thus, a second purpose ofthis chapter is to review
and evaluate the most recent controversies con-
cerning the involvement of the cerebellum in
learning that have emerged from the use of
Pavlovian conditioned reflexes, especially the
nictitating membrane reflex of the rabbit, and to
place this controversy within the historical con-
text of previous cerebellar research carried out
in the West and in the USSR.
The Western Tradition: Cerebellar
Regulation of Voluntary Movement
By the early 1900s a consensus was being de-
veloped in western Europe and the western Hemi-
sphere that the cerebellum was intimately involved
in the regulation of voluntary motor behavior.
This consensus was reached after more than 200
years of research dealing with the effects of cere-
bellar removal on sensory and motor function in
a variety of vertebrate species.
Rolando
The conception that cerebellar function is par-
ticularly related to the regulation of voluntary
motor behavior can be traced to the conclusions
of Rolando (1809-1823). In agreement with earlier
findings of Lorry (1760), Rolando noted that cere-
bellar damage profoundly disrupted the ability
to walk. However, Rolando further noted that
animals with cerebellar lesions, although ataxic,
John P. Welsh and John A. Harvey
behaved in a manner suggesting that their inten-
tion or will to perform various motor acts had
not been affected. For instance, subjects with cere-
bellar lesions would attempt to feed or avoid
obstacles even though their ataxia led to repeated
failures. Because such behaviors were attempted
despite cerebellar dysfunction, Rolando con-
cluded that the cardinal function of the cerebellum
was to reinforce the motor impulse for voluntary
movement. Since Rolando observed complete
paresis immediately after cerebellectomy, he con-
cluded that the etiology of ataxia following partial
cerebellar damage was an alteration in the bal-
anced regulation of motor impulses emanating
from the cerebral cortex. This resulted in the para-
lysis of a limited set of muscles that were nor-
mally regulated by the damaged cerebellar tissue.
Rolando's observations on the effects of cerebellar
damage were in marked contrast with the effects
of damage to the cerebrum. Lesions ofthe cerebral
cortex appeared to disrupt an animals' will, voli-
tion, or desire to perform various motor acts. The
implications of this dichotomy were clear: the
cerebrum was the source of volition and voluntary
behavior and the cerebellum ensured coordinated
behavior by reinforcing the cerebral impulses that
initiated voluntary movements. This view of cere-
bral and cerebellar function determined, for nearly
200 years, the direction of research on the role of
the cerebellum in regulating voluntary move-
ment.
Fluorens
The notion of a dominant role for the cerebellum
in voluntary behavior was also supported by the
work of Flourens (1824-1842), but he had a dif-
ferent interpretation for the etiology of the motor
deficits following cerebellar damage. In Flourens'
view, the function of the cerebellum was coordi-
nation per se, and the deficits following such
damage were specifically due to the withdrawal of
the brain center for motor coordination. Flourens
studied the effects of varying amounts of cerebellar
damage in the same animal. During the incremen-
tal removal of successively deeper layers of the
cerebellum, Flourens observed progressive deficits
in behavior. Mter complete cerebellectomy, a bird
would lose the ability to walk, stand, fly, and right
itself but would still attempt to escape from a
16. Role of the Cerebellum
threatening stimulus. However, such escape be-
ha viors were not expressed in an organized fashion
but were chaotic and uncoordinated. Flourens
reinterpreted Rolando's description of paresis to
be muscle weakness, but further noted that the
power of movements could be retained after cere-
bellar damage. However " ... the co-ordination of
movement, the ability for controlled and determined
movement was lost ..." (Flourens, 1824 as cited in
Rawson, 1932; emphasis added). Thus, Flourens
believed that deficits of voluntary movement
were not due to a lack of determination (i.e.,
volition) but rather due to an inability to control
or coordinate such movement.
Flourens' view of the cerebellum as the primary
coordinating center of the brain was also held by
Lussana (1862) and later by Lewandowsky (1903,
both cited in Luciani, 1915). These investigators
conceived of the cerebellum as a center for muscle
sense and attributed deficits in coordination of
voluntary movement to impairments in the ability
to use proprioceptive information. In this light,
Lussana recognized that " ... the muscular sense
serves in animals to make known the resistance
met with, and in voluntary movements to regulate
the forces of contraction by which the muscle is
able to overcome it" (Lussana, 1862, cited in
Luciani, 1915). Lewandowsky took as proof for
his position the fact that the elimination of move-
ment-induced feedback that occurs in tabes dor-
salis or resection of the dorsal roots of the spinal
cord produces the classic signs of cerebellar dys-
function: dysmetria of voluntary movement and
"hen's gait," a form of dysmetria in which flexion
and extension are exaggerated during walking.
The views of Sherrington (1906) were consonant
with those of Lussana and Lewandowsky. Sher-
ring ton conceived of coordinated movement as a
chain of brain stem and spinal reflexes occurring
in an organized sequence. Voluntary motor acts
occurred when the cerebral cortex engaged such
a pattern of spinal and bulbar reflex arcs. The
fluidity of any voluntary movement depended on
the reflexive regulation of muscle tone by pro-
prioceptors of antagonistic muscle groups-
Sherrington's reciprocal innervation. Sherrington
envisioned the cerebellum as having an essential
role in this process, namely to act as the " ... head
ganglion ofthe proprioceptive system ..." to ensure
that normal muscle tone was maintained through
303
the reflexive use of proprioceptive information
from the muscles, tendons, joints, and vestibular
labyrinths.
The attempt by Flourens to interpret motor
deficits produced by cerebellar damage as result-
ing from damage to a center for the coordination
of voluntary movements was soon questioned by
a number of investigators. In a review of 18 human
cases of cerebellar deficiency dating from 1839,
Dickenson (1865) concluded that the main deficit
following cerebellar pathology was the failure of
voluntary muscle power. Deficits in coordination
per se were not believed to be the etiology of the
motor syndrome evident in the clinical cases.
Rather, the cause of irregular and unbalanced
movements was thought to result from asym-
metric cerebellar damage that in turn produced
an uneven distribution of power in the volun-
tary musculature. These views were essentially
a restatement of Rolando's earlier observations.
Dickenson obtained further support for his views
from ablation studies in a variety of animals in-
cluding snakes, toads, salamanders, tortoises, fish,
pigeons, and guinea pigs. He observed that,
throughout phylogeny, the addition of the cere-
bellum in the otherwise decerebrate animal pro-
vided power to movements and allowed them to
occur more naturally. Dickenson's views were to
be strongly supported by Luciani.
Luciani
Prefacing the results of7 years' work on the prob-
lem of cerebellar function (1884-1891), Luciani
declared that " ... whatever the extent or degree
of the cerebellar lesion, ... the resulting symp-
toms are disturbances of voluntary movements"
(Luciani, 1915). Luciani provided a highly devel-
oped framework in which to view the primary
etiologies of cerebellar damage. This framework
remains valuable to the present day. Employing
freely moving dogs and apes, Luciani identified
three stages of motor behavior following cere-
bellar lesions. The first stage was the period of
"dynamic phenomena," in which the irritative
effects of the lesion on the remainder of the brain
served as the source for the consequent behavioral
deficits. Symptoms of this period following uni-
lateral cerebellectomy were unrest and agitation,
ipsilateral extensor rigidity, pleurothotonus, nys-
304
tagmus, and forced rotation along the long axis
of the bod y away from the side of the lesion. These
symptoms subsided within 10 days after surgery.
Luciani asserted that previous expetimental
work on animals had been confined to the first
stage, the early postoperative period, and had
therefore failed to observe the true deficits in
behavior resulting from withdrawal of cerebellar
function. These occurred in the second stage, the
stage of cerebellar deficiency. The summation of
the motor deficits in this period constituted the
global behavioral deficit recognized as ataxia.
Luciani devoted the majority of his attention to
this stage. Finally, the third s.tage, or stage of
"compensatory phenomena," resulted from either
the recovery of function of the damaged tissue
(organic compensation) or from changes in the
function of the cerebrum and/or remaining cere-
bellar tissue (functional compensation). Functional
compensation permitted compensatory behaviors
to counter the negative consequences of cerebellar
deficiency.
Luciani described three fundamental deficits
during the period of cerebellar deficiency: asthenia,
a loss of muscle strength; atonia, a loss of muscle
tone; and astasia, tremor and rhythmic oscilla-
tion. These three primary symptoms were pre-
sumed by Luciani to be the underlying etiology of
all movement disorders resulting from the with-
drawal of cerebellar function. Noting that dogs
with cerebellar lesions were unable to support
their weight on land but could swim virtually as
well as normal dogs, Luciani suggested that co-
ordination per se was not abolished by removal
of the cerebellum but rather that the symptoms
that appeared as deficits in coordination were
the consequence of deficits in tone (atonia) and
strength (asthenia) required for movement against
resistive forces. One component of astasia that
Luciani observed in his experimental animals was
similar to the clinical observation of titubation:
the hesitancy to initiate a voluntary movement
or the inability to execute voluntary movements
in a smooth and stable manner without tremor.
In ,agreement with Flourens, Luciani did not
ascribe these deficits to a delay in the development
of the voluntary impulse but, rather, to the in-
complete summation of neural impulses necessary
for the smooth performance of voluntary acts.
During the third stage of compensatory phe-
John P. Welsh and John A. Harvey
nomena, Luciani observed ataxic animals behav-
ing in ways that he interpreted to demonstrate the
animals' inteo,tion to avert the consequences of
atonia, asthenia, and astasia. For instance, exag-
gerated abduction of the limbs during walking
(the "drunken sailor's gate") was viewed as a
behavioral compensation for the adverse effects
of falling due to limb dysfunction. Exaggerated
abduction of the limbs, by widening the base
of support and lowering the center of gravity,
minimized the probability and severity of falling.
Another compensatory behavior exhibited by cere-
bellectomized dogs was crossing of the forelimbs
during walking. Luciani believed that this
behavior was intentionally performed to main-
tain equilibrium and thereby compensate for the
horizontal oscillations resulting from atonia and
asthenia of the vertebral muscles.
In addition to the importance that Luciani
assigned to the cerebellum for maintaining muscle
tone, strength, and stasis, he also recognized tro-
phic influences of the cerebellum on the brain and
the periphery. Direct trophic influences of the
cerebellum on central nervous structures to which
it was connected ensured normal function of sen-
sory and motor systems. Indirect trophic influ-
ences on peripheral organs were recognized when
subjects with cerebellar lesions exhibited muscle
atrophy, changes in cutaneous elements, and
diminished resistance to injury.
Holmes
An important development in our understanding
of cerebellar function was initiated by Holmes
(1917), who studied the motor deficits exhibited
by human subjects with gunshot injuries to the
back of the head. These studies were notable for
their greater attention to quantitative measures.
Holmes shared with Rolando, Flourens, and
Luciani a strong interest in the performance defi-
cits associated with voluntary movements. Con-
sistent with Luciani's description of asthenia,
Holmes' quantification of the forces of grasping,
elbow flexion and extension, supination, prona-
tion, and ankle extension revealed up to 50%
deficits on the side ipsilateral to a unilateral cere-
bellar injury relative to the unaffected side. Patients
with unilateral cerebellar lesions were described
as reluctant to move the limbs ipsilateral to the
16. Role of the Cerebellum
Figure 16.1. Effect of a gunshot wound to the right
half of the cerebellum on voluntary movement in a
man. The kymographic record is taken from Holmes
(1917). The patient was instructed to grasp calibrated
springs with each of his hands simultaneously. The
vertical bars (marked I and l' for the left and right
hands, respectively) indicate the time at which the
command to grasp was given. The left (A) and right (A')
grasping responses were followed by a command to
relax at the vertical lines marked 2 and 2'. Relaxing
responses are bibeled Band B', for the left and right
lesion, and on command these patients moved
the limbs ipsilateral to the lesion with less vigor,
force, and speed than those contralateral to the
lesion. The grasp of the hand ipsilateral to a
unilateral cerebellar lesion was demonstrated to
be delayed in initiation by 200 ms, reduced by
50% in its peak force, and doubled in its time to
peak force relative to the unaffected hand (see
Fig. 16.1). The delay in response onset was mir-
rored by an equivalent delay in the initiation of
relaxation, indicating that response onset and
offset were equally affected (see Fig. 16.1). Holmes
noted the uniqueness of this clinical presentation
because, in contradistinction to lesions of the
motor cortex, cerebellar lesions affected many
groups of muscles equally while not imparting
rigidity, affected all voluntary movements while
not limiting them in range, and left the primary
reflexes relatively intact.
305
hand, respectively. Upper markings represent time in
tenths of seconds calibrated by a tuning fork at 128 Hz.
Note that the right hand (A') showed an approximately
200-ms increase in response latency, a longer rise time
to peak amplitude and a lower amplitude as compared
with the unaffected left hand (A). Also, note that the
latency to initiate ·and achieve relaxation was longer
for the affected right hand (B') as compared with the
normal left hand (B). Reprinted with permission from
Holmes, 1917.
Holmes subdivided the cerebellar ataxic syn-
drome into five components: decomposition of
movement, asynergia, dysmetria, tremor, and
deviations from the line of movement. Decompo-
sition of movement described the break up of
continuous movements into a series of discrete
movements separated by pauses. Asynergia re-
ferred to the absence of synergistic actions of an-
tagonistic muscles required for graceful motion.
Dysmetria simply referred to alterations in the
range and force of movements. Deviations from
the line of movement described movements that
did not take the normal course of direction to
ensure the least expenditure of energy. Holmes
conceived ofthe cerebellum as a motor reinforcing
organ that did not in and of itself initiate move-
ments, but instead regulated the motor system so
that a ". .. response to a volitional stimulus is
immediate, effective and proportional to the in-
306
tensity of the cerebral impulse" (Holmes, 1917).
Cerebellar regulation of voluntary movement was
thought to be effected by continuous regulation
of postural tone as well as by phasic regulation
in the alterations of tone required for the synchro-
nous actions of muscles necessary for coordinated
movement. Later, Holmes added that, in addition
to the musculature, the tone ofthe cerebral motor
cortex and the subcortical motor nuclei were regu-
lated by the cerebellum. Accordingly, the cere-
bellum assured that the higher centers mediating
movement responded expeditiously and with nor-
mal magnitude to volitional impulses (Holmes,
1939).
Although cerebellar lesions appeared to have
their greatest effects on the performance of vol un-
tary movements, Holmes made the important
observation that involuntary (reflexive) move-
ments were also impaired after cerebellar damage.
For example, even the disynaptic knee jerk elicited
by a tap on the patellar tendon was weaker, less
brisk, and harder to elicit on the side ipsilateral
to a unilateral cerebellar lesion as compared to
the contralateral side. During the acute phase
following cerebellar injury the patellar reflex could
be entirely absent. With longer recovery time, the
ipsilateral leg would exhibit a normal amplitude
response but would continue to swing like a pen-
dulum at times when the normal leg would come
to rest. Ankle reflexes and nociceptive reflexes to
pin pricks were found to be less brisk in initiation
and maximal amplitude. As noted by Holmes
(1917, p. 471), "Even if his two hands are pricked
by a pin the patient withdraws the affected limb
less readily and less briskly than the normal."
Holmes was aware of similar reports by Luciani
(1915), who found that nociceptive reflexes in
experimental animals were also reduced by cere-
bellar lesions. However, Holmes (1917, p. 511)
noted that in humans the decrease in nociceptive
reflexes occurred with " ... no alteration in the
threshold of, or diminution in the acuity to, tactile
and painful stimuli, or even subjective differences
between the sensations similarly evoked on the two
sides ..." Thus, in Holmes' view the slowness of
movement was not due to an involvement of the
cerebellum in the processing of stimuli so as to
alter their arrival time at the cortex. The con-
clusions that Holmes (1917) reached on the role
John P. Welsh and John A. Harvey
of the cerebellum in motor movement are worth
noting:
It is an organ which has evolved on the afferent rather
than on the motor side of the central nervous system.
But it receives and integrates proprioceptive impulses
from all parts of the body, and by virtue of these it
keeps the motor mechanisms in such a state of tone
that they can react promptly and efficiently to voluntary
impulses, and thus assures the correct co-operation of
the separate motor centers that are concerned in indi-
vidual acts. (p. 528)
Although Holmes. believed that a change in
postural tone was the etiology of the deficits in
both involuntary and voluntary movements, the
implication of these findings with regard to the
long-standing notion that the cerebellum was
specifically involved in regulating voluntary motor
acts was not realized. In fact, the data provided
by Holmes strongly suggested that the deficits in
performance were global in nature, affecting both
voluntary and involuntary movements. As we
will see, this observation had great implications
for subsequent studies dealing with a possible role
of the cerebellum in learning.
The Consensus of 1927
The Combined Meeting of the Section of Neuro-
logy of the Royal Society of Medicine and the
American Neurological Association held in
London in 1927 firmly reinforced Rolando's original
notion of a specific role for the cerebellum in
voluntary movement. In summarizing the pro-
ceedings, Walshe authoritatively proclaimed that
"It is solely voluntary movement, therefore, which
is dependent upon cerebellar activity" (original
emphasis of Walshe, 1927). Feeling constrained
by the purely descriptive methodology of Luciani,
investigators at this meeting capitalized on the
findings of Sherrington and Magnus, which de-
monstrated that postural reflexes, presumed to be
the fundamental elements of coordinated move-
ments, resided at low levels of the neuraxis. This
was revealed in spinal, pontospinal, and thalamic
animals. Sherrington proposed that in order to
study the components of coordinated movement
in isolation, the higher elements of the cerebrum
should be removed. The decerebrated animal,
16. Role of the Cerebellum 307

furthermore, provided the opportunity to obtain
a clean measure of cerebellar regulation of the
postural reflexes that comprise coordinated move-
ment. Magnus (1916, as cited in Pollock and
Davis, 1927) demonstrated that walking, jumping,
and running in the-thalamic animal was indistin-
guishable from that in the intact animal. However,
the thalamic animal without its cerebellum was
no worse behaviorally than the thalamic animal
with its cerebellum intact (Magnus, 1924, as cited
in Rawson, 1932). Additionally, Pollock and Davis
(1927) found that cerebellectomy did not affect
postural and spinal reflexes of the decerebrate cat.
These outcomes signified that the basic reflex
mechanisms necessary for coordinated movement
not only resided in the spinal cord and lower
brain stem but were autonomous of cerebel-
lar function. Nevertheless, cerebellectomized but
otherwise intact animals demonstrated the pro-
minent postural alterations and motor deficits
that comprised the cerebellar syndrome so aptly
described by Luciani. These outcomes led the
London conferees to conclude that the cerebellum
effected its function only in association with the
cerebral cortex. Because Sherrington specified that
voluntary movement consisted of sequences of

FLEXIONS
EMG MOVEMENT
ONSET

CONTRDL .~

t
COOL
MEDIAL

COOL
LATERAL t
COOL 1,1'1"
MEDIAL &
LATERAL I
.....---oolllliJlllLl....--
,

CONTROL j ,&,I. .--....-z,o

too
Figure 16.2. The effect of cooling the dentate nucleus
on the onset of conditioned arm movements of monkeys
with flexions shown on the left and extensions on the
right side of the figure (Meyer-Lohmann et al., 1977).
Histograms were plotted as number of responses on
the ordinate (see calibration bar for to responses in
lower right of figure) as a function of time in ms from
CS onset (arrow) on the abscissa. Deficits in reaction
time were most prominent when both medial and lateral
aspects of the dentate nucleus were simultaneously
inactivated. During this time both EM G and movement
onsets were increased by approximately 100 ms for
flexions and 130 ms for extensions. Note that normal
performance was restored when cooling was terminat-
ed at the end of the experiment (compare control data
of top and bottom rows). Reprinted with permission
from Meyer-Lohmann, Hore, and Brooks, 1977.
308 John P. Welsh and John A. Harvey

brain stem and spinal reflexes under the guidance voluntary (instrumental) behavior. The following
and control of the motor cortex, the essential effects of cerebellar damage were repeatedly ob-
presence of the cerebrum for cerebellar dysfu~c­ served by all investigators and are of special rele-
tion provided experimental proof of the speCIal- vance for this chapter: a) a decrease in the amplitude
ized role of the cerebellum in the regulation of of instrumental responses, b) a decrease in the fre-
voluntary movement. quency of responses, c) an increase in the onset
latency of responses, d) an increase in the time
Recent Studies required to reach maximal response amplitude,
and e) an increase in the rise time of responses
The primary focus of interest in the latter part of
(i.e., the time between response onset and peak
the 20th century continued to be on the mech-
amplitude). These effects were primarily examined
anisms whereby the cerebellum regulated volun-
in instrumental acts and so the possible effects of
tary motor behavior and the primary etiologies
cerebellar damage on involuntary (reflexive) re-
of the global deficits produced by cerebellar
sponses remained uncertain. However, the obser-
damage. These studies placed an increased em-
vations of both Luciani and Holmes suggested
phasis on precise experimental measurem:nts of
that reflexive acts were also affected, but to a lesser
the instrumental behaviors of nonhuman pnmates
degree. The general agreement was that the cere-
using procedures pioneered by Evarts (1966) in
bellum was important for the performance of
combination with electrophysiologic, electromyo-
instrumental acts, but its possibly wider role in
graphic, and kinematic m~asur:s. By rever.si~ly all responding (instrumental and reflexive) was
disrupting cerebellar functIOn wIth cryogemc In-
not carefully documented by these studies. Addi-
activation, a series of experiments were able to
tional documentation dealing with these issues
'confirm and extend many of Holmes' original
was to come from Pavlovian conditioning studies
descriptions of cerebellar insufficiency (see Brooks,
conducted in Russia.
1986). Most notable of these were deficits in move-
ment onset following cerebellar dysfunction. As
shown in Figure 16.2, inactivating the dentate The Russian Tradition:
nucleus delayed the onset of movement and muscle
activity for both conditioned flexions and exten-
Cerebellar Regulation
sions of the arm (Meyer-Lohmann et aI., 1977). of Reflexive Movement
Cooling the dentate nucleus was shown to increase
reaction times for both conditioned flexions and The Pavlovian Method
extensions of the arm to approximately 130% of
By 1927, the Russian physiologist Pavlov had
normal as well as to decrease the initial velocity
already introduced the concept ofthe conditioned
of the movements. Simultaneous electrophysio-
reflex and the methods for its establishment as a
logical recordings from precentral neurons sug-
means of studying higher forms of nervous ac-
gested that profound inactivation of den~ate fun~­
tivity. A detailed treatise, translated to English,
tion delayed movement onset by remOVIng tomc
summarized more than 25 years of research
facilitation of subcortical nuclei and spinal motor
(1899-1927) on the subject (Pavlov, 1927). T~e
neurons since precentral neurons were not delayed
foundation for this research was firmly rooted In
in their onset. However, when the dentate nucleus
Descartes' concept of the nervous reflex, a funda-
was less severe1y inactivated, the activity of pre-
mental mechanism whereby the central nervous
central neurons was delayed to an extent equiv-
system converted sensory input to motor output.
alent to the increase in reaction time, suggesting
The concept of the reflex had already influenced
that dentate cooling reduced a depolarizing drive
Russian physiological thought, serving as the cor-
on the motor cortex.
nerstone upon which a deterministic theory of
cerebral activity was to be built. This notion was
Summary
advocated by Sechenov, whose "Reflexes of the
The result of the studies cited above led to a clear Brain" (1863) sought to describe the totality of
description of the effects of cerebellar damage on cerebral function through reflex action.
16. Role of the Cerebellum
In the course of his physiological studies on
digestion, Pavlov observed that salivary reflexes
were not always elicited by oral stimuli but could
come under apparently "psychic" control. The
fundamental observation was that salivary re-
flexes were not only elicited unconditionally by
food in the mouth but would come to be elicited
by a neutral stimulus if its occurrence preceded
the receipt offood. These neutral stimuli included
those commonly present during an experiment
such as the food bowl and the experimenter as
well as novel auditory and visual stimuli such as
whistles and light bulbs. Reluctant to yield to
a subjective terminology that labeled these re-
sponses as "psychic secretions" or "voluntary re-
sponding," Pavlov sought to determine objectively
the conditions under which salivary reflexes could
come to be elicited by previously neutral stimuli.
Following the lead of Sechenov, Pavlov consid-
ered these "psychic secretions" to represent the
formation of new reflexes.
The Pavlovian method employed a biological
reflex for which there was a known eliciting stim-
ulus, the unconditioned stimulus (UCS), and a
definable reflexive response, the unconditioned
response (UCR). From approximately 1898 to
1930, Pavlov directed his research efforts at exam-
ining the conditions under which stimuli that
were initially neutral with respect to a biological
reflex could come to elicit that reflex. Such stimuli
were termed conditioned (actually conditional)
stimuli (CSs) and the response they elicited the
conditioned response (CR). Of special interest
were the temporal relationship between the CS
and UCS, the number and frequency of paired
presentations of CS and UCS, the modality and
intensity of the CS, the intensity of the UCS, the
health ofthe animal, and the context in which the
stimuli were delivered.
Two conditioning procedures were typically
employed in the studies to be described below.
The first represented what we now consider to be
the strict Pavlovian method in which the animal
was restrained and the delivery ofthe stimuli was
independent of the animals' behavior. For example,
in this method a tone CS would be paired with a
shock UCS applied to the foot of a dog by means
of a cuff electrode. Although the shock UCS
would elicit a UCR ofleg flexion, the duration of
the shock was not affected. Similarly, although
309
the CS would come to elicit a CR of leg flexion,
the shock UCS would still be delivered at the
specified CS-UCS interval and for the specified
duration. This method will be referred to as
Pavlovian conditioning or, simply, conditioning.
The second contained an instrumental (voluntary)
component and was methodologically identical
to the conditioned avoidance response. It will
therefore be referred to as the avoidance method,
or avoidance conditioning. It should be recalled
that Pavlov (1927) considered instrumental be-
havior to be based on the establishment of condi-
tioned reflexes.
The avoidance method employed the general
procedures of Pavlovian conditioning except that
the shock UCS could be delivered only when the
dog's paw made contact with the floor. Thus,
flexion of the leg would immediately terminate
the shock. For example, an animal could com-
pletely avoid the shock UCS by flexing its leg to
the CS before UCS onset or it could escape from
the shock by flexing its leg after UCS onset. It
should be obvious that the Pavlovian method
was always used in the study of autonomic func-
tions such as salivary, pancreatic, gallbladder,
and stomach secretions as well as heart rate
and blood pressure (Bykov, 1957; Gantt, 1989),
whereas both methods were employed for leg
flexion.
Pavlov considered the cerebral cortex to be the
site of associative learning and the conditioned
reflex to be a tool for uncovering cerebral (mental)
function. These views determined the experimental
questions in Pavlov's laboratory during his
active period of research on the conditioned
reflex (1898-1930). It was only near the end of
this period that one of his students (Popov,
1929b) examined the role of the cerebellum.
However, while Western scientists were soon in-
troduced to the method of Pavlovian conditioning
(e.g., Yerkes and Morgulis, 1909), they remained
largely unaware of either the physiological and
pharmacological studies on the conditioned reflex
that were being carried out by Pavlov and his
students or of the subsequent studies on the cere-
bellum. By contrast, Russian scientists were well
aware of Western studies on the cerebellum and
employed Pavlovian conditioning to obtain more
objective measures of cerebellar pathology. The
following is a summary of these studies.
310
Popov
In 1929, one of Pavlov's students, N.F. Popov,
reported detailed observations of one dog with
complete cerebellectomy, one dog with unilateral
cerebellectomy, and one cat with removal of the
vermis (Popov, 1929a). In large part, Popov's
observations of motor deficits up to 18 months
after surgery were in complete agreement with
Luciani. Popov reported changes in sensory and
motor function of dogs and cats with cerebellar
damage by employing an array of neurological
tests. This initial study verified many of the highly
documented findings of Luciani and attempted
to place the observed deficits in sensory and motor
function within the context of a Cartesian frame-
work based on reflex activity.
Immediately after cerebellectomy, the clinical
presentation was clonic extension of the forelegs,
slight flexion of the hind legs, and opisthotonos.
The hind legs were characterized by marked hy-
pertonia by the second postoperative day but this
was not a constant phenomenon. Hypertonia gave
way to hypotonia by the eighth day, which, in
turn, persisted until 2 months after the surgery.
At this point, hypertonia returned. Chronic
deficits were general agitation and unrest, head
oscillations, hen's gait, falling backward, and com-
pensated ambulation characterized by marked
abduction of the limbs. In agreement with previous
observations by Luciani and Holmes, Popov also
observed deficits in reflexive behaviors. For
example, there was an abolition of the response
to pin prick, and a general increase in the threshold
for evoking responses to painful stimuli. These
effects were noted up to 1 month after the surgery.
Popov observed that this dog showed a marked
inability to swim.
Many ofthe same deficits were observed on the
side of the lesion in a dog with unilateral cere bel-
lectomy: ipsilateral extensor rigidity, hypertonia
of the ipsilateral foreleg, weakness of the ipsilat-
eral body reflexes, llgitation, and unrest. The hemi-
cerebellectomized dog also demonstrated stra-
bismus, pleurothotonos, and forced rotation. As
with a complete cerebellectomy, a unilaterally
cerebellectomized dog demonstrated marked
hypertonia on the side of the lesion immediately
after the surgery; this was replaced by hypotonia
on the seventh postsurgical day. Normal tone
John P. Welsh and John A. Harvey
returned in this animal 10 weeks later. This dog
could not stay afloat after the surgery. Removal
of the vermis in a cat induced opisthotonos, hyper-
tonia of the vertebral muscles, agitation and un-
rest, forced rotation, astasia of the head and limbs,
clonic movements of the hind leg, dysmetria of
foreleg reaching, and marked extension of the
hind limbs while walking.
In interpreting these outcomes, Popov noted
that the dominant symptom of cerebellar damage
was marked hypertonia of the extremities, neck,
and back, which was most prominent during ac-
tivity. Be described the extremities during walking
as appearing as if they were unyielding sticks and
noted the greater and more enduring deficits in
the hind legs as compared to the forelegs. He
could not clearly demarcate Luciani's three stages
of deficits after cerebellar removal. Instead, he
believed that all of the aforementioned manifesta-
tions of cerebellar damage were constantly present
from. the operation until death, simply varying in
intensity throughout the animal's life.
Popov was particularly intrigued by what ap-
peared to be a paradoxical effect of cerebellar
damage on complex voluntary movements in con-
trast to the effects on reflexive movements. For
example, the movements comprising the complex
motor acts of walking, reaching, and swimming
appeared to be intensified, while comparatively
simple, reflexive movements to pin prick and pain-
ful stimuli could be attenuated or sometimes
abolished.
Popov believed that the primary function of
the cerebellum was to inhibit the reflexive activity
of centers in the midbrain that were responsible
for relaying motor commands ofthe cerebrum to
the spinal cord. Popov accepted Sherrington's
positions that complex movements depended on
the analytic and synthetic capabilities of the
cerebrum and that the principal means whereby
the cerebellum exerted its function was through
the integration of proprioceptive information. The
deficits in complex motor acts after cerebellar
damage suggested to Popov that motor centers
of the midbrain were in a state of uncoordinated
hyperexcitability and could not relay the cere-
beral commands for flexion to the limbs. In
Popov's view, the extensor rigidity after decere-
bellation was functionally similar to the profound
extensor rigidity that occurred after decerebra-
16. Role of the Cerebellum
tion, both resulting from the inability of the
cerebrum to express flexion and adduction. The
reflexive activity of midbrain motor centers,
unchecked by regulatory inhibition of the cere-
bellum after cerebellar damage, would not permit
the passage of descending motor commands but
instead would express a dominant predisposition
for excitation of extensor motoneurons. Complex
movements would lose their fluidity and become
slow as a result.
Popov believed that a second function of the
cerebellum was to intensify the "correcting and
focusing" impulses of the cerebrum that were
essential for the proper maintenance of reflex
function. In this way, Popov could explain the
apparent paradox of the simultaneous occurrence
of hypometria of the reflexes and hypermetria of
more complex motor acts. Popov also believed
that the cerebellum had an important function
that was independent of the cerebrum. This addi-
tional role, as he put it, was essential for preparing
an "essential ground" for the execution of move-
ments by ensuring a proper state of tone prepara-
tory to muscular activity.
The detailed description of Popov's findings
have been presented in order to emphasize that
he obtained the same effects of cerebellectomy
as had scientists in the West. Moreover, his obser-
vations and interpretations were essentially ident-
ical with those of Western scientists, notably
Luciani and Holmes. However, Popov was not
satisfied with the primarily observational method
on which his observations and those of others
were based. This is clearly stated in his second
report (Popov, 1929b), which began with the fol-
lowing comment:
Destruction of control of voluntary muscles by means
of complete or partial extirpation of the cerebellum
presents an extremely complex picture. Because the
usual method of observation is completely subjective,
a large number of theories arose, giving rise to myriad
interpretations and a wholly unscientific terminology
in which to express them. Thus, it is not surprising that
the study of cerebellar function has remained exactly
where the classical experiments of Luciani left it, half
a century agb.
For these reasons, Popov applied Pavlovian
conditioning procedures to the study of cerebellar
function. I t is clear that Popov considered the CR
elicited by a CS to be analogous to voluntary
311
movements of humans. Because of his extensive
work on the effect of cerebellectomy on move-
ments of the hind limbs (Popov, 1929a), Popov
studied the defensive reflexes of the hind limbs of
cerebellectomized dogs. In a first study that
employed Pavlovian conditioning, a flashing light
served as the CS and preceded the shock UCS
delivered to a leg cuff on each trial. Normal dogs
readily acquired a CR oflimb flexion to the flash-
ing light. Cerebellectomized dogs also acquired
pronounced CRs to the CS within eight parings
of CS and UCS. However, these CRs differed
from control dogs in the direction of movement.
Instead oflimb flexion as a response to the flashing
light, cerebellectomized dogs responded with limb
extension. This was observed on each trial in
which the light was paired with paw shock
because in the Pavlovian method shock delivery
was independent of whether the animal flexed,
extended, or did not respond to the light. Cere-
bellectomy did not affect the unconditioned limb
flexion elicited by the shock UCS, which could
be observed on each conditioning trial. The cere-
bellectomized dogs would extend their leg to the
light CS and then flex their leg to the shock UCS.
These dogs were unable to acquire leg flexion as
the CR even after 100 pairings of the flashing
light CS with the shock UCS.
To capture further the pronounced effects of
cerebellar dysfunction on CRs ofthe limb, Popov
subsequently employed the avoidance method of
conditioning. The CS was a metronome beating
at 168 beats per minute and having a duration of
approximately 40 s. Popov's original recordings
are presented in Figure 16.3. Normal dogs readily
flexed their limb to the metronome, maintained
flexion for the duration of the stimulus, and
thereby avoided the shock (top tracing in left half
of Fig. 16.3). However, as in the previous experi-
ment, cerebellectomized dogs displayed a pro-
nounced limb extension as the CR in response to
the metronome CS (top tracing in right half of
Fig. 16.3). This brought the limb in contact with
the floor, resulting in the delivery of the shock
UCS and, consequently, elicitation of leg flexion
as the UCR. Flexion was not maintained, pre-
sumably due to the continued presentation of
the metronome CS, which would elicit extension
as the CR. Thus, for as long as the metronome
was on, the cerebellectomized dog would vacil-
312
Figure 16.3. The effect of cerebellectomy on condi-
tioned avoidance responding in a dog. The kymo-
graphic tracings, from top to bottom, represent motor
reactions of a dog's hind limb (upward and downward
deflections indicate flexion and extension, respectively),
presentation of the metronome CS (downward deflec-
tion of tracing 1 indicates CS onset and duration),
delivery of the DCS (each upward deflection of tracing
2 indicates delivery of shock to the paw), and time (each
upward deflection of tracing 3 marks 1 s). The left
half of the figure presents a typical CR in a normal dog.
Note that the dog demonstrates a robust flexion CR
(upward deflection of pen) in response to the metronome
late between flexion and extension of the limb
while the normal dog would maintain a pro-
nounced flexion response (see legend to Fig. 16.3
for details).
In interpreting these outcomes, Popov consi-
dered the fact that a pronounced result of cere-
bellectomy was extensor rigidity. Popov viewed
the cqange in CRs following cerebellectomy as a
shift from a propensity for flexion to one for
John P. Welsh and John A. Harvey
CS. The dog maintains this flexion CR for the entire
duration of the CS and thereby avoids onset of the
paw-shock DCS. The right half of the figure presents
a typical conditioning trial after cerebellectomy. The
cerebellectomized dog responds with a prominent limb
extension (downward deflection of pen) at metronome
onset, which brings its paw in contact with shock
(upward deflection of pen on tracing 2). The shock
elicits a flexion response that permits the limb to escape
the shock. For the duration of the metronome stimulus,
flexion responses elicited by shock are followed by
extension responses that bring the limb back into con-
tact with the shock. (Reprinted from Popov, 1929b.)
extension. due to the removal of the cerebellum's
inhibitory regulation of the reflexive mechanisms
that intensified the propensity for extension.
Popov believed that limb flexion to a CS required
finer regulation by the cerebral cortex through
indirect pathways that coursed through the cere-
bellum in order to supplement the direct pathways
of the pyramids. In the presence of cerebellar
damage " ... only a strong unconditioned peri-
16. Role of the Cerebellum 313

pheral stimulus ... [could] ... elicit a simple de-
fensive reflex in the form of withdrawal." In
conclusion, Popov stated that " ... the cerebellum
plays a large role in the formation of conditioned
motor reflexes." It is clear, however, that Popov
did not conceive this role to be one of serving as
the site where learning occurred but rather one
of regulating the reflexive activity of the brain
stem so that the output ofthe motor cortex could
produce a normally executed CR.
Gambaryan
The effect of cerebellectomy on conditioned
leg flexion was reexamined 30 years later by
Gambaryan (1960) and Fanardjian (1961).
Gambaryan employed the avoidance procedure
in a manner similar to Popov's second experi-
ment where the dog could avoid paw shock if it
maintained flexion in the presence of the CS.
Gambaryan permitted the symptoms of the im-
mediate postoperative period to subside before
subjecting the animals to the conditioning para-
digm. This period ranged from 2.5 to 10.5 months.
In two puppies with complete cerebellectomy,
conditioned leg flexions were acquired within 90
presentations of a bell CS that signalled shock to
the hind limb. The absence of the cerebellum in
one ofthese puppies (Odinokaya) is documented
in the top panel of Figure 16.4, whereas the bottom
panel illustrates conditioned leg flexions elicited
by the bell CS with no evidence of the leg exten-
sion that had been noted by Popov. However,
Gambaryan did report that the leg flexion CRs
were frequently preceded by leg extension, and
this occurred even after 130 trials when condi-
tioning had been reliably achieved. Gambaryan
attributed these differences in experimental out-

Figure 16.4. Top panel presents the posterior

view of the brain of a puppy named Odinokay
in which the cerebellum was completely re-
moved. The bottom panel presents condi-
tioned avoidance responses of the hind limb
ofthis puppy after cerebellectomy. CRs ofleg
flexion are elicited by a positive CS (bell + )
but not by a CS of another frequency that
had never been paired with the UCS (bell- ).
The tracings, from top to bottom, indicate
hind limb CRs (upward deflection represents
flexion), CS onset and duration, UCS of paw-
shock delivery, and time in seconds. Note
that the CRs were successfully maintained so
that no shock was delivered. (Reprinted with
permission from Gambaryan, 1960).
314
come to the shorter postoperative period em-
ployed by Popov. However, conditioned flexion
responses were acquired at times when the
animals were severely ataxic and demonstrated
virtually all of the chronic motor deficiencies
noted by Luciani and Popov.
Puppies with partial removal of the cerebellum
acquired the flexion response three times more
rapidly than those with total cerebellectomy. A
puppy that had 10.5 months to recover from
cerebellectomy acquired the flexion response even
more quickly than normal. In addition, a dog
with a lesion of the dorsal columns and whose
A
,1' -, ~._ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _J
2~~f~--~~~~~--------'-~--------------------~-4~---­
+
J~~----------------------------~----
4 ~'-------------_,--J
5
6
5,~_==~====~~~;;~~~======~
= --=------t
85
r AI .. '-.~_ _ _ _ _ _ _ _ _
4 ..
~~( ________________________--JL_______
J",.Ld. ~'-_________--~
Figure 16.5. Conditioned motor reaction before (A)
and after (B) ablation of the paramedian lobe bilaterally
and of the vermis (save lobules I - III). The numbered
tracings, from top to bottom, indicate 1) movements
of left rear limb, 2) movements of right rear limb, 3)
movements of left front limb, 4) movements of right
front limb, 5) pneumographic recording of respiration,
6) mark of CS delivery, 7) mark of UCS delivery,
and 8) time in seconds. Note that the CS was only
paired with shock to the left rear limb. Limb flexion is
indicated by an upward deflection ofthe pen. A: Before
John P. Welsh and John A. Harvey
cerebellum was removed after acquisition training
retained the conditioned flexion response. In
summary, Gambaryan's experiments indicated
that cerebellectomy did not block the acquisition
or retention ofleg flexion CRs even in the presence
of marked motor deficits.
Fanardjian
Fanardjian (1961) examined the effects of cere bel-
lectomy on conditioned leg flexion in 10 dogs.
The Pavlovian method was employed to measure
simultaneously the responses of all four limbs
J /.
1
•
J
----
jrfM '''L
......._---.J.f'..,._ __
the lesion the flexion response of the reinforced limb
(tracing 1) is rapid and flexion is maintained for the
duration of the CSt B: After cerebellar damage the
animal initiates CRs but demonstrates prominent
astasia of the limb before onset of the shock UCS.
Delivery of an inhibitory stimulus (CS-) that was never
paired with shock (second deflection in tracing 6)
occurred between the two presentations of the positive
CSt The CS- failed to elicit a CR. (Reprinted, with
permission, from Fanardjian, 1961.)
16. Role of the Cerebellum
during the pairing of visual or auditory CSs with
a shock UCS to one hind limb. The UCS was
delivered independently of the animal's behavior
by a cuff attached to the paw. In 4 of the 10 dogs,
CRs were established before surgery. The primary
result obtained with all 10 dogs was that cerebel-
lectomy did not block the retention or prevent
the de novo acquisition of CRs. This can be seen
in Figure 16.5, which compares the preoperative
performance of CRs in the top portion of the
figure with the postoperative performance ap-
proximately 1 month after surgery in the bottom
portion (see figure legend for details). Conditioned
flexion responses, although smaller in amplitude
and duration, could be elicited reliably even on
the second day after surgery, a time when severe
extensor rigidity and opisthotonos were present
(Fig. 16.6; see figure legend for details). However,
in agreement with Gambaryan and consistent
with the findings of Popov, cerebellectomized
dogs exhibited a greater tendency to extend their
hind limb in response to the CS, especially in the
immediate postoperative period. Normal dogs
sometimes showed a slight conditioned extension
response preceding conditioned flexion. How-
ever, the frequency of extension in the intact
dog was less than in the cerebellectomized dog.
------~~--~--------------~--------~~--.~----~----
J (
-~~., ... .....
Figure 16.6. All conventions are the same as in
Figure 16.5. Conditioned reflexes of a dog's hind limb
on the second day after cerebellar ablation. Virtually
all of the cerebellum was removed in this dog except a
small portion of the inferior vermis. Only two condi-
tioning trials are presented. On the first trial, the CS
elicits a small flexion CR of short duration. However,
315
Fanardjian concluded that the normal CR con-
sisted of two components: a weak tendency for
extension and a stronger tendency for flexion.
The effect of a cerebellar lesion, then, was to
strengthen the extension component of the CR at
the expense of the flexion component. In agree-
ment with Gambaryan and Popov, the tonic
component of the conditioned flexion response
was more susceptible to cerebellar dysfunction
than the phasic component. At early postopera-
tive periods, the tonic component of limb flexion
was abolished in some cases while the phasic
component remained intact. Thus, whereas cere-
bellectomized dogs initiated conditioned flexion
responses, they had difficulty maintaining the limb
in a maximally flexed position for the duration
of the CS. This can be seen in the oscillatory
behavior of the CR in Figure 16.5 and its short
duration in Figure 16.6 (see legends for details).
This may have in part accounted for the oscil-
latory behavior of limb flexion and extension
noted by Popov when employing the avoidance
method (see Fig. 16.3).
By simultaneously measuring all four limbs,
Fanardjian (1961) found that normal dogs acquired
the flexion response of the reinforced limb while
gradually reducing movements in the other limbs.
........... " ...• , .......... , ... , '1'.'
onset of the UCS is followed by a large flexion UCR.
On the second trial, the CS elicits a CR characterized
by a long onset latency and small amplitude. This
initial CR is followed by a second, large flexion CR
that is not maintained. As on the first trial, onset ofthe
UCS elicits a robust UCR. (Reprinted, with permission,
from Fanardjian, 1961.)
316
Some cerebellectomized dogs, however, were un-
able to inhibit the three nonreinforced limbs; the
CS elicited flexion responses in all of the limbs.
Fanardjian concluded that an inability to inhibit
responding in the three nonreinforced limbs was
a manifestation of the removal of a specific func-
tion of the cerebellum which normally acted to
suppress inappropriate and superfluous move-
ments. Such a role for the cerebellum was seen by
Fanardjian as enabling an animal's behavior to
become "specialized" and thus more prepared to
meet the demands of the environment. Fanardjian
concluded that:
... insufficiencies of the cerebellum are hard to detect
by observing contraction of one muscle or even a limb,
since the disorders affect all levels of the central nervous
system, eventually causing changes in space and in
time of the delicate and interrelated activity of the
motoneurons of the spinal cord.
Accordingly, greater deficits following cerebel-
lar lesions were more easily observed by taking
simultaneous measures of multiple behaviors and
determining changes in their relation with one
another. Fanardjian recognized that deficiencies
of cerebellar function and the centripetal effects
of cerebellar lesions on the rest of the central
nervous system were difficult to distinguish from
one another on the behavioral level. Moreover,
abnormal function of spinal motor neurons could
not be discounted as an important factor causing
irregular conditioned limb movement following
cerebellar damage.
Orbeli
New notions of cerebellar function were intro-
duced by the Russian physiologist Orbeli (1940).
Summarizing 12 years of work (1928-1940),
Orbeli confirmed the classic findings of Luciani
and extended the role of the cerebellum into the
realms of sensory and autonomic function. Re-
garding motor function, Orbeli noted changes in
the excitability of motor nerves and muscles fol-
lowing cerebellar damage. Orbeli noted that the
time required for excit'ation of motor nerves and
muscles was often extended after cerebellar
damage. Changes in chronaxy of muscles and
motor nerves were not isomorphic so that the
ratio of the excitability of the muscle and the
nerve deviated from normal in the animal with
John P. Welsh and John A. Harvey
a cerebellar lesion. Additionally, Orbeli noted
changes in the ratio of chronaxy in antagonistic
muscles and the breakdown of reciprocal inner-
vation (Sherrington, 1906) in movements elicited
by stimulation of afferent nerves. In addition to
the well recognized role ofthe cerebellum in motor
function, Orbeli viewed the cerebellum as an
important regulator of sensory function. He
referred to studies of his own, employing external
stimuli, that demonstrated increases in the stimu-
lus thresholds for eliciting various unconditioned
reflexes. He thus corroborated the previous
observations of Luciani, Holmes, and Popov
concerning the susceptibility of unconditioned
reflexes to cerebellar damage. Noting that the
thresholds for eliciting reflexes of cerebellecto-
mized animals varied greatly from day to day,
Orbeli likened these alterations in sensory func-
tion to a "sensory ataxia."
Orbeli also noted changes in digestive function
following cerebellar extirpation. These changes
included retardation of intestinal motility, attenua-
tion in secretory activity of organs of digestion
to pharmacological agents, and changes in pan-
creatic function to glycemicchallenge. Alterations
in basal metabolism, delays in thermoregulation,
and delays in the initiation of baroreceptor re-
flexes to changes in blood pressure were other
changes in autonomic function noted after cere-
bellar extirpation. Orbeli concluded that the
cerebellum had an adaptive and trophic influence
over all bodily tissues. The outstanding question
for Orbeli was the mechanism whereby the cere-
bellum exerted its trophic function on the sensory
and motor systems. Orbeli proposed that the
change in autonomic function following cerebel-
lar extirpation was a consequence of widespread
alterations in the "vegetative" function of the
entire body. Generalized alterations in vegetative
function were considered to be the primary etio-
logy of the decrements in excitability of the central
nervous system following cerebellar extirpation.
Livshits
Orbeli's ideas served as the basis for a series of
investigations by Livshits (1947) and Krasusky
(1957) on cerebellar involvement in salivary CRs.
It was believed that conditioned salivation pro-
vided a better measure of the role of the cerebel-
 16. Role of the Cerebellum
lum in the formation of associations because basal
salivary activity was far less disrupted than the
skeletal motor system following cerebellar damage.
Livshits (1947) provided a detailed account of
two dogs, Barbos and Kulak, whose conditioned
salivary activity was measured before and after
complete cerebellar removal. CSs that preceded
the presentation offood were a metronome beat-
ing 100 times per minute, a bell, a tone, and a
light. The CSs preceded the presentation of the
food by 30 s. Before the cerebellar lesion, Barbos
normally salivated from two to five drops to the
metronome, tone, and bell CSs (Fig. 16.7). Very
little responding occurred to the light. Later, the
entire posterior vermis, most of the hemispheres,
and all of the deep nuclei were removed. The only
remaining cerebellar tissue after the surgery was
the flocculus and portions of the anterior lobe.
Ten days after surgery, there was no salivary
responding to the metronome, bell, or light, and
a mean level of responding to the tone of only 0.5
drop. However, 5 months after the lesion, Barbos'
salivary responding approached its preoperative
norm. These data, along with the observations of
BARBaS
EFFECT OF CEREBELLAR EXTIRPATION
6 6
5 5
v
VI
4 4 VI
a. v a.
o v
0::
o
3
v 3
o
v salivation was further examined by Krasusky
2 v 2
v (1957). This study employed nine dogs that had
v
v acquired salivary CRs before cerebellectomy. In
o
v
v
M T L 8
n
MT L B
tl
MT L B
o
PREOP 10 DAYS 5 MONTHS
6/10-13/33 4/5/34 9/1-23/34
Figure 16.7. Effect of cerebellar damage on conditioned for up to 35 months after the surgery. The CRs
salivation of the dog Barbos. Ordinate presents the
mean number of drops of saliva to four different CSs:
M, metronome beating 100 times per sec; T, tone; L,
light; B, bell. Preoperative data were taken from experi-
ments 124 and 126, June 1933. Postoperative data were
taken from experiment 193, April 1934 (10 days), and
experiments 274 and 278, September 1934 (5 months).
See text for details. (Data from Livshits, 1947.)
317
Gambaryan and Fanardjian, clearly indicated
that the length of the postoperative period is an
important variable in assessing the effects of cere-
bellar extirpation on conditioned reflexes.
In the second dog (Kulak), profuse conditioned
salivary responding was observed before surgery
to each of the CSs paired with presentation of
food (Fig. 16.8A). The entire cerebellum was
removed except for a portion of the posterior
vermis and a small portion of the left hemisphere.
As in the case of Barbos, Kulak demonstrated a
virtual absence of conditioned salivary respond-
ing to each of the CSs when tested on the tenth
day after cerebellar extirpation (Fig. 16.8A). How-
ever, some recovery of conditioned salivation
was observed by the 25th day after the lesion
(Fig. 16.8A). Despite the absence of the cerebel-
lum, food deprivation, which would be expected
to increase the response-evoking properties of
the CS, restored conditioned salivation to pre-
operative levels for all stimuli except the tone
(Fig. 16.8B; see legend for details). The restoration
of conditioned salivation produced by food
deprivation was not due to an increase in the
baseline level of salivation because responding to
conditioned inhibitory stimuli (CS-) remained
absent. This was in contrast to Kulak's behavior
before the lesion when food rationing enhanced
responding to both conditioned excitatory and
inhibitory stimuli.
Krasusky
~ The effect of cerebellar lesions on conditioned
the immediate postoperative period, conditioned
salivary activity of every dog was significantly
depressed. Some dogs remained depressed at this
postoperative level for many months. Krasusky
measured conditioned salivation in these dogs
of every dog showed some recovery from their
immediate postoperative levels. However, the rate
of recovery was highly variable among the nine
dogs. At the time of each dog's sacrifice, four re-
sponded at levels below their preoperative norm,
two regained their preoperative level, and two
exceeded their preoperative level of performance.
318 John P. Welsh and John A. Harvey

A B
16 16

14 14

12 12
10 III
a.. 10
III
a..
0 0
a:: 8 8 a::
0 0
6 6

4 4

2 2
0 0
t.4TLB TLB
t.4 t.4TLB TLB
t.4 TLB
t.4 t.4TLB
PREOP 10 DAYS 25 DAYS PRE FAST 3/4 FAST FAST
6/19/3" 7/3/3" 7/11-18/3" 2/10/35 2/11/35 2/15/35

Figure 16.8. Data presented and abbreviations as in
Figure 16.7. A: Effect of cerebellar extirpation on con-
ditioned salivation of the dog Kulak. Preoperative
data were taken from Experiment 380, June 1934. Post-
operative data were taken from Experiment 381 (10
days) and Experiments 388-394 (25 days), July 1934.
See text for details. B: Effect of food deprivation on
conditioned salivation in the cerebellectomized dog,
Kulak. Before fasting (PRE FAST), responding to each
of the CSs was quite low (data presented as means from
Experiment 473). Restriction of Kulak's food to one
fourth of normal (~ FAST) enhanced conditioned
salivation (data presented as means of Experiment
474). Total food deprivation for 40 hours (FAST)
restored conditioned salivation to preoperative levels
for each of the stimuli except the tone (data from
Experiment 477). Fasting did not enhance responding
to conditioned inhibitory stimuli (data not shown),
suggesting that an increase in baseline responding did
not contribute to the increases in salivation elicited by
the positive CSs. (Data from Livshits, 1947.)

Averages offive dogs whose conditioned salivary
activity was measured up to 15 months after the
surgery are presented in Figure 16.9. This figure
shows that conditioned salivary activity was re-
duced to 30% of its preoperative level in the
month following cerebellar lesions. However, over
the course of 15 months, conditioned salivation
returned to a level within the range of preoperative
values. Dogs with cerebellar lesions demonstrated
changes in the pattern of salivation to CSs of ex-
tended duration. Before surgery, each dog sali-
vated in a characteristic way to a CS of 3 minutes'
duration. For instance, the magnitude of saliva-
tion could be the same for each minute of the CS,
the magnitude of responding could be maximal
in the first minute and decrease to a minimum in
the third, or relatively low during the first minute
and increase to a maximum in the third. For all
dogs tested in this manner at 20 to 29 months
after the surgery, the pattern of salivation was
changed so that salivation was relatively low dur-
ing the first minute, increased during the second
minute, and reached a maximum in the third
minute.
The characteristics of the responses following
cerebellar damage, an absolute reduction in sali-
vation immediately after the surgery and a reduc-
tion ofthe magnitude of salivation at early relative
to late latencies during the CS, signified to
Krasusky that the animals were demonstrating a
generalized attenuation in the excitability of the
nervous system.
Karamyan
Because of his interest in the theory of brain
evolution proposed by Sechenov (1852, cited in
Karamyan, 1959), Karamyan carried out an ex-
tensive series of studies to examine the evolution
of cerebellar function. He did so by examining
the effects of cerebellectomy on motor functions,
including CRs, in fish, amphibians, reptiles, birds,
16. Role of the Cerebellum
140
120
1
LJ
100
en
a.. • produced a wide range of effects. All fish
I ]1--./1 i 1
0 80
a:::
0 60
.c.o
20
.iT 1
.I.
0123 6 12 15
MONTHS POSTOPERATIVE
Figure 16.9. Effect of cerebellar extirpation on con-
ditioned salivary responses as a function of post opera-
tive interval. Data are presented as mean ± standard
error of the mean of the cumulative records of salivary
drops to CSs. Each point represents the mean of the
five dogs named Melin, Smirny, Sery, Tourist, and
Naida. See text for details. (Data from Krasusky, 1957.)
rodents, and cats (Karamyan, 1959, 1962, 1968;
Karamyan et aI., 1969).
Severe motor disorders were noted in dogfish
after extirpation of the cerebellum: disturbance
of gill movement, attenuated reaction to pin prick,
erratic swimming movements, circling toward the
side of the lesion. The disorders were less severe
in skates and consisted of increases in motor
activity and changes in posture. Karamyan com-
pared these effects in the chondrichthyes (dogfish
and skate) with removal of the cerebellum in
in osteichthyes fish (goldfish, pike, carp, and
perch). Lesions in the latter class resulted in many
sensory-motor disorders that varied with the
species studied. Reduction in pain sensitivity and
disorganized swimming behavior were noted in
all species. Perch and pike were most susceptible
to cerebellar damage, lying on the surface with
arched back, and swimming upside-down in an
uncoordinated manner. Intact carp developed
defensive CRs to visual and auditory CSs paired
with electric shock to the tail as the UCS. Defen-
sive CRs were not retained from one day to
319
another but were readily acquired each day after
7 to 10 pairings. Removal of the cerebellum in
carp and goldfish prevented acquisition of condi-
tioned defensive responses even after 20 days of
conditioning. Partial lesions of the fish cerebellum
demonstrated profound reductions in CRs.
Whereas most retained permanent deficits in CRs,
some reacquired their preoperative level of per-
formance 1 month after the lesion. Removal of
the forebrain and optic tectum did not affect CRs
acquired to visual or auditory CSs. Karamyan
et aI. (1969) concluded that:
the fish are the first representatives in the phylogenetic
scale of vertebrates, where a new system of integration,
the cerebello-mesencephalic, is developed, which
provides for a high motor activity composed of both
conditioned and nonconditioned components of reflex
activity. (p. 640)
Of the class amphibia, frogs and toads exhibited
changes in tone and posture at rest and during
swimming, abolition of rhythmic motion during
swimming, and extensor rigidity after removal of
the cerebellum. Defensive CRs of the hind leg
were established by pairing a bell or light CS with
a shock UCS to the hind leg. Normal frpgs did
not attain stable levels of conditioning. Toads
were found to be better than frogs and could
acquire stable CRs over 20 days oft raining. Cere-
bellectomy transiently abolished the CRs of toads
but they were reacquired on the third day after
the operation. Karamyan concluded that the
acquisition of CRs in amphibians was not asso-
ciated with the cerebellum.
Of the class aves, Karamyan studied pigeons
and poultry. Confirming the findings of Flourens,
cerebellectomized pigeons exhibited profound
alterations in motor function characterized by
swaying of the head, nystagmus, agitated behav-
ior, retraction of the head, increase in extensor
tonus during movements, and reduction of muscle
tone at rest. Abduction of the limbs during walk-
ing, astasia of the body, and undirected flying
additionally characterized pigeons with partial
cerebellar extirpation. Cerebellar damage did not
profoundly alter the motor behavior of ducks
and roosters as compared to pigeons. Cerebellar
extirpation caused alterations in tone of the neck
320 John P. Welsh and John A. Harvey

A A

f ",II" II ",tl ,in" II "",,"'_'M~"hI"" hi.......,........

Figure 16.10. Effect of cerebellectomy on

cardiac and respiratory conditioned reflexes
J in the pigeon. A: Top three tracings present:
14.7 1) EKG with the mark of the electric shock
UCS superimposed on it, 2) pneumographic
Iltll' ifni IIl""'" " . i 11th' I f1 h ,If" i iti' rli iii i Iii i i rr i r-rt\, i nhli C" recording of respiration, and 3) time marker
in seconds with CS onset indicated by inter-
ruption oftime marks. Note that onset of the
CS elicits a marked tachycardia and increase
B 5 in respiration. B: Bottom three tracings de-
monstrate that a stimulus (CS-) not paired
~
with the UCS fails to elicit any changes in
heart rate or respiration. (Reprinted, with
Ju permission, from Karamyan, 1959.)

A A

2 .!.l.LlJj , ] I I II I , I 1l.J I , I ( J I ( I I I I Ie l , I LJ ; i.' I !J ill 111 '''':' I

B 6

i l l I ,! ! ( I !J , II II II I I L I I II I ] I I ! I: I ! I , ! I,I : I , l'lJl] III

Figu.re 16.11. The effect of cerebellectomy on con-
ditioned respiratory reflexes of the cat. A: The top three
tracings represent: I) plethysmographic recording of
respiration, 2) EKG with mark of the electric shock
UCS superimposed on it, 3) time base in seconds with
CS presentation superimposed on it. Note that a con-
ditioned reduction in respiration is elicited by the posi-
tive CS. B: A stimulus (CS-) that was never paired with
shock also elicits a respiratory CR indicating an inability
to inhibit responding after cerebellectomy. (Reprinted,
with permission, from Karamyan, 1959.)
[6. Role of the Cerebellum
and limb muscles. In contrast to the motor agita-
tion observed in pigeons, roosters and ducks
became unnaturally quiet after cerebellar extirpa-
tion. Cerebellectomy weakened but did not
abolish respiratory and cardiac CRs in pigeons
(Fig. 16.10).
In mammals, Karamyan removed the entire
:::erebellum in rats and cats. Deficits in rats after
removal of the cerebellum were extensor rigidity
during walking and atonia of the neck and limb
muscles during rest. The deficits were greater and
more enduring in the hind legs as compared
to the forelegs. Behavioral compensation was
characterized by prominent astasia and ataxia.
Increased sensitivity to pain and tactile stimuli
characterized the cerebellectomized rat. Removal
of the cerebellum in cats weakened withdrawal
reflexes and enhanced scratching and licking
reflexes. Cerebellectomized cats extended their
limb and tail more forcefully to a hammer tap
than normals. Removal of the cerebellum induced
more severe and long-lasting deficits in CRs of
cats as compared to the birds. For 1 month after
extirpation of the cerebellum, cats demonstrated
highly variable levels of CRs to both excitatory
CSs paired with an electric shock and inhibitory
CSs. During the second month after the surgery,
A
•
======================r===~~~==J
________________~________________________~~~.~t______
.~t~__ •
------------------~~~--------------~---------
« • ,t"" 4 t'
, . f n tIt' I , "f I • t t,
Figure 16.12. Conditioned respiratory reflexes of an
acerebellarcat acquired to a metronome stimulus paired
with ammonia vapor to the nose. A: Conditioned re-
duction in breathing to a positive CS. From top to
bottom: 1) pneumographic recording of respiration,
321
cats manifested a distinct increase in excitability
as demonstrated by enhanced responding to both
excitatory and inhibitory CSs. By the third month
after cerebellar ablation, positive CRs regained
their preoperative level of performance whereas
inhibition of CRs to a CS - did not (Fig. 16.11).
Similar results were obtained by pairing a metro-
nome CS with the presentation of ammonia vapor
to the nose as the DCS (Fig. 16.12). In this para-
digm, an apnea CR was acquired to the CS after
30 pairings whereas stable differentiation could
not readily be acquired in cerebellectomized cats.
Summary
This section has presented the results obtained
by seven investigators from 1929 to 1962 using
the Pavlovian method. The effects of cerebel-
lectomy that they noted through observation
of organisms ranging from fish to mammals were
in essential agreement with those reported by
Western investigators. More importantly, all
seven investigators were agreed that cerebellar
damage did not abolish an animal's retention of
conditioned reflexes nor did it prevent an animal
from acquiring conditioned reflexes. These results
were based on studies employing a wide range of
dill.
t t t t , • , , • t t , , t
'" r t ' t I • t
2) mark of the CS, 3) mark of the VCS, 4) time in
seconds. B: Apnea during CS - indicates failure of
differentiation after cerebellectomy. (Reprinted with
permission from Karamyan, 1962.)
322 John P. Welsh and John A. Harvey

species (toads, pigeons, cats, and dogs) and a wide been well documented by Holmes in his studies
variety of conditioned reflexes (leg flexion, body on voluntary movements.
withdrawal, salivation, cardiac, and respiratory).
Cerebellar lesions did impair the ability of animals
to perform CRs appropriately or with the same Learning, Memory,
frequency and vigor as they had preoperatively. and the Cerebellum
However, many of these performance deficits Types of Learning
gradually abated with time after surgery. The
and Memory
effects of cerebellectomy were the same whether
the conditioned reflex was established by the strict J. Hughlings Jackson proposed that higher mental
Pavlovian method or by the avoidance method, functions are not indivisible abilities but are
which allows for voluntary responses. Finally, as composed of more basic skills. The ability to
would be anticipated from Western studies, both learn, for example, depends not on a "learning
instrumental responses and conditioned reflexes center" but rather a combination of interrelated
were more vulnerable to the effects of cerebellar functions. Depending on the learning task, these
damage than were unconditioned reflexes. How- functions could include some combination of
ever, these Russian studies suggested that this visual, auditory, and/or somesthetic perceptions
increased vulnerability might be more apparent and their interactions, fine motor coordination,
than real. spatial localization, and kinesthetic-proprio-
First, there was some evidence of deficits in ceptive feedback. Thus, learning or memory and
unconditioned skeletal defensive reflexes and in their expression in behavior require the co-
autonomic reflexes. This suggested that the per- ordinated activity of sensory systems, associa-
formance deficits in the CRs may have been due, tion cortex, motor cortex as well as the cere-
in part, to a deficit in the final pathway through bellar and extrapyramidal systems that allow for
which both the CRs and UCRs were expressed. the proper integration and modulation of motor
Unfortunately, the Russian studies never closely responses. Impairment of any of these functions
examined this possibility. However, the similar could either interfere directly with the occurrence
effects of cerebellar lesions on unconditioned and of learning and memory or impair the optimal
conditioned leg flexion noted by Popov and performance of what had been learned. This point
Fanardjian strongly supported this view. of view is consistent with current evidence ob-
Another possibility raised by the Russian tained from both clinical and experimental studies
studies was that the CS employed to elicit the indicating that learning and memory are not
CR was not strong enough to overcome the unitary phenomena. There are many different
decrease in excitability resulting from cere- forms of learning and many different types of
bellectomy. Food deprivation is known to in- memory (Squire, 1987). Presumably different
crease the strength of the unconditioned as well forms of learning and memory occur because
as the conditioned salivary reflex. The finding by differences in the nature of the tasks required
Livshits (1947) that food deprivation in a cere- of an organism result in a different degree of
bellectomized dog returned the salivary CR to involvement of various neural systems. Conse-
normal strongly suggested that deprivation had quently, damage to any particular brain region
increased the response-evoking properties of the can leave some forms of learning and memory
CS sufficiently to overcome the effects ofthe cere- intact while apparently eliminating others.
bellar lesion. This interpretation is strengthened Recent studies in humans and other primates
by the observation that initiation of salivary CRs has led to the suggestion that there may be two
was delayed in cerebellectomized dogs (Krasusky, broad categories oflearning and memory: decla-
1957). In short, the Russian studies suggested that rative and procedural (Squire, 1987). Declarative
cerebellectomy had increased the time taken for or explicit memory allows for knowledge about
the onset of a response, as well as the frequency the world around us, and is available to conscious
and amplitude of the response-effects that had recollection. Remembering a person's name or
16. Role of the Cerebellum
face would be an example of declarative memory.
Procedural or implicit memory allows for knowl-
edge of "how," and current thinking (Squire, 1987)
is that it would include learned motor skills
acquired through instrumental or Pavlovian
conditioning, cognitive skills (e.g., remembering
the rules for addition), and the phenomenon of
priming (the improvement of memory through
the prior presentation of certain stimuli).
Human amnesia has been linked with damage
to the medial temporal region and to the dien-
cephalic midline. However, the amnesia that is
associated with such damage is only for declara-
tion memory and not for procedural memory.
Since the loci of procedural memory are not
known, there has been a great deal of speculation
concerning the possible neural systems involved
(Squire, 1987). A recent study of patients with
Huntington's or Parkinson's disease has led one
group of investigators to suggest that motor skill
learning may be mediated by a corticostriatal
system (Heindel et aI., 1989).
An older line of research has focused on the
cerebellum as the site of motor learning. Lashley
and McCarthy (1926) provided one ofthe earliest
experimental studies on the role of the cerebellum
in learning and memory. Using maze learning,
they found that cerebellar damage did not affect
the ability to remember the correct sequence of
responses in a maze. Their report on one rat with
a large cerebellar lesion is worth quoting:
Number 4. Adult female. The performance in initial
learning was: Time, 2,388 seconds; Errors, 33; Trials,
35. Preliminary retention tests gave errors in only first
two trials. Time, 229 seconds; Errors, 7; Trials 12. After
operation there was extreme contraction of the left
hind leg and extension of the right. In walking there
was rotation to the left and frequent backward
somersaulting. When picked up the animal clutched
wildly at anything within reach and gave indications
of vertigo. For the first days she was force-fed, then
began to eat spontaneously but showed little improve-
ment in symptoms. Thirty days after operation the
motor syml'toms were still pronounced. She walked as
if drawing a heavy weight, with fore and hind leg
extended forward and dragging her along in a series of
lunges. At this time she was tested in the maze and
made a perfect retention record: time, 801 seconds; errors,
0; trials 10. (emphasis added)
323
Nevertheless, beginning with the publication
of a theoretical paper by Marr (1969), there have
been increasing speculations that some forms of
motor learning such as the vestibular ocular reflex
might be acquired and stored in the cerebellar
cortex (see Ito, 1984). However, there is no direct
evidence that the theoretical speculations (e.g.,
Albus, 1971; Houk et aI., 1990; Moore and Blazis,
1989) or the results of in vitro studies (e.g.,
Sakurai, 1989) are relevant to the in vivo situa-
tion. On the contrary, recent findings suggest
that the locus of learning and memory for the
vestibular ocular reflex (Lisberger, 1988; Weiser
et aI., 1989), for other forms of vestibular com-
pensation (Llinas and Walton, 1979), and for
long-term habituation (Lopiano et aI., 1989) is
within the brain stem.
A renewed interest in the involvement of the
cerebellum in learning and memory was gener-
ated when McCormick etai. (1981) reported that
Pavlovian conditioning of the nictitating mem-
brane reflex in the rabbit was impaired by
hemicerebellectomy. Given the rich history of
Pavlovian conditioning studies on cerebellar
involvement in the performance of conditioned
reflexes reviewed above, the susceptibility of yet
another conditioned reflex to cerebellar damage
was not unexpected. What was unexpected was
the subsequent claim that learning and memory
underlying the conditioned nictitating membrane
response (NMR) was located in the cerebellum
(e.g., Thompson, 1986).
Cerebellar Regulation
of the Conditioned N M R
The unique aspect of the deficits in nictitating
membrane CRs following hemicerebellectomy
was that the CRs appeared to be abolished and
could not be reacquired (McCormick et aI., 1981).
Furthermore, this effect could be reproduced by
relatively small lesions in the area of the anterior
aspects of the interpositus nucleus with no
obvious sensory or motor dysfunction in the reflex
pathway of the UCR or in the animal in general
(McCormick and Thompson, 1984; Yeo et aI.,
1985). Unilateral lesions in the interpositus
nucleus abolished CRs of the nictitating mem-
brane on the side ipsilateral to the lesion without
324
affecting CRs of the contralateral side. These
effects could be obtained with either visual or
auditory CSs.
In the final analysis, the conclusion that the
interpositus nucleus was the locus of memory
essential for learning and retention of the condi-
tioned NMR was based on two arguable pro-
positions: the assumption that deficits specific to
learned responses indicated a role of the damaged
area in learning or memory, and the belief that
learning and memory were localized in discrete
brain areas (see Thompson, 1986). More recently,
Welsh and Harvey (1989a) reexamined the effects
of electrolytic lesions in the anterior aspects of
the interpositus nucleus on Pavlovian condition-
ing of the rabbit's NMR. The results of this and
subsequent studies employing intracerebellar
lidocaine to produce reversible lesions (Welsh
and Harvey, 1989b, 1991) clearly demonstrated:
that the previous findings summarized by
Thompson (1986) were due to performance de-
ficits and not to effects on learning and memory.
These studies will now be described in greater
detail.
In the first study (Welsh and Harvey, 1989a),
conditioning of the rabbit's NMR was accom-
plished using standard procedures. Each condi-
tioned trial consisted of the paired presentation
of a tone and an air-puff UCS. The UCS was de-
livered to the right cornea 285 ms after CS onset.
In accordance with common practice, a response
was defined as at least 0.5 mm of nictitating
membrane extension and was scored as a UCR
if it occurred after UCS onset and as a CR if it
occurred within the 285 ms between CS and UCS
onsets. It should be noted that conditioning trials
do not allow one to observe any CRs that might
have occurred with latencies longer than the CS-
UCS interval of 285 ms, since the UCR elicited
by the UCS would mask their presence. Con-
cerned with the possibility that animals with
cerebellar damage might have problems in the
initiation ofCRs, Welsh and Harvey (1989a) used
test trials that were interspersed among the
conditioning trials. During a test trial, the tone
CS was presented to the animal as before, but the
UCS was omitted. This allowed for the observa-
tion of responses having latencies in excess of
285 ms. Thus, the performance of each animal
could be compared between conditioning and
John P. Welsh and John A. Harvey
test trials. Animals were first trained to post-
asymptotic levels of responding (greater than 90%
CRs) and then tested for CR retention 3 weeks
after surgery.
In agreement with Yeo et aI., (1985), four groups
of animals could be distinguished during postop-
erative testing on the basis of the nonoverlapping
behavioral effects as measured by the frequency
ofCRs occurring during the CS-UCS interval of
conditioning trials. Also, in agreement with Mc-
Cormick and Thompson (1984) and Yeo et al.
(1985), one group of 15 animals with unilateral
cerebellar lesions that included the dorsolateral
aspects of the anterior interpositus nucleus dem-
onstrated abolition of ipsilateral CRs during
conditioning trials. A second group of 47 animals
whose lesions never encroached on the anterior
interpositus nucleus showed no retention deficits
during conditioning trials. There were two inter-
mediate groups that demonstrated substantial
decrease in CRs on the first retention day. One
group of 14 animals recovered to normal levels,
while another group of 6 animals did not.
The use oftest trials allowed Welsh and Harvey
(1989a) to obtain a precise measurement of CR
topography and this in turn revealed that cere-
bellar lesions did not simply abolish the occur-
rence of CRs, but rather induced a continuum of
deficits in response frequency, latency, rise time,
and amplitude. These deficits all acted to reduce
the probability of observing CRs within the limit-
ed windows of observation employed during con-
ditioning trials. Thus, 11 of the 15 animals that
had demonstrated abolition of CRs during paired
CS-UCS trials revealed a substantial number of
CRs when given the opportunity to respond on
the tone-alone test trials. No differences could be
detected between the placements oflesions in the
11 animals that did demonstrate CRs during test
trials and the 4 that did not.
Figure 16.13 presents topographical represen-
tations of CRs of the four groups of lesioned
animals and a group of normal controls during
test trials when no UCS was presented. This figure
shows that the CR of normal animals is uniphasic,
reaching a peak amplitude of approximately 5 mm
of membrane extension at 350ms after CS onset.
The 47 animals that showed a good retention of
CRs postoperatively also demonstrated normal
CR topopraphies during test trials, maintaining
16. Role of the Cerebellum
5
4
3 Dr·metric (n
E
E
2
o 250 500
msec
Figure 16.13. The effects of unilateral electrolytic lesions
in the region of the deep cerebellar nuclei on the topo-
graphy of ipsilateral conditioned NMR of rabbits. Each
curve represents the amplitude of nictitating membrane
extension elicited by a 250-ms tone CS for 1800-ms
after tone onset on trials in which the tone was presented
alone. Rabbits of the impaired and dysmetric groups
had equivalent amounts of damage to the interpositus
nucleus with damage consistently sustained to the
dorsolateral aspects ofthe anterior interpositus nucleus
and adjacent white matter. Rabbits of the recovered
group on average sustained less damage to the inter-
normal amplitudes and rise times (no-deficit
group of Fig. 16.13). However, 11 of the 15 ani-
mals that showed a total abolition of CRs during
paired trials demonstrated dysmetric CRs during
test trials that were characterized by low ampli-
tudes, longer onset latencies, and increased rise
times (dysmetric group of Fig. 16.13). Two groups
demonstrated intermediate effects of cerebellar
lesions on CR topography. The six animals that
demonstrated impaired CR frequency and did
not recover (impaired group of Fig. 16.13) also
demonstrated CRs that were as deficient in ampli-
tude as the dysmetric group but retained normal
onset.latency and rise time. The group of 14
animals that recovered normal CR frequency after
325
----- Normal (n = 82)
r
----- No - Deficit (n = 47)
Recovered (n = 14)
Impaired (n = 6)
=15)
1000 1500
positus nucleus while the anterior aspect of the inter-
positus nucleus of virtually all rabbits of the no-deficit
group remained intact. The topography of the CR of
normal rabbits represents the average of all rabbits in
the lesion groups on the day before cerebellar lesions.
The vertical line at 285 ms after tone onset represents
the time that the DCS would have been delivered on
conditioning trials. Compare the topography of nicti-
tating membrane responses ofthe normal and dysmetric
rabbits to the topography of the hand grasps in the
human with cerebellar damage in Figure 16.1.
cerebellar lesions continued to display deficits in
CR amplitude (recovered group of Fig. 16.13). It
should be noted that the changes in response
topography demonstrated by the dysmetric group
are quite similar to those obtained by Holmes in
a human patient with unilateral damage to the
cerebellum (compare Figs. 16.1 and 16.13).
In each of the groups affected by cerebellar
lesions, tone-alone trials revealed greater levels
of responding than was observed on conditioning
trials. The reason for this can be more easily seen
in Figure 16.14, which presents in greater detail
CR topography during the 285 ms after CS onset.
This time period is identical to the interval be-
tween CS and UCS onsets employed on condi-
326
4 ,-
3.5
3 /
2.5
E 3
E 2
1.5
0.5
0 0
0 50 100
msec
Figure 16.14. A detailed view of conditioned nictitating
membrane extension on tone-alone trials for 285 ms
after tone onset. Each curve represents the average
topography of the CRs of normal rabbits and the four
lesion groups depicted in Figure 16.12. The 285-ms
time period corresponds to the interval between CS
tioning trials. Accordingly, the amplitude of each
response at 285 ms after tone onset represents the
extent to which one could observe CR in each of
the lesion groups during paired CS-UCS condi-
tioning trials. The remainder of the CR could not
be observed on conditioning trials because the
UCR elicited by the corneal air-puff obscured its
measurement.
Membrane extension of normal rabbits and
lesioned rabbits in the no-deficit group reached
approximately 4 mm of extension by 285 ms after
tone onset. The response of the recovered group
reached, on average, 2.5 mm of membrane exten-
sion by 285 ms. Membrane extension for the
impaired and dysmetric groups at 285 ms only
reached 0.75 and 0.25 mm, respectively (Fig. 16.14).
It is important to note that the definition of a
response uniformly employed among researchers
studying nictitating membrane conditioning is a
0.5-mm or greater extension of the nictitating
membrane. Accordingly, only a small difference
in CR frequency (less than 2%) was observed
between conditioning and tone-alone test trials
John P. Welsh and John A. Harvey
L
4
,-
,- 3.5
3
/
/ 2.5
2 3
1.5
1
0.5
150 200 250
and UCS onsets employed on conditioning trials. Verti-
cal marks extending downward from each curve
represent the time at which 0.06 mm of membrane
extension of the average topography was attained. This
measure represented the best estimate of the time of
response onset.
in the no-deficit and recovered groups because
more than 98% of these responses exceeded the
0.5 mm criterion by the time the UCS was pres-
ented. However, greater responding was detected
on tone-alone trials in the impaired and dysmetric
groups because a significant percentage of re-
sponses did not reach the amplitude criterion by
the time the corneal air puff was presented on
conditioning trials. This was most dramatically
demonstrated by the dysmetric group, in which
less than 4% of the responses reached the criterion
in the limited 285-ms window of observation used
during CS-UCS pairings on conditioning trials,
as compared with an average of 21 % CRs during
test trials. Thus, the combined effects of a reduc-
tion in amplitude and an increase in rise time
made it possible to overestimate the effect of cere-
bellar lesions on CR frequency when only limited
windows of observation were employed. This is
so because cerebellar lesions brought conditioned
membrane extensions closer to the threshold of
exclusion from the response category. For a more
detailed analysis of response topography and
16. Role of the Cerebellum
VIII
CS" CN
CR
UCR
Figure 16.15. Schematic illustration of current views
concerning the pathways of the conditioned and un-
conditioned NMR reflex (filled arrows) and the path-
ways through which the cerebellum may modulate
these reflexes. V, VI, and VIII, 5th, 6th, and 8th cranial
nerves, respectively; ACC, accessory abducens nucleus;
CN, cochlear nucleus; GC, granule cell of cerebellar
cortex; IC, inferior colliculus; 10, inferior olive; IP,
kinematics in these animals before and after cere-
bellar lesions see Welsh (1992).
Cerebellar Regulation
of the Unconditioned N M R
One of the advantages of the NMR is that the
CR and UCR are expressed through a common
final pathway. Thus, a tone CS and air-puff
UCS are capable of activating retractor bulbi
motor neurons located in the accessory abducens
nucleus (Gray et ai., 1981; Harvey et ai., 1984;
Holstege et ai., 1986). Activation of the accessory
abducens nucleus leads, sequentially, to contrac-
tion of the retractor bulbi muscle via the Vlth
cranial nerve, retraction of the eyeball into the
orbit, and extension of the nictitating membrane
(Prince, 1964). Retraction of the eyeball is the
necessary and sufficient condition for occurrence
of the NMR when tactual stimulation of the cornea
serves as the UCS (Marek et ai., 1984). The audi-
tory system is prewired into the unconditioned
nictitating membrane reflex. Although auditory
stimuli cannot evoke an overt response before
learning, they do activate motor elements of this
reflex (Berthier and Moore, 1983), thereby pro-
327
interpositus nucleus; PC, Purkinje cell; PN, pontine
nuclei; PO, pars oralis ofthe trigeminal sensory complex;
RFJI>H, the preblink area of the reticular formation;
RN, red nucleus. Note that there is a direct projection
from pars oralis to both IP and GC as well as the
projection from 10 via climbing fibers. There is no
evidence for projections from PN to IP.
ducing reflex facilitation as measured by a tone-
induced increase in the amplitude of the UCR
(Harvey et ai., 1985). The filled arrows in Figure
16.15 represent a simplified diagram of the CS
and UCS projections onto a preblink area located
in the reticular formation (RF/PB). The RF/PB
might be the point at which the CS can activate
the motor system ofthe UCR (Harvey et ai., 1984;
Holstege et ai., 1986).
Acquisition of CRs to a tone CS appears to
involve synaptic facilitation that converts a pre-
viously weak, subliminal stimulus into one that
can recruit a sufficient number of retractor bulbi
motor neurons to elicit an overt response. As
learning proceeds, the CS acquires the ability to
elicit CRs having higher amplitudes and shorter
onset latencies. These CRs can thus appear to be
as robust as the UCR. However, even after ex-
tended training, the response-evoking properties
of the CS always remain less than that of the
UCS, whose intensity in conditioning studies
(approximately 2.2 kg/cm 2 source pressure) is su-
pramaximal, approximately 25 times greater than
threshold. In this regard, even after 16 days of
conditioning at optimal stimulus parameters, the
force of the NMRs elicited by a tone CS is only
328
equivalent to the force of a UCR elicited by a
corneal air puff of 0.55 kgjcm 2 , an intensity that
is one quarter that employed in conditioning
studies (Welsh, 1992). These data suggest that, at
the level of the motor substrate for NMRs, a
well conditioned tone has only one quarter the
response-engendering power ofthe UCS employed
in conditioning studies.
Previous claims that cerebellar lesions impair
the ability of the CS to evoke CRs with no impair-
ment of the ability of the UCS to elicit UCRs
have assumed that the response-evoking proper-
ties of CS and UCS are equivalent. In order to
test the hypothesis that CRs were being differen-
tially affected because the CS was a weaker sti-
mulus than the UCS, Welsh and Harvey (1989a)
measured the UCRs elicited across a 30-fold range
of air-puffUCS intensities. This procedure clearly
indicated that the deficits observed in CRs follow-
ing lesions of the area of the interpositus nucleus
were not specific to the learned response. Thus,
UNCONDITIONED RESPONSE
5 Normol
4
/
Cerebellar
.3
E
E 2
a ~~-----------------------------------
a 250 500
Figure 16.16. Interpositus lesions alter the topography
of unconditioned NMRs especially at low intensities
of corneal air puff. Each dotted line represents the
average topography of the UCR of normal rabbits for
nine intensities of corneal air puff. Increasing the air-
puff intensity increases the unconditioned NMR but
does not affect the rise time. Each solid line represents
the average topography of UCRs of interpositus
lesioned rabbits across the same range of air-puffinten-
John P. Welsh and John A. Harvey
dysmetric rabbits including those that failed to
demonstrate CRs during either conditioning or
test trials also demonstrated impaired UCRs as
compared with controls. These deficits were most
prominent at low UCS intensities (Fig. 16.16).
The UCRs of normal rabbits, indicated by the
dotted lines, progressively increased in amplitude
as UCS intensity was systematically increased
from 0.09 to 2.93 kgjcm 2 . The dysmetric animals
(solid lines) also demonstrated an increase in am-
plitude and rise time with increasing UCS inten-
sity, but their UCRs were consistently lower in
amplitude and had longer rise times as compared
with the UCRs of normal rabbits at equal UCS
intensities. At the lowest UCS intensity, dysmetric
rabbits demonstrated deficits in the UCR that
approximated those of the CR.
Welsh and Harvey (1989b) reexamined the
equivalent effect of anterior interpositus lesions
on the regulation of CRs elicited by a tone CS
and UCRs elicited by a low intensity UCS. This
1000 1500
msec
sities. The subtle differences in topography between
normal and interpositus-lesioned rabbits become ex-
aggerated as the intensity of the air puff is lowered so
that significant changes in topography are easily noticed
at the lowest intensities. Compare the topography of
UCRs elicited by the lowest intensities of air puff in
this figure with the topography of the CRs of the
dysmetric group in Figure 16.13.
16. Role of the Cerebellum
was accomplished through the use of reversible
lesions produced by microinjections of the local
anesthetic lidocaine into the region of the anterior
interpositus nucleus. Lidocaine injections revers-
ibly abolished CRs acquired to a 100-dB tone
CS and at the same time significantly impaired
the UCRs elicited by a low intensity UCS (0.09 kg/
cm 2 ). UCRs elicited by this low intensity UCS
were reduced in frequency and amplitude, and
increased in rise time. These effects of lidocaine
on CRs and UCRs were completely reversible.
Because lesions or reversible inactivation of
the interpositus nucleus produced similar disrup-
tions of the CR and UCR, one can conclude that
the cerebellum is important in the regulation of
both the learned and unlearned reflexes and that
this regulation is most likely on the retractor bulbi
motor neurons, which form a final pathway for
both responses. This hypothesis is supported by
the additional fact that all NMRs, whether they
are elicited by visual or auditory CSs (Yeo et aI.,
1985), whether they are augmented UCRs result-
ing from the pairing of two UCSs (Skelton et aI.,
1988), or whether they are conditioned under
Pavlovian (McCormick et aI., 1981) or instru-
mental (Polen char et aI., 1985) contingencies, are
disrupted by interpositus lesions. All of the experi-
ments reviewed above suggest to us that the inter-
positus lesion has little to do with the disruption
of a specific sensory system or the supposed
memorial processes specific to a particular form
of conditioning. Reduced excitability of the acces-
sory abducens nucleus and/or retractor bulbi
muscle, however, is consistent with all ofthe data
and could be the etiology of the performance
deficits of conditioned NMRs following inter-
positus lesions.
Learning During Inactivation of Interpositus
While we have concluded that the CR deficits
seen after interpositus lesions were a manifestation
of a general performance deficit, one could still
argue that the CR impairments were due to the
combined effects of a performance and learning
deficit. For this reason, a final set of experiments
was undertaken to resolve this issue by examin-
ing the retention oflearning in the absence of per-
formancedeficits (Welsh and Harvey, 1991). Such
a study was possible because of two factors. First,
329
the previous studies of Welsh and Harvey (1989b)
demonstrated that lidocaine infused in the region
of the interpositus nucleus could reversibly
abolish CRs. Second, it is well known that pre-
venting response occurrence in a reversible fashion,
by pharmacological or physiological means, does
not prevent learning during Pavlovian condition-
ing. For example, atropine was used to block
salivation during conditioning ofthe salivary res-
ponse. When tested later, without atropine, the
animals produced salivary CRs to the CS, indi-
cating that learning had occurred (Crisler, 1930;
Finch, 1938). Similarly, crushing the motor nerves
to produce paralysis of the leg did not prevent
the acquisition ofleg-flexion CRs, since such CRs
could be evoked by the CS after the nerve had
regenerated and movement was possible (Beck
and Doty, 1957; Kellogg et aI., 1940; Light and
Gantt, 1936).
We therefore examined whether rabbits could
acquire CRs during the time that their ability to
perform CRs was impaired by infusions of lido-
caine into the interpositus nucleus. If the inter-
positus nucleus was important for learning, then
animals should not acquire CRs during inter-
positus inactivation by lidocaine infusion and
they should not demonstrate any retention of
CRs when tested without lidocaine. On the other
hand, if the interpositus was only involved in the
performance of CRs through an action on the
motor side of the reflex, then animals would be
able to learn even though they would not be able
to perform the learned response. Consequently,
these animals should demonstrate full retention
ofCRs when subsequently tested with a normally
functioning interpositus.
The results from the first animal used in such
a study were reported by Welsh and Harvey at a
conference on the olivocerebellar system in motor
control held in Turin, Italy, October 1987 and are
presented in Panels A, B, and C of Figure 16.17.
This animal was first trained to make nictitating
membrane CRs to a light CS that was paired with
an air-puffUCS. On a subsequent day the animal
demonstrated 88% CRs to the light CS (Panel A,
open circle above the letter C). The animal was
then injected with 1 III of lidocaine into the inter-
positus nucleus at time zero (arrow in Panel A),
which resulted in a total abolition of CRs to the
light CS for a period of 15 min followed by a rapid
330
recovery to 100% CRs by 25 min after injection.
The animal was then given a second injection of
lidocaine (first arrow of Panel B), which produced
an abolition of CRs to the light CS over the next
2.5 min (first open circle in Panel B), indicating
the reliability of the effects of lidocaine. Begin-
ning 2.5 min after this second injection, the animal
was given a 70-min conditioning session during
which a tone CS was paired with the air-puff
UCS. During this session, 1 pI of lidocaine was
injected into the interpositus nucleus every 15 min
as indicated by the arrows in Panel B. The rabbit
showed little evidence of CR acquisition to the
tone CS as CRs never exceeded 11 % within any
10-min block of trials during this session (Panel
B, solid circles). In order to monitor the degree
of inactivation of the interpositus nucleus by lido-
caine, light-conditioning trials were interspersed
with tone-conditioning trials during this 70-min
session. The infusion of lidocaine was effective in
maintaining a consistent impairment of CRs to
the light CS as indicated by the low level of re-
sponding on light-conditioning trials, with overall
responding to the light CS being 14% (Panel B,
open circle between brackets). Two days later this
animal was again returned to the conditioning
A 8
100
80
0
I
0
1/1
0::
u
I-
z
w
60
I
0
U
0:: 40 I I
w I
0-
I I
....
I .... .... .... .... ....
co w ~
Figure 16.17. Effects oflidocaine inactivation of inter-
positus on learning. This experiment was carried out
in March/April, 1987. Open circles represent CRs to
John P. Welsh and John A. Harvey
session and exposed to light-conditioning trials
(Panel C, open circle). In the absence oflidocaine,
the animal gave 100% CRs to the light CS, indi-
cating that the effects of previous lidocaine injec-
tions had completely dissipated. More impor-
tantly, even though lidocaine injected into the
interpositus nucleus had blocked the performance
of CRs during acquisition (Panel B, solid circles),
this animal demonstrated an average of98% CRs
to the tone CS during retention (Panel C, solid
circles). In short, the interpositus nucleus was not
essential for learning. In addition, these results
indicated that inactivating the rostral interpositus
had not blocked the sensory effects of either the
CS or UCS since any such effect would also have
retarded learning. Therefore, one can conclude
that the interpositus is neither part of a pathway
involved in learning nor a site of learning.
Using a modification of this procedure, Welsh
and Harvey (1991) have replicated and extended
this basic finding with the inclusion of controls
receiving infusions of saline and controls for non-
associative responding. The results of this most
recent study confirmed that the interpositus is
essential for performance but not for acquisition
or retention of learned responses.
C
00 o e-e'e_e
0 - 0 CR TO LIGHT CS:
e-e CR TO TONE CS i
:1-
:11)
:w
:0::
II)
I I I I
~
a
I I I I I
N
I I I I.
I I I I I
0 W ~ ~ ~ 0 W ~
TIME (MIN)
the light CS and closed circles to the tone CS. Arrows
indicate the injection of 1 flilidocaine into the anterior
interpositus. See text for further details.
16. Role of the Cerebellum 331

VIII~
CS"~ SOC
~ AUD
~ CT

VI \
CR ~.-/RF\ ~ ~
UCR~~~~

ucs"
v
PO

Figure 16.18. Schematic illustration of pathways that

may be involved in the conditioned NMR reflex. The
solid lines indicate established connections for the un-
conditioned NMR and open lines some ofthe possible
pathways for the CS and UCS. This figure illustrates
only two of the possible sites at which associative
strength might accrue for acquisition of CRs. In this
case we have simply indicated how a brain stem circuit
and some neocortical circuit might represent sites (e.g.,
RFIPB and association cortex) at which the interaction
L~~~M
of CS and UCS could lead to synaptic facilitation
required for learning in order to stress the role of the
motor cortex and thus emphasize another site at which
cerebellar influences might be brought upon the per-
formance of the CR. AUD CTX, auditory cortex; IC
MG, inferior colliculus and medial geniculate nucleus;
MOT CTX, motor cortex; SOC, superior olivary com-
plex; SOM CTX, somatosensory cortex; VN, ventral
thalamic complex;? CTX, unknown site in association
cortex. Other abbreviations are as in Figure 16.15.

Use of the Nictitating Membrane cerebellum may receive information concerning

Reflex to Study Cerebellar Function CS and UCS onsets and modulate motor systems
involved in the NMR (filled arrows; see legend
There are several advantages in the use of the for details). Figure 16.18 emphasizes that there
conditioned NMR for the study of learning and are multiple systems involved in learning. Each
memory, which have been described in detail of these systems contains elements through which
(e.g., Gormezano et aI., 1983). The conditioned the cerebellum is known to influence movement.
NMR has also been shown to be a powerful tool Thus, the cerebellum could directly affect the pre-
for studying the effects of drugs on learning and blink area through its projections to the red
memory under conditions that require one to nucleus (Fig. 16.15), as well as its influence over
distinguish between sensory, associative, and the thalamus and motor cortex (Fig. 16.18).
motor effects (Harvey, 1987). The results of Welsh There are some unique aspects to the NMR
and Harvey (1989a-1991) indicate that the condi- that are relevant for the study of motor functions.
tioned NMR can also be used to distinguish be- First, it provides a measure of cerebellar regula-
tween the possible effects of cerebellar damage on tion of a cranial reflex that involves a small popu-
sensory processes, associative learning, and motor lation of motor neurons (approximately 100 re-
functions with a high degree of resolution and tractor bulbi motor neurons in the rabbit, Gray
reliability. Thus, the NMR can provide an addi- et aI., 1981), a final pathway via the Vlth nerve
tional tOQl for examining cerebellar function. that is well defined, and a single muscle (the re-
Figures 16.15 and 16.18 emphasize the multiple tractor bulbi) that is overwhelmingly responsible
systems through which cerebellar influences might for producing the NMR. In addition, the NMR
be able to modify the performance of the CR and is a behavior that is produced only through an
UCR. Figure 16.15 presents some possible brain agonist system, because there are no antagonist
stem pathways (open arrows) through which the- muscles for eyeball retraction. Despite the simpli-
332
city of this reflex and response system, cerebellar
lesions produced many of the same effects on
measures of the NMR as compared with more
complex voluntary movements (compare Figs.
16.1 with 16.13 and 16.16). Another advantage of
the NMR preparation is that it provides a high
level of control over stimulus presentations so
that psychophysical measures of sensory processes
can be readily accomplished. A third advantage
ofthe NMR is that conditions can be arranged so
that both a learned and an unlearned response
may be measured from the same effector system,
increasing the probability that the same muscles
and motor neurons will be recruited for both the
CR and UCR. Any changes in the performance
of the UCR can provide an index of the viability
of the final pathway for the CR.
Conclusions
Current thinking on cerebellar function agrees
with, and has not challenged, Rolando's earliest
viewpoint that the cerebellum has a profound role
in ensuring optimal performance of the voluntary
motor act. However, this review of the literature,
especially the recent findings on the NMR, has
indicated the need for a reappraisal of the role of
the cerebellum in behavior. The common belief
that the cerebellum has an exclusive role in volun-
tary behavior should be replaced with the re-
cognition that the cerebellum regulates both
voluntary and involuntary movements. Once re-
cognized, the reason why the voluntary move-
ment is so highly susceptible to cerebellar damage
can be reevaluated.
A primary difference between reflexive and
learned movements is the nature of the eliciting
stimuli, the strength of which may determine the
relative importance ofthe cerebellum in ensuring
optimal performance of movements that they
elicit. The strength of the involuntary or uncondi-
tioned movement is directly proportional to the
intensity ofthe eliciting stimulus and the motiva-
tional/arousal state of the organism. However,
the ability of a CS or a voluntary command to ini-
tiate movement depends on three factors: stimu-
ulus intensity, motivational/arousal level, and
the amount of associative strength that has devel-
oped through learning. The statement of cere-
bellar patients that they cannot promptly initiate
movements despite any degree of effort epitomizes
the inability of the motor system to utilize the
John P. Welsh and John A. Harvey
voluntary command when the cerebellum is
damaged, and this is directly analogous to the
decreased ability of CSs to elicit CRs in experi-
mental animals.
One can conclude that the purpose ofthe cere-
bellum is to ensure that motor nuclei respond
with a strength proportional to the response-
evoking properties of the eliciting stimuli as
determined by their associative strength, intensity,
and biological significance (motivational/drive
state of the organism). Cerebellar damage does
not affect the ability to learn or remember motor
skills but rather shifts the excitability thresholds
of motor nuclei so that they no longer depolarize
to an extent proportional to the afferent volleys
carrying the commands for movement.
Acknowledgments. We thank Gertrud G. Champe,
Director of the University of Iowa Translation
Laboratory, and her colleagues, for their excellent
translation of the Russian works. We especially
thank Drs. Ramon A. Grigorian and Victor V.
Fanardjian for their generous donation of many
Russian works on the issue of the cerebellum and
Pavlovian conditioning.
This work was supported by United States
Public Health Service Grant MH16841. 1.P.W.
was supported by United States Public Health
Service Training Grant MH 15773.
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Index
A-amino-3-hydroxy-5-methyl-4-isoxazole-propionate
(AMPA),64
Acetylcholinesterase (AChE), 23-24,31-33,45,213,
256,258,261,264
Afferent convergence, 149,161-162
D-2-amino-5-phosphonovaleric acid (APV), 62-64
Amino acid-activated channels, 196-198
ARG, see Autoradiography
Aspartate, 116, 120, 125-129
Autoradiography (ARG), 15-17
Axis cylinder, 72-73
Babinski, 283
Basilar pontine nuclei (BNP), 1, 135-138, 140-141,
144, 149-162
functional operations in rat, 149-158
local-circuit neurons of, 159-161
noncortical afferent systems of, 136-141, 158-159
GABA-ergic components in, 136,138-141
synaptic features in, 141-149
Brachium pontis (BP), 140
Ca channels, 189
Calcium-binding protein (CaBP), 7-11, 18
CaM kinase,
in mediating tropic effect of EAA, 67
Central nervous system (CNS), neurons of, 116, 121,
124-125
biophysical techniques in study of, 182-183
loss of during CSN development, 56-68
patch-clamp approach to, 198-199
Cerebellar compartmentation,
conserved across phylogeny, 41
Cerebellar connectivity,
events leading to, 41,43-48
Cerebellar cortex connections,
topographical organization of, 5-19
Cerebellar development, 5, 84
Cerebellar grafting,
long-term survivals after, 85-99
short-term survivals after, 99-111
Cerebellar granule cells,
in culture, 56-57
survival requirements of, 57-62
chronic depolarization in, 57-58
effect of NMDA in, 58-62
Cerebellar module, 40-41
Cerebellar morphogenesis,
micro tubule-associated protein in, 72-81
principal events in, 99
Cerebellar neurogenesis,
description by Ramon y Cajal, 72
putative role of cytoskeleton in, 72-73
Cerebellar organotypic cultures,
cellular and tissue differentiation in, 185
preparation of, 184-185
Cerebellar output,
defining control signals from, 283-292,296
dentate, 283, 285-288, 291
fastigial, 283, 287-288, 290-291
interposites, 283, 286-288, 291, 293-294
Cerebellar sagittal zones,
functional basis of, 267-279
functional importance of, 275-277
historic descriptions of, 267-268
Cerebellum,
and cartography, 48-51
compartmentation in,
higher levels of organization than, 38-40
molecular markers in, 22-51
learning and memory in, 322-330
during inactivation of interpositus, 329-330
types of, 322-327
335
336 Index
Cerebellum (cont.)
with ordered organizational pattern
role in voluntary and reflexive movements, 301-
332
Climbing fibers (CF), 22,46-50,96, 106, 108-109,
113,125,127-129,178-179,213-214,268,
270,274,278
activation of, 179
and bands of Zebrin 1 PCs, 16-17
in motor function, 268-271
in motor learning, 267, 271-273
operations in cerebellar cortex, 268-273
and patterns of Zebrin 1 immunoreactivity, 18
precerebellar,
capability to signal peripheral events, 226-227
connection with mossy fiber system, 226-252
as conveyor of phasic information, 226
as conveyor of tonic information, 226, 229
projection terminals of, 34
relation between anatomical departments
and aiTerents of, 261
visual, 255-256,263-264
system, 226-232,237
CO, see Cytochrome oxidase
Complex spike (CS), 214,226-227,229-244
Conditioned response (CR), 309,311-332
Conditioned stimuli (CSs), 309,311-322,324-332
CS, see Complex spike
Cyclic guanosine monophosphate (GMJ)-dependent
protein kinase (cGK), 7-9
Cyclic guanosine 3': 5' phosphate-dependent
protein kinase (cPK), 41,44,46
Cysteine sulphinic acid decarboxylase (CSAD), 24-
25, 33, 121-122, 124
Cytochrome oxidase (CO), 31-33,45-46
DAB, see Diamino benzidine
DAO, see Dorsal accessory olive
Davis, L., 301,307
Deep cerebellar nuclei (DCN), 5, 156,297
Descartes, 308
DHP, see Dihydropuridine
Diaminobenzidine (DAB) reaction, 16, 18, 141
Diamidine Yellow Dihydrochloride (DY, 2HCI), 243-
244, 248, 251
Dickenson, W.H., 303
Dihydropuridine (DHP) calcium effectors, 58, 64, 68
Dorsal accessory olive (DAO), 6
Dorsal column nuclei (DorCN) cells, 135, 155-156,
161
Durcopan, 116
Dynamic selection hypothesis, 267-279
preliminary experimental evaluation of, 277-278
EAA, see Excitatory amino acids
Eccentricity (ECC), 220-221
EGL, see External granular layer
Electromyography (EMG), 285-287
EMG, see Electromyography
EPSP, see Excitatory postsynaptic potential
Ethylene glycol tetraacetic acid (EGTA), 173
Excitatory amino acids (EAA), 56, 62-68,
maturational stage
dependence of effects of, 64-66
neurobiology of, 56-68
Excitatory post synaptic potential (EPSP), 96
External granular layer (EGL), 5
Eye movements,
evoked, 262-263
Fast Blue (FB), 243, 248
Flourens, 283, 292, 302-304, 319
FTX, see Funnel-web spider toxin
Function,
theories of, 267
Funnel-web spider toxin (FTX), 174-175
GABA, see Gamma aminobutyric acid
GAD, see Glutamic acid decarboxylase
Gamma aminobutyric acid (GABA), 24, 67
110-111, 116-122, 124-125, 129, 165,
177-179, 193-195, 198
-ergic channels, 165, 198
-ergic neurons, 135-137,140-141,144,147,
149-152,159-162,211,250
Gamma-D-glutamylaminomethyl sulphonic acids
(GAMS),62
Gaze shift (GS),
saccadic, 215-216, 218-224
Gaze-sustained deviation (GSD), 218-219, 222
Glutamate, 116-117,120,124-125,127
Glutamate (Glu) receptors, 58, 62-66
Glutamatergic channels, 196-198
Glutamic acid decarboxylase (GAD), 24, 33,
116-117, 121, 138-139, 141, 160
Glutaminase, 129
Glutamine, 116, 120, 128-129
Glutamine synthetase, 129
Glycine, 116-121
Golgi cells, 5,23,30,40,76,117-19,121,126,135, 159
Grafted Purkinje cell (GPC),
dendrites of, 92,94-95,97,105-106,108,110-112
dendritic differentiation of, 105-107
growth and navigation ofaxons
originated from, 96-99
and host cortical cerebellar circuits, 95-96

location and dendritic arrangement of, 92-95
migratory routes of, 101-105, 112
in nervous mutant mice, 92
in normal cerebellum, 89
in pcd mice, 89-92,95-96,98-102, 104, 109-110
procedure of 85-86
proliferation period of, 101
selective invasion of, 85-96
immunological distinction of categories in, 87-88
mechanisms involved in, 87-88
nonrandomness of, 87
starting point of, 113
synapse formation of,
procedure of, 55
resulting in integration of, 108-111
synaptic integration of, 92-96
Grafted Purkinje cell dendrite (GPD), 110-111
GS, see Gaze shift
GSD, see Gaze-sustained deviation
Haggqvist method, 22
Holmes, G., 283,292,294,301,304-306,
308, 310-311, 316, 322, 324
Horseradish peroxidase (HRP), 6, 261
conjugated to wheat germ agglutinin
(WGA-HRP), 6-7,13,34-35, 136-141,
155-156, 158-159, 162,243-247,248-252
Huntington's disease, 323
Inferior olivary nucleus, rhythmogenesis in, 201-211
Inferior olive (10) neurons, 165, 213, 242-245, 247,
251
oscillatory activity of, 165, 203-211
analog simulation of, 203-205
hybrid system to determine extent of, 205-210
preferred frequencies of, 207-209
Inhibitory post-synaptic potentials (IPSPs), 96, 159,
177, 179, 273
Internal granular layer (IGL), 58, 65
Intracerebellar compartments,
development of, 41,43
matching connections to, 46-51
10, see Inferior olive
IPSPs, see Inhibitory post-synaptic potentials
Jackson, J. Hughlings, 322
K channels, 191-193
Kainate (KA), 62-63, 67, 195
Kynurenic acid (KYN), 62
Index 337
Lashley, K.S., 323
Lewandowsky, M., 303
Low thoracic-high lumbar (LTHL),
spinocerebellar projections, 34
terminal fields, 35-36, 48
Low threshold Ca spikes (LTS), 203, 206-208, 210
LTHL, see Low thoracic-high lumbar
Luciani, L., 301,311-313,314,316
Lussana, 303
Magnus, R., 306-307
Main cuneate nucleus (MCN) neurons, 242-252
MAPs, see Microtubule-associated proteins
Marr, D., 323
MCN neurons, see Main cuneate nucleus
MF, see Mossy fiber
Medial accessory olive (MAO), 6
Microtubule-associated proteins (MAPs), 72-81
chemical structure of, 81
evolutionary conservation in properties of, 75
expression of in cerebellum, 75-77
expression of in cerebellum-derived cultures, 77-79
-mRNA in the developing cerebellum, 80-81
putative role of in axon and dendrite growth, 73-74
Mossy fiber (MF), 22, 34, 36, 40, 47-51, 58,
96, 119, 125~126, 128-129, 175, 177, 179,213,
226, 242
-granule cell activation, 40
somototopic organization of, 268
terminal fields,
mediolateral compartmentation due to, 36-38
Motilin, 24, 33
Movement coordination, multiple maps in, 283-298
Myotomes, in the coronal dimension, 296-297
Na channels, 190-191
Nerve growth factor (NGF), 56
Nervous mutations (NR), 10
Neural grafting, see Grafted Purkinje cell
Neuroactive amino acids, see Purkinje cell, cerebellar
Neurogenesis, see Cerebellar neurogenesis
Neuronal differentiation, 84
Neuronal replacement,
degree of, 85
when effective, 84
Neurotropic effect, positive, 92
Neurotransmitter amino acids,
microscopic immunocytochemistry of, 116-129
Nictitating membrane response (NMR), 323, 328
conditioned, 323-325,327,329,331-332
cerebellar regulation of, 323-327
338 Index
Nictitating membrane response (cont.)
unconditioned,
cerebellar regulation of, 327-329
use in study of cerebellar function, 331-332
N-methyl-D-aspartate (NMDA), 58-68, 165,
194, 196-198
NMR, see Nictitating membrane response
Nucleus reticularis tegmenti pontis (NRT), 1-2
OCP, see Olivocerebellar projection
Olivary neurons,
electroresponsive properties of, 201-202
as limit cycle oscillators, 203
Olivocerebellar projection (OCP), 36
development of, 6
to Purkinje cells, 268
subdivisions of, 13-16
Orbeli, L.A., 316
P channel, 165
Parallel-fiber, activation of, 179, 296-297
Parkinson's disease, 323
Pavlov, I.P., 301, 308-310
Pavlovian conditioning, 301-302, 308-309, 311,
324, 329, 3;23
PC, see Purkinje cell
PEP 19,7-9
Peristimulus time histograms (PSTH), 227,229-
232,235-236,240,242-243
Peroxidase anti-peroxidase (PAP), 141
Perturbed locomotion, 267,273-278
Perturbed volitional movement, 276
Pinseau Terminal, 177
Post saccadic drift (PSD), 220-221
Post synaptic potential (PSP), 273
P-PATH, as marker for Zebrin-negative PC, 33
Presynaptic parallel fibers, stabilization of, 84
Protocolumnar organization, 7
PSD, see Post saccadic drift
PSTH, see Peristimulus time histogram
Purkinje cell (PC), see also Grafted Purkinje
cell (GPC)
cerebellar,
compartments of, 36
corticonuclear projection and, 38
competition with grafted, 85-92
dendritic inhibition in, 177-178
dendritogenesis in, 112
electrophysiology of, 165, 167-179
electroresponsiveness of, 167
dendritic action potentials in, 174
dendritic conductances in, 170
ionic conductances in, 167-170,185-198
cell-attached recordings of, 188-189
in outside-out patches, 190-193, 198
transmitter-activated, 193-194
voltage-gated, 185-188
P channels in, 174-175
plateau potentials in, 170-174
sensitivity to neuroactive transmitters, 194-
195
synaptic input in, 175-177
GABA-ergic responses and, 177-178
heterogeneity of, 5-19
in adult cerebellum, 9-12
as disclosed by cerebellar mutations, 9-10
level of, 11-12
in prenatal development, 7-9
relation between cerebellar connections
and, 12-18
host, 85-94
immuno reactivity, 12-13
relationship between CS and SS of, 233-239
motor cortex effect on, 239-252
response properties of saggital strips of, 273-275
tridimensional arrangement of dendrites in, 84
voltage- and transmitter-gated channels in 182-
199
Purkinje cell degeneration (PCD), 10
Purkinje cell-specific glycoprotein (PSG), 7-8, 41
Quisqualate (QA), 62-64, 66, 195
Rabbit flocculus,
eye movements of, 255-264
topographical organization of, 264
zonal structure of, 255-257
Ramon y Cajal, 1, 72-73, 81, 84, 89, 97, 99, 105,
108, 112-113, 116, 135, 159, 177, 279, 283
Real time postsynaptic response (RTPR), 273-275,
277-278
Reflexive movements,
role of cerebellum in, 301, 308-332
Russian tradition of, 308-322
Rolando, 302-304, 306, 332
RTPR, see Real time postsynaptic response
Saccadic eye movements
cerebellar control of, 215-224
effect ofthe flocculus-paraflocculus on, 222-224
effect of inferior olive lesion on, 216-222
effect of the lesion of the posterior vermis
on, 222-224
Saccadic system, 213,215-216

Sechenpv, 301, 308-309, 318
Sherrington, c., 303,306-307,310,316
Simple spike (SS), 214,226,233-236,238-239,
241-244, 270, 274-278
Sperry's hypothesis, 11
Spinocerebellar projection,
development of, 6-7
pattern of in agranular cerebella, 7
SS, see Simple spike
Synaptic adhesion warning, of Larramendi, 111
Synaptophysin, 30-31
Tambaleante (TBL), 10
Taurine, 116-117, 120-125
TEA, see Tetrathylammonium
Tetramethyl benzidine (TMB) method, 13
Tetrodotoxin (TTX), 169-172,174-175,
186, 190, 194-195,203,208
Tetrathylammonium (TEA), 185-186, 188-189, 192
TTX, see Tetrodotoxin
Tubulin isotypes, in cerebellum, 79-81
Unconditioned response (UCR), 309,311,322-
324, 326-329, 332
Index 339
Unconditioned stimulus (UCS), 309,311-315,
319-321, 324, 326-331
Vestibular ocular reflex (VOR), pathways, 213,
256, 262, 264
Voltage-sensitive Ca2+ channels (VSCC), 58, 64,
68
Voluntary movement,
role of cerebellum in, 301-332
western tradition of cerebellar regulation in, 302-
308
VOR, see Vestibular ocular reflex
WGA-HRP, see Horseradish peroxidase, conjugated
with
Wheat germ agglutinin, see Horseradish peroxidase,
conjugated with
Zebrin I , 12-19,25-41,43-46
immunoreactivity pattern of, 18
not regulated by afferent input, 44-45
Zebrin II, 25-32,43-45
Zebrin III-V, 30,32,43