J. comp. Physiol.

121,349-366 (1977)

Journal
of Comparative
Physiology- A
9 by Springer-Verlag 1977

The Anatomy and Motor Nerve Distribution

of the Eye Muscles in the Crayfish
DeForest Mellon, Jr.
Department of Biology, University of Virginia, Charlottesville, Virginia 22901, USA

Summary. The gross anatomy of the muscles in the ci:ayfish compound

eye and the distribution of brain oculomotor neurons were studied by a
variety of anatomical and physiological techniques. There are 11 major muscles in each eye. These vary considerably in size and influence upon eye
movements and in their source of motor innervation. Muscles that cause
defensive eyestalk withdrawal are controlled by axons of a giant motor
neuron cluster. Muscles that move the eyecup in vertical planes are innervated
by cells of an anterior motor cluster, as well as by cells in the medulla
terminalis. Muscles which move the eyecup horizontally are supplied by
neurons of the lateral motor cluster. The separation of the oculomotor
system into different neuronal groups that supply different sets of muscles
thus reflects functional specializations of the component divisions.

Introduction
The eyes of decapoda and some other Eumalacostracan crustaceans are mounted
on movable stalks which articulate with the head. Eye movements are effected
by a variable number of muscles which can move either the entire eyestalk
or its parts. The eye muscles are under control of an oculomotor system within
the supraoesophageal ganglion. Neurons of this system are responsive to selected
environmental stimuli, and their impulse output can initiate and maintain
detailed and specific variations of eye positions in space.
Oculomotor systems of the stalk-eyed crustacea are useful objects for neurophysiological investigation, since they have operational features that epitomize
several general problems in the nervous control of behavior. These include
the generation of detailed motor patterns in the absence of proprioceptive feedback, the integration of diverse modes of sensory input in establishing specific
motor commands, and the elaboration of perceptual memory. Thus, it is not
surprising that crustacean eye control has come under intensified study in recent
years.

350

DeF. Mellon,Jr.

Predictable changes in position adopted by crustacean eyestalks in response

to different sensory inputs have been the subject of several of these studies.
Sch6ne (1951, 1954) was concerned with the relationship of eye position to
both statocyst input and the background illumination perceived by the compound eyes. His results, obtained primarily with shrimp, but including some
observations with crayfish, indicate that vertical deviation of the eyestalks from
their preferred resting orientation is a function of the interaction between both
visual and statocyst input. The separate reflexes act to compensate for perceived
rotation of an animal around the longitudinal body axis (roll).
Recently, Sch6ne and his colleagues (Stein and Sch6ne, 1972; Sch6ne et al.,
1976) and Fay (1973) have demonstrated that eye position is under additional
sensory influence from proprioreceptors within the walking legs. Tilt of a
platform which supports the legs is perceived by the animal as would be
tilt of the body in the opposite direction. Interaction occurs between this third
avenue of sensory influence and those discussed above; all three summate algebraically (but not necessarily to equal extents) to generate the final position
of the compound eyes (Fay, 1973).
A cellular analysis of crustacean eye responses to body tilt has been
performed by Burrows and Horridge (1968a-c). Compensatory movements of
the compound eyes of the crab result whenever the animal is tilted about either
the roll axis or the transverse body axis (pitch). In either case, eye movements
tend to compensate for the imposed rotation of the crab in space by counterrotational behavior. Intracellular recordings showed that some reflexes depend upon
eight of the nine muscles present in the eyecup, and these are driven to different
extents by neurons of the oculomotor system during the various experimental
conditions.
Predictable eye movements in crustaceans are generated by at least three
additional categories of sensory input. These include rotation about the dorsoventral body axis (yaw), which evokes statocyst-mediated movements of both
eyes in a direction opposite to the imposed rotation (Cohen and Dijkgraaf,
1961; Sandeman and Okajima, 1972, 1973a, b). Sandeman and his colleagues
(Sandeman and Okajima, 1973 b; Silvey and Sandeman, 1976 a-d) have recorded
electrical activity from single oculomotor neurons during stimulation of statocyst
hairbeds. The functional connections between the statocyst hair sensory neurons
and ipsilateral oculomotor neurons appear to be monosynaptic. Individual
motor neurons receive input from sensory hairs having a preferred direction
of excitation by fluid movement within the statocyst horizontal canal, and
they appear to be inhibited by sensory hair neurons having an opposite stimulus
preference.
Rotation of the visual environment about a stationary animal evokes optokinetic nystagmus, characteristic sequelae of eye movements in which both compound eyes tend to track movements of the visual surround. The tracking
activity is periodically interrupted as the eyes reach their extreme mechanical
limit, at which time both simultaneously flick back to their starting position.
Optokinetic nystagmus has been examined extensively in crabs (Horridge and
Sandeman, 1964; Horridge, 1966a, b, c; Burrows and Horridge, 1968a, b,
c, d; Horridge and Burrows, 1968a, b; Sandeman, Erber and Kien, 1975;

Eye Muscles in the Crayfish

351

Sandeman, Kien and Erber, 1975). Like the statocyst-evoked "vestibular" nystagmus, optokinetic reflexes function to stabilize retinal images. The reflex occurs
in response to perceived relative motion of the animal and the visual field,
regardless of whether this is caused by forced movements of the eye itself
or by movements of the environmental features. The only exception to this
occurs when a crab voluntarily generates eye movements during retraction or
extension of the eyestalks (Horridge and Burrows, 1968c), or while the animal
executes a change in direction of locomotion (Horridge and Sandeman, 1964).
It is clear from the foregoing account that the crustacean oculomotor system
possesses a high degree of detailed functional complexity which is, nevertheless,
amenable to experimental approach at more than one level. I have chosen
to examine this system in the crayfish because of the presence of cave-dwelling
forms with reduced visual structures. It will be interesting to determine the
extent to which selective pressures have brought about changes in the eye motor
system of such forms. The goal of the present study is to establish an anatomical
and functional basis for future comparison with troglobitic species. This and
the following paper provide descriptions of the major crayfish eye muscles,
and their mode of innervation by specific groups of motor cells within various
brain regions. A preliminary account of the spatial distribution of some oculomotor cells has been published (Mellon et al., 1976).

Materials and Methods

Crayfish studied were Procambarus clarkii obtained from a biological supply house and kept until
used in large stainless steel holding tanks. Animals were used within two weeks of arrival and
usually were not fed.
Anatomical studies of the eye musculature were performed by fine dissection in crayfish Ringer
solution (Van Harreveld, 1936) to which, in some instances, 0.01% methylene blue was added.
On a few occasions, the eyestalk musculature was fixed in alcoholic Bouin's solution and dissected
in 70% ethyl alcohol.
The n u m b e r and position of oculomotor neurons was determinated by back-filling the peripherally-transected motor axons with 300 m M cobaltous chloride. An animal was chilled on crushed
ice for about one-half hour and then decapitated. The brain, optic tracts, and appropriate eye
muscles were dissected with watchmakers' forceps and fine scissors and were isolated in Ringer
in a small Sylgard-lined petri dish. The brain was pinned to the floor of the dish and a well
was constructed of petroleum jelly around the appropriate muscle, so that its motor nerve protruded
through the wall of the well. Ringer solution was then replaced with distilled water and the muscle
nerves were cut. After a period of about 45 s, the distilled water within the well was replaced
with 0.3 M cobaltous chloride. The dish was covered and placed in a refrigerator at 5 ~ for
24 to 48 h. Development was performed in fresh Ringer solution to which 2.2% a m m o n i u m sulfide
was added. After development, the preparations were fixed in alcoholic Bouin's solution, dehydrated
in ethyl alcohol, and cleared in methyl salicylate. In most cases, the brain was desheathed just
prior to being placed in the clearing bath.

Physiology. Electrophysiological examination of motor nerve function involved en passant extracellular recording of impulse traffic from restrained animals. A crayfish was chilled on ice for 30 min
and clamped in a recording chamber. A small hole drilled through the dorsal exoskeleton into
the pericardiai cavity served to perfuse the circulatory system with chilled Ringer. After most
of the animal's blood had been replaced by Ringer, the rostrum was removed and one or both
eyes were immobilized by clamps. Muscles, oculomotor and optic nerves were then exposed by
fine dissection.

352

DeF. Mellon, Jr.

Intracellular electrical recordings from eye muscles were obtained by dissecting the cuticle
and hypodermal tissue away from the appropriate area on the eye stalk. Glass micropipettes,
filled with 2.5 M KC1 and having resistances of i 5 4 0 M e g o h m s were used. Signals were led to
a high input impedance amplifier and displayed oscillographically for observation and photography.
In most instances, sufficient " s p o n t a n e o u s " impulse activity within the oculomotor axons
was observed in the absence of specific stimulation to obtain positive correlations between electrical
activity in nerves and muscles. In other cases tactile stimuli were delivered to the eyes and orbital
region using a fine camels hair brush. At times, brief (0.5 ms) electrical shocks were also used
to excite nerves supplying specific muscles.

Results

Gross Anatomy of the Eye Muscles

Figure 1 is a dorsal view of the external eye structure of P. clarkii in a posture
of repose. Each stalked compound ey~ extends anterolaterally from the protocephalon at an angle of about 75 ~ with the animal's midline and roughly
10~ above a horizontal plane passing just below the ventral wall of the eyecup.
Structural support for the suspension of each eye is provided by the median
ocular plate, to which are firmly attached the proximal ends of sub-cylindrical,
calcified members of the eyestalks, the basophthalmites. Only the musculature
of these and the distal eyecups have been examined in detail.
Descriptions of the eye muscles are available for Procambarus (Sugawara
et al., 1971) and for at least two other genera of crayfish: Astacus (Schmidt,
1915) and Orconectes (Robinson and Nunnemacher, 1966). In some respects,
the previous anatomical studies were not found to be sufficiently detailed for
purposes of this paper. In addition, discrepancies occur between those accounts
and the present discription, which identifies eleven separate muscles in the
entire eyestalk of P. clarkii by an arbitrary numbering system. This is unrelated
to the previous descriptions in which muscle names were assigned on the basis
of assumed functions that, in at least some instances, appear to be in error.
At any rate, since more than one eye muscle probably is active during most
eye movements, and because the pivot point of the eye is liable to change
during horizontal eye movements (Burrows and Horridge, 1968a) it appeared

],-..

Fig. 1. Dorsal view of the top head region of Procambarus clarkii with the rostrum removed,
illustrating the normal posture of the c o m p o u n d eyes

Eye Muscles in the Crayfish

353

Fig. 2. Diagram of the supraoesophageal ganglion of Procambarus. The three major groups of
oculomotor neurons are indicated on both sides, and the axons of representative neurons are
shown exiting the ganglion. Major regions of dendritic arborization of the different neuronal groups
are included. ON, optic nerve; OCM, oculomotor nerve; ONMB, optic nerve motor bundle; DMB,
distal motor bundle; AMC, anterior motor cluster; LC, lateral motor cluster; GC, giant cell cluster

safest to designate the individual muscles by number until a more thorough

study could be made of their specific involvements during eye movements.
The muscles of the eyestalk are innervated by motor axons which run within
the optic nerve and a separate oculomotor nerve (Fig. 2) (Hisada and Higuchi,
1973 ; Mellon et al., 1976). Axons of the giant oculomotor cells (GC) and those
of the anterior motor cluster (AMC) run within the optic nerve and then,
peripherally, within two small branches: the optic nerve motor bundle (ONMB)
and the distal motor bundle (DMB). The oculomotor nerve (OCM) contains
axons of cells in the lateral motor cluster (LC). It emerges from the dorsal
surface of the supraoesophageal ganglion and passes lateral to the optic nerve
as both exit the head through the epimeral foramen. Along part of its route
within the eye, the OCM becomes intimately associated with branches of the
ONMB.
Figure 3 shows diagrammatic dorsal views of the right eye. In Figure 3A,
the two arteries and two nerve supplies of the eye are indicated in cutaway.
All four structures reach the eye through a large median foramen in the anterior
epimeral plate, beneath the base of the rostrum.

L Descriptions of Individual Muscles

Muscle 11. Movements of the basophthalmite relative to the head are accomplished mainly through the action of muscle 11. This muscle has its origin
on an apodeme close to the lateral margin of the epimeral foramen. It is much
narrower at its origin than at its insertion, which occupies an elongate area
on the dorsal part of the basophthalmite (Fig. 3B). Muscle 11 appears to be
the homologue of the Attractor muscle described for Orconectes by Robinson
and Nunnemacher (1966).

354

DeF. Mellon, Jr.

\
Fig. 3A-D. Dorsal view of the right eye. A the major arteries and nerves to the eye shown through
the partially dissected basophthalmite. ON, optic nerve; OCM, oculomotor nerve; A.O., optic
artery; A.OM., oculomotor artery; EP.F., epimeral foramen. B-D muscles of the eye, as described
in the text

Muscle 12. Muscle 12 is a flat sheet and has its origin dorsally on an apodeme
at the anterior edge of the basophthalmite (Fig. 3 B). This muscle runs parallel
to the long axis of the eye and inserts directly on the dorsal wall of the eyecup.
It appears to be the homologue of the Dorsal Retractor described by Robinson
and Nunnemacher (1966). Because of its dorsal attachments, muscle 12 comprises
a major suspensory element of the eyecup. Vertical movements of the eyecup
relative to the basophthalmite are partly controlled by the degree of tension
in this muscle. J
Muscle 13. Muscle 13 has three distinct and separate branches which originate
in close proximity to one another from a sclerite near the distal termination
of the basophthalmite. The three separate branches of this muscle insert in very
different regions of the eyecup.
Muscle 13a is a slender structure which inserts at a point on the dorsal
eyecup proximal to the insertion of muscle 12 (Fig. 3 B). This muscle is apparently the homologue of the dorsal head of the Lateral Retractor muscle in

Eye Musclesin the Crayfish

355

Orconectes. Its position and attachment suggest that, like muscle 12, it provides
for the suspension of the eyecup.
Muscle 13 b inserts on the lateral wall of the eyecup (Fig. 3 B). It is a short
stout muscle and undoubtedly is involved in laterally-directed movements of
the eyecup relative to the basophthalmite. Muscle 13 b is probably the homologue
of the ventral head of the Lateral Retractor muscle in Orconectes.
Muscle 13c is a slender muscle, the probable homologue of the Lateral
Rotator described for Orconectes. This muscle inserts on the floor of the eyecup,
posteriorly and near the ventral midline (Fig. 3 C).
Muscle 14. Muscle 14 is composed of two slips, 14a and 14b, which originate

from separate adjacent sclerotized regions of the eyestalk membrane medial

and distal to the origin of muscle 12 (Fig. 3 C). They are the homologues of
the Medial Retractors of Orconectes. Both slips insert close by one another
on the medial wall of the eyecup. From the placement and attachment points
of muscles i4a and 14b, it is tentatively concluded that both are involved
in medially-directed movements of the eyecup.
Muscle 15. This is the largest muscle in the eye. It is provided with two points

of origin (Fig. 3 C). The most proximal of these is an apodeme connected to

the lateral boundary of the epimeral foramen. The more distal origin is a
sclerotized region of the ventromedial eyestalk membrane. The fibers of muscle
15 are grouped in fascicles which are helically arranged along its length in a
manner reminiscent of the deep flexor muscles of the crayfish abdomen. All
fibers of this muscle insert on the dorsolateral wall of the eyecup, just proximal
to the corneal region. This muscle is undoubtedly the homologue of the
Abductor muscle of Orconectes. It should be noted, however, that muscle 15
of Procambarus is surely not responsible for abduction of the eyes, but, instead,
mediates the rapid reflex withdrawal of the eyestalk.
Muscle 16. Muscle 16 is a long, rather slender muscle having its origin near

the ventral margin of the epimeral foramen (Fig. 3D). It inserts on the floor
of the eyecup at a point near the insertion of muscle 18. Muscle 16 retracts
the eye and, like muscle 15, swings the eyestalk medially toward the midline
position beneath the rostrum. This muscle may be the homologue of the lateral
Ventral Depressor described by Robinson and Nunnemacher (1966).
Muscle 17. This muscle originates on the ventral eyestalk membrane near to

one of the lateral terminations of the basophthalmite (Fig. 3 D), and it inserts
on the ventromedial eyecup just proximal to the corneal margin. It appears
to be the homologue of the Orconectes Ventral Retractor. Both muscles 17
and 18, described below, are instrumental ill eyecup depression during protective
withdrawal.
Muscle 18. Muscle 18 is a short broad sheet which originates on the ventral

eyestalk membrane distal to the origin of muscle 17. It inserts on the ventral
floor of the eyecup (Fig. 3 C). This muscle is probably homologous with the
medial Ventral Depressor of Orconectes.

356

DeF. Melion, Jr.

II. Innervation of the Eye Muscles

A. Anatomical Results
Methylene blue staining provided a consistent, if incomplete, picture of the
distribution of OCM, ONMB, and DMB axons to the eye musculature. All
muscles of the eye except 13b, 13c, and 14b are at least partially innervated
by axons which run within the O N M B and/or the DMB. In addition, OCM
axons supply muscles 13b, 13c, 14a and 14b. In only two cases was the number
of axons supplying any one muscle easily apparent in stained preparation:
muscle 13 a receives terminations of only three axons, from the ONMB. These
are sufficiently small to be identified as belonging to neurons of the AMC
rather than those of the GC. Two large axons supply muscle 15, and their
relative diameters indicate that they do belong to GC neurons.
More precise information about the distribution of the individual A M C
and G C axons was obtained by the technique of axonal backfilling with cobaltous chloride. Figure 4 illustrates typical results obtained when this procedure
was used on the motor supplies of muscles 11, 12, 13a, and 17/18. Muscle
11 is innvervated by five identified A M C cells (Fig. 4A). These are clearly
cells A through E as previously catalogued by Mellon et al. (1976). They include
the medial group of three neurons (A-C) and the two large dorsally located
somata, D and E.
Muscle 12 is supplied by four laterally situated A M C cells, F - I (Fig. 4B).
The precipitated cobalt is less intense in both this photograph and that of
Figure 4 C, possibly due to the longer distances required for migration of cobalt
from these more distally situated muscles.
The motor supply of muscle 13 a is shown in Figure 4C. The three AMC
neurons filled appear to be cell D and two of the medial cluster. Although
precise identification of individual cells within the medial group is difficcult,
these two neurons are probably A and C. Muscle 13a thus shares its entire
motor supply with muscle 11.
Axons of four ventrally placed A M C neurons, cells J-M, exit the optic
nerve within the DMB near the medulla terminalis, and they are probably
distributed only to muscle 18. Figure 4 D shows the results of backfilling the
DMB with cobalt. The difficulties encountered in attempting to selectively fill
the axons to individual muscles of this group precluded a more precise anatomical determination of the motor neuron distribution.
The three cells of the giant cluster were briefly noted in a previous paper
(Mellon et al., 1976). The dendritic arborization of each of these three neurons
is sufficiently unique to allow individual identification on morphological grounds
alone. Figure 5A shows a cell-designated G l - o n the right hand side of a
preparation in which it was the only giant neuron visualized by backfilling
the ONMB. The axon, neuritic segment, and two stout club-like dendrites which
give this cell a characteristic appearance diverge from a common junction.
The dendrites course ventrally across the medial margin of large fiber tracts
comprising rostral extensions of the circumoesophageal connectives. The dendrites of G1 are further characterized by profuse tufts to which they give rise
along their course.

Eye Muscles in the Crayfish

357

Fig. 4A-D. Visualization of selected AMC neurons produced by axonal backfilling of the motor
supplies to (A) muscIe 11, (B) muscle 12, (C) muscle 13a, (D) muscle 17/18
Figure 5 B is a photograph illustrating cells G2 and G3. The dendritic trees
of these two neurons from the same side have very similar branching configurations. These spread laterally and posteriorly from the junction of the axon
and neuritic segment and appear to wrap around the rostral extensions of
the circumoesophageal connectives. There are no apparent dendritic tufts such
as occur on cell G~. The primary difference between Gz and G3 is in dendritic
branch diameter those of G2 being roughly twice as large as the corresponding
processes of G3. In addition, the plane of the branch distribution of G2 is
positioned dorsal and ouside to that of cell G3. Selective axonal backfilling
indicates that cell G t supplies muscles 15 through 18. Cell G2 innervates only
muscle 15, while cell G3 supplies all of the other three (Fig. 5C-D).
B. Electrophysiology
Confirmation of the anatomical observations was obtained by recording electrical activity simultaneously from different efferent pathways and specific eye

358

DeF. Mellon, Jr.

Fig. 5A-D. Dendritic morphology of cobalt-filled GC neurons. A Dorsal view of Ga on the right
side of brain. Soma at right; neuritic segment runs ventrally and out of focal plane. B G2 and
G3 backfills from a fortuitous preparation in which G 1 did not fill. C visualization of G1 and
G3 obtained by backfilling the motor supply to muscles 16/17. D G1 and G2 backfills of the
motor supply to muscle 15

musles. During the course of these studies, it was found that, as in the crab
(Burrows and Horridge, 1968c), individual fibers of the different muscles are
not always physiologically homogeneous. While some muscle fibers clearly receive terminals from all axons constituting the motor supply to a particular
muscle, others do not.
Figure 6 illustrates electrical records obtained simultaneously from intracellular electrodes within fibers of muscles 11, 12, and 13a, and from a suction

Eye Muscles in the Crayfish

359

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Fig. 6A-C. Correlation between electrical activity in selected fibers of muscle 12 (A, top trace),
muscle 11 (B, bottom trace), muscle 13a (C, bottom trace), and the ONMB. In each case, identified
pairs of nerve and muscle events are numbered for clarity. Calibrations: 200 ms: 20 mV

electrode placed on the ONMB. Five distinct amplitude classes of excitatory

junction potentials (EJP'S) obtained from a fiber of muscle 11 (Fig. 6) are
temporally correlated with five classes of nerve impulses, supporting the predictions based upon the anatomical data for this muscle. These recordings were
obtained in a fiber from the medial portion of muscle 11. As in the eye muscles
of the crab (Burrows and Horridge, 1968c) phasic and tonic motor systems
exist in parallel and the individual muscle fibers clearly fall into either one
category or the other. In muscle 11, laterally placed muscle fibers are supplied
by fewer axons, and they exhibit little, if any, tonic activity.
Electrical records from muscle 12 are more complex. In the general, the
lateral portion of this muscle is tonically active, some individual fibers exhibiting
EJP's which are correlated with four different impulse heights from the O N M B
(Fig. 6B). Other fibers, however, are appreciably different in the nature of
their activity. Tonic EJP's are present, but no more than two classes of these
can be positively correlated with distinct O N M B impulses. At least one EJP
type persists even following section of the entire optic tract and the OCM
(Fig. 7D). The motor neuron responsible for this category of junctional activity
9 has its soma in the medulla terminalis, as visualized by backfilling the motor
supply of muscle 12 with cobalt, and its activity is uniquely affected by illumination of the compound eye (Mellon and Lorton, 1977).
The branch of the O N M B which innervates muscle 13a is sufficiently long
that its activity can be directly recorded by suction electrode. Three sizes of
motor impulses are recorded from this nerve, and all are temporally correlated
with EJP's in individual fibers of the muscle (Fig. 6 C). These data agree numerically with findings from axonal backfilling experiments on muscle 13 a. However;
in order to verify that some of the motor axons which supply muscles 11
and 13a are shared, simultaneous extracellular records were obtained in pairs
from both of these structures and from muscle 12. These data are shown in
Figure 7A-C, and confirm that some portions of muscle 11 and muscle 13a

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360

DeF. Melon, Jr.

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Fig. 7. A-C paired extracellular records from muscles 11, 12, and 13a as described in the text.
D intracellular records from a fiber of muscle 12 (bottom trace) and extraceliular records from
its motor supply, in an isolated eye. Nerve record retouched for photographic clarity. E intracellular
records from a fiber of muscle 18 (bottom trace) and extracellular records from the DMB. Correlations between at least one impulse type and EJP's are indicated by asterisks. Calibrations: 200 ms;
20 mV

are a c t i v a t e d in concert, while the m o t o r s u p p l y to m u s c l e 12 is distinctly

s e p a r a t e f r o m t h a t o f the o t h e r two.
E l e c t r o p h y s i o l o g i c a l r e c o r d i n g s s h o w e d t h a t o n l y muscle 18 is s u p p l i e d by
a x o n s o f the D M B . F i g u r e 7 E shows s i m u l t a n e o u s r e c o r d s o b t a i n e d f r o m a
m u s c l e fiber a n d f r o m the D M B . O n e A M C a x o n clearly supplies this fiber,
a l t h o u g h r e c o r d s f r o m o t h e r s m a y s h o w either no activity o r c o r r e l a t e d E J P ' s
f r o m u p to three different axons.
C o n f i r m a t i o n o f giant cell a x o n d i s t r i b u t i o n was o b t a i n e d b y s i m u l t a n e o u s
electrical r e c o r d i n g s f r o m the O N M B a n d muscles 15 t h r o u g h 18. Evidence
t h a t cell G3 supplies muscles 16, 17, a n d 1 8 - t o the exclusion o f m u s c l e 1 5 - i s
s h o w n in F i g u r e 8. A large O N M B axon, g e n e r a t i n g i m p u l s e s with at least
twice the a m p l i t u d e o f t h o s e in a n y A M C a x o n is p h a s i c a l l y driven b y m o d e r a t e
tactile s t i m u l a t i o n o f the h e a d , especially in the o r b i t a l region. I m p u l s e s in
this cell are c o r r e l a t e d o n e - f o r - o n e w i t h discrete, facilitating E J P ' s in fibers
f r o m muscles 16, 17, a n d 18 (Fig. 8 A , E - F ) b u t n o t with E J P ' s f r o m muscle
15 (Fig. 8 B, D).
I n t e n s e tactile s t i m u l a t i o n o f the head, i n c l u d i n g the eye itself, evokes activity
in a n o t h e r very large O N M B n e u r o n . I m p u l s e s in this cell a r e c o r r e l a t e d with

Eye Muscles in the Crayfish

361

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[Lt,X,

[.. iL . . . . .

: 15 .....

'"

Jl'[[

J'['

....

~"

'"

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.L

l'l ""

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:

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17

D
N .... [~ .....
. . . . -. . .

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~

Ll~t,,,k . . . .
l~t~'i ....

it ,,
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"1

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r'

1
17

17
16

Fig. 8. A - D simultaneous records from fibers in muscles 15 and 17 and from the ONMB during
strong tactile stimulation of the eyes and anterior head. In A, the large spike in the nerve (N)
record is from the axon of cell G3. In B, eye stimulation evokes responses not only from G3
but from a larger axon as well, cell Gz. Records in C and D from another preparation. Arrows
mark pair of spikes in G2 which generate EJP's only in muscle 15. E - F intracellular record
pairs from muscles 16, 17, and 18 to demonstrate common innervation by cell Ga. Calibrations:
200 ms; 50 mV

junction potentials in muscle 15 alone (Fig. 8B, D). The identity of this giant
motor axon, therefore, must be G2, since the only alternative, cell Gt, supplies
muscles 16-18 in addition to muscle 15 (cf. Fig. 9). Cell G1 could not be
activated by tactile stimulation of the h e a d or eyes. However, the presence
of a functioning axon in addition to G3 supplying the fibers of muscle 15
was demonstrated by electrically stimulating the O N M B (Fig. 9A-D). Paired
intracellular electrical records were obtained from individual fibers in muscle
15 and each of the other three withdrawal muscles while the optic nerve was
stimulated with 0.5 ms electrical shocks. At minimal stimulus intensities an
EJP was evoked from the fiber in muscle 15 alone. When a slight increase
in stimulus intensity was effected, an EJP was generated in each of the fibers
from muscles 16, 17 and 18. Simultaneously, a step increase occurred in the
amplitude of the response recorded from muscle 15. These results show that
a single low threshold O N M B axon innervates muscle 15. A second motor
neuron, exhibiting a slightly higher stimulus threshold, is distributed to all
four of the muscles under examination. It is concluded that the axon with
the lowest threshold is G2, and the cell common to all four of the muscles
is G1.

362

DeF. Mellon, Jr.

16

17

~
'=

~
-i

D A ~ I

18

(3

H
'
1l

f ~ .

-f

~..

,J

Fig. 9. A - C simultaneous intracellular record pairs from muscle 15 and muscles 16, 17 and 18
in response to electrical stimulation of the ON. D simultaneous records from muscle 15 and
16 in response to maximal brief, electrical stimulation of the ipsilateral circumoesophageal connective. Similar records in response to (E) subthreshold and (F-H) suprathreshold electrical stimuli
in another preparation. Synaptic failure occurred shortly before record H was photographed, Calibrations: A-D, 50 ms and 50 mV; E-H, i0 ms and 25 mV

What is the normal pathway for excitation of G1 ? The intimate anatomical

association of dendritic branches from this neuron with fiber tracts in the circumoesophageal connectives suggested an obvious possibility, namely, ascending
giant fiber activity. This expectation was born out by results of experiments
in which the ipsilateral connective was electrically stimulated while recording
simultaneously from muscles 15 and 16 (Fig. 9E-H). Junction potentials in
fibers from both muscles appeared simultaneously at threshold voltage for an
exceedingly large action potential in the electrical records from the connective,
possibly originating in the medial giant axon. Whatever the identity of the
presynaptic neuron, the synapse with the motor neuron (G~) is very labile
in the radically dissected preparations required by these experiments. This property proved useful in determining which of the following alternatives offered
the most reasonable explanation of the results. Simultaneous generation of
junction potentials in muscles 15 and 16 might be caused either by excitation
of a single shared motor neuron (G1) or by two separate motor neurons (G2
and GB) excited in common by a single presynaptic neuron. To decide which
of these two alternatives best accounted for the experimental observations, a
continuous train of single shocks, suprathreshold for the large connective impulse, was delivered to the ipsilateral connective at a frequency of one Hz.
When failure of synaptic transfer occurred after several minutes, responses in
both muscles disappeared together. This finding strongly implicates a common
final efferent path and supports the contention that cell G1 is normally activated
by a neuron or neurons within the ipsilateral connective.
Identification of LC motor neurons on an individual cellular basis has not
yet been attempted. Nonetheless, we wished to confirm electrophysiologically
the methylene blue staining data that suggested LC axons supply muscles 13 b,
13c, 14a, and 14b. Accordingly, various fibers in these four muscles were
sampled by intracellular electrode while simultaneously recording from the ipsilateral OCM. The results are shown in Figure 10. In each case, some EJP's

Eye MuscIes in the Crayfish

363

A
.... IL. Ii.T ,
--r. r',-"

t.,
r+

L .Li[I+I ....
F
+r+~r-

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-i ~

~,JU,,
-.n

.il,
-+~

, l,tih,Ltl,
+~m . . . .

t....
,~'

li

' ~''

,*
~

ii,.iil-'..i
t,
"T'r-,-+"

l" - I ' [ . . . . .

F[

?i

r r l --

"t

"--

-rq~-+?p

~ rr'[

"-TVW+I[

L :2
Ir

i+~r r-'al' ~ .;.i. I:"

~"

rl'.,
, . L .t Ja_

iJ

//

.a

+"

~='-~:"1 rl

. . . .

,r

"U'"n-~ ....

"-+j(~''lrl .....

,r-,,,r'?-], .... l r-r-1 ....

,1-+,l,-c,~,-:

E
i'i;,~,T~

Irql

', ''T '

I1~ Ir'l

; ;' / "*;

,.:,

['"'""'["
.+lu.+l]*

Ir t[

. ...J. ...... U rLr ,'

,*~
~
,, ... Lre,

; '- .*, , "" rlTl'~' i T ' j , I~l~[P."~, [ " ' '~' 'J

Fig. 10. A - D s i m u l t a n e o u s electrical records from O C M (top trace) a n d single fibers in muscle
13b (A), 13c (B), 14a (C) a n d 14b (D). E records f r o m O N M B a n d a fiber of m u s c l e 14a. A t
least two O N M B a x o n s e v o k e E J P ' s in 14a (asterisks). C a l i b r a t i o n s : 200 m s ; 50 m V

evoked in individual muscle fibers are correlated with a particular class of

nerve impulse in the OCM record. The included records also demonstrate that
muscle 14a is supplied by at least two axons of the O N M B (Fig. 10E). These
data support the anatomical results and implicate LC neurons in the control
of horizontally-directed eye movements.

Discussion
In the crayfish, axons of motor neurons that influence eye movements are
distributed to ipsilateral muscles, which they supply via three anatomically distinct tracts: two branches of the optic nerve and a separate oculomotor nerve.
This anatomical separation may reflect a segregation of the eye muscles into
functional subsets. The oculomotor nerve supplies, apparently to the exclusion
of others, four muscles which, on anatomical grounds, must move the eyecup
horizontally. The non-giant motor axons of the optic nerve supply muscles
which probably are responsible for vertical movements of the eyecup. An apparent further subdivision of this group is the distal separation of axons which
provide for, respectively, eyecup elevation (ONMB) and eyecup depression
(DMB). Finally, axons of the giant motor cluster (GC) undoubtedly effect
both depression and withdrawal of the entire eyestalk during protective reflexes
by which the eyes are withdrawn to a secure position beneath the rostrum.

364

DeF. Mellon,Jr.

These anatomical arrangements appear to be at variance with the organizational framework of the crab oculomotor system in which, according to Burrows
and Horridge (1968a), motor axons from both optic and oculomotor nerves
intermingle and innervate all muscle blocks in the eye. However, knowledge
of the distribution of individual motor neurons within the crab eye is about
as incomplete as our present understanding of the involvement of different
crayfish eye muscles in the various compensatory reflexes. It is thus possible
that the differences are more apparent than real. The mechanisms of crustacean
eye movements are complex. As Burrows and Horridge point out, no hinge
is present at the eyecup-eyestalk joint, and the eyecup, which is suspended
by the tonic activity of several muscles, is consequently free to move within
three planes of freedom. They note, " I t is possible that a muscle with no
detectable change in impulse frequency may, nevertheless, contribute to a movement as the balance at the flexible joint is changed by other muscles." These
observations emphasize the need for caution in the interpretation of anatomical
data alone when making conclusions about the contributions of individual eye
muscles to different eye movements.
The anatomy of the giant motor axons which drive protective eye withdrawal
reflexes is interesting. Two types of dendritic branching morphology are exhibited by specific cells of this group, suggesting questions which concern the
electrophysiological consequences of specific structural arrangements. For example, what is the functional significance of the paired, club-like dendrites of
G1 as opposed to that of the more open branching configuration which characterizes G2 and G37 Is this an obligatory function of the large size of GI
axon, or of the specific nature of the transmission process at this synapse?
The tufts which occur in profuse numbers at the termination of the G1 dendrites
have a superficial resemblance to the dendritic spines of cells in the vertebrate
central nervous system. Dendritic spines have been viewed as a mechanism
for injecting postsynaptic current into the main axis of large dendritic trunks
(such as those on cerebellar Purkinje cells) without severely lowering their input
impedance. The advantages gained by this sort of morphological arrangement
are a greater longitudinal spread of synaptic potentials within the dendrite
and an increased safety factor for the generation of dendritic action potentials
(Llinfis and Hillman, 1969).
The dendritic morphology of cell G1 also superficially resembles that of
the crayfish motor giant axon at the latter's synapses with the giant fibers
in the ventral nerve cord. These are rectifying electrical synapses, and were
first studied physiologically by Furshpan and Potter (1959). Using cobalt iontophoresis, Mittenthal and Wine (1973) have recently been able to visualize the
synaptic connections made by the motor giant axon with both the lateral and
medial giant fibers. The morphology of the postsynaptic fiber at the region
of functional contact is characterized by fine projections and tufts that appear
to be involved in intimate association with the presynaptic axon. It is probable
that the postsynaptic projections penetrate invaginations of the presynaptic membrane, as originally shown by Robertson (1953). On morphological grounds,
therefore, it seems as if theGx dendrites are suitably designed for the injection
of large amounts of synaptic currents and the passive or active transmission

Eye Muscles in the Crayfish

365

of resultant potential change along the paired dendritic branches with minimal
amplitude decrement. It is hoped that electrophysiological studies now underway
with cell G 1 will provide sufficient functional evidence to understand the anatomy of this interesting neuron and the basis for those differences in dendritic
branch configuration exhibited by cells G2 and G3.
I am grateful to Mr. Gene Lorton for his technical assistance with some phases of this work.
I also thank Sharon Greene for executing the illustration in Figure 1 and Susan Snarez for illustrating
Figure 2. This work was supported by USPHS research grant NS04989.

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