Documente Academic
Documente Profesional
Documente Cultură
2-1-IEF
STARTPAGE
PEOPLE
MARIE CURIE ACTIONS
PART B
“Circuit-hubs”
Table of Contents
mechanisms controlling the onset of synchronisation in immature networks. Therefore, the overall
aim of the project is to test the existence of “circuit-hubs” in developing cortical networks and
validate the hypothesis that the initiation of synchronous network events is dependent on precise
connectivity patterns between neurons. ’Circuit-Hubs’ is organised along three main objectives: (1)
understanding the functional topography of hippocampal circuits (2) proposing a morpho-
physiological description of “circuit-hubs” (3) analysing the function of “circuit hubs”. In the
following lines we will describe the methodological approaches implemented to meet these
objectives.
Objectives against the state of the art - The first coherent activity patterns in developing cortical
networks have been described almost twenty years ago but the mechanisms leading to neuronal
synchronization are still unknown. One possible explanation relies on the complexity of cortical
networks (variety of morpho-physiological cell types, of communication devices between neurons,
etc), a property even emphasized at early developmental stages. Indeed, immature cortical networks
gather not only different types of neurons but also cells at different developmental stages.
Furthermore, developing networks are by definition highly dynamic and plastic. So far two
hypotheses have been proposed to drive the onset of early network oscillations: (1) a “network
theory”: synchrony is generated by the build-up to a threshold of random local perturbations in a
highly recurrent synaptic network (de la Prida et al., 1999; la Prida et al., 2006, Bolea et al., 2006,
Marchionni 2007), and/or (2) a “cell theory” where rhythmicity is provided by the autonomous
activity of a group of pacemaker neurons (Sipila et al., 2005, Sipila et al., 2006, Zheng et al., 2006,
Strata et al. 1997). Therefore, the understanding of the mechanisms leading to population coherence
in developing cortical structures requires having simultaneously and dynamically access to two
levels of observation: the network and the cell. The considerable progress made by imaging
techniques now offers this possibility. Our goal is to localize and characterise “network hubs”
based on the analysis of spontaneous network dynamics without any a priori assumption.
Expected results - We expect by the end of the project to reach the following results:
(1) By reconstructing the temporal dynamics of the network we expect to identify “hub cells”
capable to drive its activity, i.e. showing high correlation with global network firing and
anticipating synchronous events (GDPs).
(2) By performing targeted electrophysiological recordings of spontaneous and evoked activity
in hub neurons we expect to characterize their functional properties and confirm that they drive the
oscillatory activity of the network. We hope to be able to understand the cellular mechanisms by
which these cells influence the network state.
(3) Based on morphological reconstruction of biocytin-filled targeted hub-cells we expect that,
although still immature, hub neurons display spread and wide morphological features. This
observation would support the theoretical model underlying this project.
(4) Immunohistochemical characterization of these cells should narrow the functional family of
hub neurons to specific cell types.
(5) Demonstration that inter- and multi- disciplinary strategies bridging mathematic and physics
to neurobiology are strongly appropriate in brain research.
Expected outcome beyond the scope of the proposal – By reaching the above mentioned point 4,
we will open a wide field of investigation that goes far beyond questions of developmental
neurobiology. Indeed, if high-connectivity neurons are of a specific cell class and drive network
synchrony, the next logical step following the outcomes of this project would be to perform
experiments on genetically engineered mice where the specific neurochemical marker for these
neurons would be tagged with GFP. That way, it will be possible to perform a full description of
these cells, study their role in mature networks and eventually test whether they also contribute to
other types of oscillations such as epileptiform activities. Finally our approach to study the
functional topography of networks will be of obvious interest for biologists interested in complex
biological systems even outside the field of Neurobiology.
Feasibility - The feasibility of the project relies on the perfect combination of: 1) the scientifical
expertise and unique experimental facilities offered by the host institution and team testified by
their publication records 2) the interdisciplinary background of the host supervisor: Dr. Cossart
received a double undergraduate training in mathematics and physics at the Ecole Centrale Paris- a
French Grande Ecole- and a doctoral and postdoctoral training in neurobiology of GABAergic
networks, 3) the financial support for the equipment of the host team, 4) the computational
background and approach of the applicant testified by his education and previous research
experiences on living neuronal networks and 5) solid preliminary experimental data (see B4
“feasibility and credibility”).
Conclusion - The relevance of this project relies on its multi- and interdisciplinary aspects to
address a fundamental question of developmental neurobiology. Consequently, the scientifical
outcomes of the project and the interdisciplinary pioneering strategy used will definitely be of
interest for a very broad spectrum of scientists working on 1) development 2) pathologies related
to development (like epilepsy) 3) neuronal networks 4) complex systems. We believe that the
above reasons represent solid scientifical, technological and socio-economic arguments to support
such project in Europe, a project that represents also an essential step in the research carrier of the
applicant towards his establishment as independent researcher.
• Research methodology
To schematize the overall project we can distinguish three main aims: 1) description of the
functional topography of hippocampal circuits 2) morpho-physiological and chemical description of
“circuit-hubs” and 3) function of “circuit hubs” and simulation of network dynamics. Each aim is
described in more details below. In the initial part of the project we will develop appropriate data
analysis tools to accomplish the aim 1. During the central part of the project all the experiments will
be run to provide a morpho-physiological description of “circuit-hubs” (aim 2). In addition, during
this period experiments will be coupled to the analysis of the network dynamic during hub
stimulation. This will allow to better design experiments capable of unrevealing the function of
hubs for simulating the network dynamic (aim 3).
Choice of the preparation - We will describe the functional topography of the developing
hippocampus and experiments will be performed on hippocampal slices from rats (P3-P7). This
cortical structure was chosen because the host team has recently described the maturation sequence
of population coherence in this region from embryonic stages to the second postnatal week, thus
providing a solid framework to the proposed project (Crépel et al. 2007). We will focus on the CA3
region of the hippocampus since this area has the highest propensity to generate Giant Depolarizing
Potentials (GDPs, Ben-Ari, 2001). GDPs are the early patterns of network activity in the
hippocampus and were first described two decades ago in the host institute (Ben-Ari et al. 1989,
Ben-Ari, 2002). These coordinated activities are synaptically-driven mostly by GABAergic
transmission, which matures earlier and is excitatory during the first post-natal week (Ben-Ari,
2002, Tyzio et al. 2006).
Imaging network dynamics - The activity of the network will be monitored with single-cell
resolution using two-photon calcium microscopy. This unique method recently developed by the
host team (Cossart et al., 2005, Crepel et al., 2007, Tyzio et al. 2006, Goldin et al. 2007, see also
below), represents a high-resolution and non-invasive imaging strategy to visualise neuronal
networks in action in thick biological samples. Two-photon laser scanning microscopy has several
advantages over conventional confocal scanning techniques. Photo-damage and bleaching, which
are the two major limitations for imaging of living specimen, is practically negligible. Also, the
pulsed IR LASER penetrates much deeper in living tissue at depths where the network connections
are preserved (from the slicing procedure for example). However, the major limitation of such
method based on conventional scanners is time resolution (typically~1sec/frame). In order to
circumvent this limitation, the host team has already successfully used a pioneering system based
on a multi-beam scanning of the preparation; such system achieves millisecond resolution and
reliably detects single action potentials when imaging the entire field (Crepel et al., 2007, Goldin et
al. 2007, Tyzio et al. 2006). The time resolution of the imaging system is in the order of 100 ms
corresponding to the readout time of the CCD camera used for image acquisition. The first
limitation of this experimental approach could be the slow intrinsic dynamics of calcium signals and
the possibility that some fluorescence events might not correspond to action potential firing. Still, at
early developmental stages calcium is probably an even more reliable indicator of neuronal activity
than sodium spikes which can be often poorly developed. The second limitation would be the
description of network activity in slices and not in more integrated preparations. However, we
believe that a description of slices is a good first step in understanding network dynamics since (1)
the problem is simplified; (2) at these stages the same patterns described in vitro are observed in
vivo (Adelsberger et al., 2005; Leinekugel et al., 2002); (3) similar experimental conditions are not
available in vivo at present and (4) ultimately the same approach can be applied in vivo when
appropriate tools will be available.
Conclusion - Given the overall objective of the project and the three different aims to reach it, it is
essential to carry on an interdisciplinary and multidisciplinary approach which includes: 1)
mathematical analysis of the network dynamics; 2) multibeam two-photon calcium microscopy 3)
electrophysiology and 4) morphological and immunohistochemical description of hubs. This
interdisciplinary project is based 1) on the double background education of Dr. Bonifazi (Degree in
Physics, PhD in Neurobiology) 2) on the expertise of the supervisor in imaging/electrophysiology
and 3) on the additional support for morphological studies provided within the host institution.
At the end of the project we expect to describe the morpho-functional and chemical properties of
specific cell types and how these cells are capable to drive oscillatory network activity. These
results will open new research perspectives not only regarding brain development but also the
understanding of GABAergic circuits, mechanisms of network synchronization and pathological
disorders associated to hyper synchrony.
• Originality/innovative nature of the project, & relationship to the appropriate 'state of the art'
One of the main challenges of modern neuroscience is to understand how dynamic oscillations are
related to the wiring structure of the anatomical connectivity. This is an ambitious aim, but we
believe it can be achieved now because of the convergence of new experimental and theoretical
developments. More specifically, as described above, the aim of the project is to test the existence
of “cell-hubs” in the network and validate the hypothesis that the initiation of synchronous network
events is dependent on precise connectivity patterns between neurons.
An innovative methodological approach - Multibeam two-photon calcium microscopy will be
used to monitor the activity of the network with single-cell resolution. This unique method has been
recently developed by the host team (Cossart et al., 2005, Crepel et al., 2007, Goldin et al. 2007, see
also below) and represents a high-resolution and non-invasive imaging strategy to visualise
simultaneously the activity of hundreds of cells in neuronal networks. Combining these pioneering
imaging tools with cutting-edge statistical data exploration is by definition innovative. Furthermore,
our preliminary data (see B4) indicates that it is indeed feasible.
Exceptional conditions - Therefore the originality and innovative nature of the project lie in the
link between network dynamics and single-neuron properties. As such, it requires an inter-
multidisciplinary approach that will be provided by extensive daily interactions between the
candidate and the neurobiologists in the host team. The ambition to gather different fields of
expertise and in particular to bridge the gap between theoreticians and neurobiologists in order to
solve complex problems related to network studies has motivated several research groups. We
believe that this interaction becomes really fruitful when the same person gathers multiple fields of
competence (without necessarily being an expert in each domain). Both the postdoctoral applicant
and the host team leader meet this requirement.
Originality of the scientific question - Our work hypothesis is original because it addresses a
fundamental question of neurobiology based on theoretical predictions. Indeed the existence of
“circuit-hubs”, i.e. neurons with spread and/or much larger number of connections influencing
strongly the state of the network, is based on models of complex systems and networks, like Self-
Organized Criticality (SOC) paradigm, small-world and scale-free networks .
Novel concepts - Although extensively studied, the factors that determine the timing of rhythmic
activities during cortical development are not completely understood, in particular because firm
evidence on this point requires having both a dynamic and global vision of the network but also
assessing the unitary properties of its key components in order to describe activity generators
without any a priori assumption. Our research plan should reveal both the elements and dynamic
connectivity of cortical microcircuits without a priori assumption. In that sense, the significance of
the proposed project goes far beyond the simple comprehension of developing networks, as, by
extension, we believe that a similar approach can be generalized to the analysis of other
synchronous population discharges such as epileptiform bursts. Reaching the objective of the
proposal will open a wide field of investigation that goes far beyond questions of developmental of
neurobiology. Indeed, if high-connectivity neurons are of a specific cell class and drive network
synchrony this project will pave the way for a broad spectrum of investigations. For example, it will
be possible to study genetically engineered mice where the specific neurochemical marker for these
neurons will be tagged with GFP in order 1) to perform a full description of these cells 2) study
their role in mature networks and 3) eventually test whether they also contribute to other types of
oscillations such as epileptiform activities. Finally such an approach to study the functional
topography of networks will be of obvious interest for other biologists interested in complex
biological systems even outside the field of Neurobiology.
Therefore, given the state of the art, the aim of our project to link network dynamics to the
morphological properties of neurons represents an innovative and original strategy to address a
complex biological question within a theoretical framework.
The hypothesis at the basis of our research proposal is that synchronous network events are
dependent on precise connectivity patterns between neurons. In particular we will test the
existence of “circuit-hubs” that is “super-connected” cells providing developing networks with
fast transmission of information. This is a fundamental question that is largely supported by
theoretical models and experimental observations. The methodological approach to be pioneered in
the proposed project will be of great value for the European Community since it will completely
change the way network studies are pictured. This approach of network studies is attractive to
several fields in biology outside Neuroscience (ex: cardiology, endocrinology). The development of
such tools in France in the recently created Institut de Neurobiologie de la Mediterranee is essential
for the European Union to keep Europe as competitive and dynamic knowledge-based in the field of
brain development. This is also crucial to increase the attractiveness of Europe for researchers.
Anyway, such interdisciplinary strategies require efforts at European level first, because of the big
economical effort (e.g. fast two-photon imaging is a very expensive technique) and secondly,
researches of excellence carried on different countries in different fields must be combined (i.e.
research on development in INMED, France; theoretical advanced studies at SISSA, Italy – where
the applicant did his PhD).
Post doctoral fellows (10) from a wide range of countries are present.
Outstanding results - INMED has made a series of pioneering observations in the developing
hippocampal network from rodents and primates, summarized by these 5 fundamental features:
1. GABAergic synapses are excitatory because of a different chloride gradient. This enables
GABAergic synapses to activate Voltage-gated Calcium channels, to remove the Mg2+ blockade
from NMDA channels and to increase the intracellular Ca2+ concentration, providing a signal
that stimulates neuronal growth and differentiation (Ben-Ari et al., 1989; Barbin et al., 1993;
Ben-Ari et al., 1997).
2. During the first post-natal week, the GABAergic network provides the main source of the early
patterns of network activity called Giant Depolarizing Potentials (GDPs) (Ben-Ari, 2002)
3. There is a sequential expression of GABA and glutamate synapses in neonatal rodents and in
utero primate hippocampi in interneurons and CA1 pyramidal cells (Tyzio et al., 1999;
Khazipov et al., 2001).
4. Somatic and dendritic GABAergic circuits are established in a sequential manner during the
first post-natal week: dendritic projecting interneurons develop before peri-somatic projecting
interneurons (Hennou et al., 2002).
5. Oxytocin a maternal hormone initiating labour, triggers a transient inhibitory switch in GABA
signaling in the fetal brain during delivery and control the emergence of coherent calcium
plateaus at birth which are progressively shut down a few days later by the synapse-driven giant
depolarizing potentials (GDPs) that synchronize the entire network. (Crepel at al., 2007; Tyzio
et al., 2006)
Facilities and funding - The group has priority access to facilities that include (i) two setups for
patch-clamp recordings under visual control performed with an infra red microscope that also
include a fluorescence system; (ii) a recently acquired TrimScope confocal system coupled to a
pulsed IR LASER (Chameleon, Cohérent) and double-patch clamp amplifiers. It receives funding
from the Agence Nationale de la Recerche (Jeunes Chercheurs).
B2 TRAINING
• Clarity and quality of the research training objectives for the researcher
Since April 2007 I am postDoc in the host institution and my contract will end at the end of the
year. Indeed, considering my previous studies on information processing in neuronal networks and
the interest of Dr. Cossart to understand the development of neuronal circuits, my short stay at
INMED was aimed to explore the possibility to converge our different expertises and backgrounds
for an interdisciplinary pioneering approach to the study of networks. Remarkably, in the few
months we spent working in collaboration, we were able to collect very strong evidences of the
scientifical solidity and feasibility of the proposed project (see B4). Given this very promising
starting point, this Marie Curie fellowship will allow us to carry on all the required studies and
deepen the experimental work. In addition, the publications which we will be able to produce
during the duration of the fellowship will be a strong positive impact for our young carriers. In the
case of Dr. Cossart this fellowship would push and strengthen her independent research carrier,
bringing new expertise in her team due to my original scientific profile. In my case it would
represent the last final big step to reach my independent research maturity and position. The
possibility offered by this fellowship to achieve my on-going project, write publications and present
the results of our research at international conferences will allow me to establish long-lasting
contacts which will launch my independent carrier.
Within the next two years my training objectives are (1) to deepen my experience on new
experimental methodologies for studying neuronal networks (standard electrophysiology combined
to fast two-photon calcium imaging) (2) to work in close collaboration with neurobiologists and
address fundamental issues in the field of network development that are also clinically relevant for
the understanding of brain dysfunction (3) to strengthen my scientific independence as the only
onsite expert in complex systems (4) to work in the interdisciplinary environment of INMED
(clinicians, medical council research centre (INSERM-U29), educational centre (TousChercheurs),
three biotech companies). As an Italian citizen, my ultimate goal is to start my own
multidisciplinary research group in Italy or in a Mediterranean European country, hence the
importance of broadening my networks in neurosciences.
Background- Studying properties and dynamics of living neuronal networks is the real “driving
force” of my postgraduate studies. Due to my strong mathematical and theoretical background
interact with clinicians, educational centre and biotech companies. This will be certainly useful to
develop my own independent carrier.
• Host expertise in training experienced researchers in the field and capacity to provide
mentoring/tutoring
As previously described, the host Institute, “Institut de Neurobiologie de la Méditerranée” is
composed of a medical council research centre devoted to neurobiology (Inserm-U29, 9
independent teams), an educational centre devoted to teaching experimental sciences to high school
students& residents (TousChercheurs) and three biotech companies. INMED is now an appreciated
centre of training as reflected by the growing number of candidates (10 postDoc and 12 PhD
students – number limited at 1 per permanent or established researcher) including from the US and
European countries and the venue for long stays of highly recognized researchers (Drs Mc
Naughton, Segal, Holmes and Baram). INMED offers an impressive number of experimental
facilities (which include 12 set ups for in vitro patch-clamp recordings (cultures, slices and intact
brain structures), 2 fast two-photon imaging set ups, 1 confocal microscope, molecular and
cellular biological tools, surgery, culture rooms and animalerie) to carry on experiments on
electrophysiology, immunohistochemistry, molecular biology and morphology and it is a rare
opportunity in the European context. In fact, all these facilities are shared by 9 different research
teams which are strongly collaborating. This approach allows investigating scientifical problems
with complementary expertises and from different points of view. Therefore the research quality is
of top level as recognized by the publication in best scientifical journals (e.g. Tyzio R, Cossart R,
Khalilov I, Minlebaev M, Hübner CA, Represa A, Ben-Ari Y, Khazipov R., Science December
2006).
Mentoring/Tutoring capacity of the host team- Although at an early stage of her career as a team
leader, Rosa Cossart has successfully trained undergraduate students and a postdoctoral fellow (Dr.
Goldin, Marie Curie fellow; Goldin et al. 2007); at the moment she is supervising a first year PhD
student (Camille Allene).
B3 RESEARCHER
• Research experience
Before starting any research experience my educational background was strongly mathematical and
theoretical. In fact, as an undergraduate, I studied Physics, in particular, Condensed Matter
Physics a discipline that focuses on many-body interaction systems like atoms with many electrons
or lattices and crystals. These systems are composed of many interacting units and the global state
of the system is dependent on the strength and spatiotemporal properties of their interactions, e.g.
systems in critical states show long-distance scale-free interactions. Analogously, neuronal
networks can be viewed as many-body interaction systems, where units are neurons and interactions
are synaptic connections. Such view of neuronal networks has already shown very interesting
results (Hopfield, 1982; Schneidman et al., 2006). Therefore, I do think that my mathematical
skills represent an essential but also very original educational background which allows me to
investigate neuronal networks on a novel manner.
During my research experience, although I often focused more on data analysis, I became more
and more familiar with experimental neurobiology. Indeed, performing accurate experiments on
neuronal networks is still very difficult and just in the last years multi-electrode recordings and
optical imaging allowed more precise measurements. Therefore studying neuronal networks require
the right knowledge for being able to evaluate feasibility and relevance of measurements.
In this context my undergraduate & graduate experience as a trainee in the lab. of prof. Fromherz
(Max Planck Institute for Biochemistry, Martinsried, Germany; January 2000 - January
2001; June 2001 – July 2001) was extremely formative. The experimental work carried on during
this period was the object of the thesis for my Degree in Physics. The aim of the project was to
develop a direct electrical communication between two disconnected nerve cells via a
Proposal Part B Page 13/34
The Marie Curie Actions circuit-hubs FP7-PEOPLE-2007-
2-1-IEF
semiconductor chip. During this dense experience, I learnt how to culture snail neurons, to perform
electrophysiological recordings with double intracellular electrodes, to record extracellular voltages
with planar silicon electrodes and to design and develop in the clean room a silicon chip for
extracellular recording. The result of my short but intense research brought to the development of a
chip for direct electronic communication between two disconnected nerve cells via a semiconductor
chip (a neuroelectronic prosthesis; Bonifazi P. and Fromherz P., 2002).
I next started my PhD studies in neurobiology under the supervision of prof. Vincent Torre at the
International School for Advanced Studies (March 2002 - January 2006, ISAS/SISSA, Trieste,
Italy). The major aim of the research during my PhD was to investigate how information is
processed by neuronal networks and to identify their basic and general mechanisms. Therefore as a
possible general model of neuronal networks, I performed experiments on dissociated neuronal
cultures. Experimental measurements were carried on using multielectrode arrays (MEAs) which
allow to deliver electrical stimuli to the neurons and to monitor their electrical activity. I focused
on: (1) the variability of firing in single neurons and in neuronal ensembles in response to different
stimuli; (2) neural coding mechanisms based on the firing rate and on the first spike latency of
neurons; (3) the role of excitatory and inhibitory transmission. The ability of different neural coding
schemes to convey information was measured and compared using information theory and
classification analysis. Therefore, I acquired experience for carrying on experiments on networks
and I learnt how to develop appropriate mathematical tools to characterize the dynamic of networks.
As a next step as a postdoctoral fellow in the laboratory of Dr. Hugh P.C. Robinson (February
2006 - March 2007, Department of Physiology, Development and Neuroscience, University of
Cambridge, UK), I studied synchronization and oscillations in neuronal networks. In particular,
I used my experimental experience with MEAs to measure network activity and investigate the
dynamics of gamma oscillations in acute cortical slices. During this period, I learnt to prepare acute
cortical slices from mice. Additionally I learnt and developed mathematical tools for describing the
spatiotemporal dynamic of oscillations in neuronal networks.
Therefore, the research experience proposed within the INMED will be the next logical step to
complete my cursus. This training will represent an essential step in my carrier since 1) it will allow
me to learn new experimental methodologies for studying neuronal networks (standard
electrophysiology combined to fast two-photon calcium imaging) 2) working on development in
very close collaboration to neurobiologists will be a complementary experience for my education
and 3) working on an interdisciplinary (Neurophysiology/Physics) and multidisciplinary (Two-
Photon Network Imaging/Electrophysiology/Morphology) project will definitely widen my
knowledge, research interests and scientific contacts. The team of Dr. Cossart within the INMED is
one of the only research groups in Europe gathering the above conditions, with such excellent
facilities, while offering me the possibility to also contribute, by training people in mathematical
analysis and developing data analysis software, to the life of the institution.
EDUCATION
PhD: PhD (cum laude) in Neurobiology, December 2005, SISSA/ISAS, Trieste,
Italy. Title of the thesis: “Information processing in dissociated neuronal
cultures of rat hippocampal neurons” (supervision of prof. V. Torre)
Academic Degree: Degree (Laurea) in Physics, 110/110 cum laude, May 2001, University of
Perugia, Italy. Title of the thesis: “Studies for the development of a neural
electronic prosthesis” (supervision prof. F.S. Pavone, prof. P. Fromherz)
RESEARCH EXPERIENCE
From 04/2007 : PostDoc in the group of Dr. Cossart at INMED, INSERM U29, Parc
Scientifique de Luminy, Marseille (France)
02/2006 – 03/2007: PostDoc in the lab. of Dr.Hugh P.C. Robinson in the Department of
Physiology, Development and Neuroscience, University of Cambridge
(UK).
03/2002 – 01/2006: Ph.D. student in the lab of prof. Vincent Torre in the Neurobiology Sector
of the International School for Advanced Studies (ISAS/SISSA), Trieste
(Italy).
01/2000 – 01/2001: Trainee in the lab. of prof. Fromherz at the Department of Membrane and
Neurophysics of the Max Planck Institute for Biochemistry, Martinsried
(Germany)
PUBLICATIONS
1. Mazzoni A., Broccard F., Garcia E., Bonifazi P., Ruaro M.E. and Torre V.. On the dynamics of
the spontaneous activity in neuronal networks. PLoS ONE. 2007 May 9.
2. Ban J., Bonifazi P., Pinato G., Broccard F., Studer L., Torre V. and Ruaro M.E.. ES-derived
neurons form functional networks in vitro. Stem Cells. 2006 Nov 16.
3. Bonifazi P.1, Ruaro M.E.1 and Torre V.. Statistical properties of information processing in
neuronal networks. Eur J Neurosci. 2005 Dec. 22 (11): 2953-64 (1 Paolo Bonifazi and Maria
Elisabetta Ruaro equally contributed to the paper).
4. Ruaro M.E.2, Bonifazi P.2 Torre V.. Towards the neurocomputer: Image processing and pattern
recognition with neuronal cultures. IEEE Transactions on Biomedical Engineering, vol. 52, pp.
371-383, 2005 (2 Paolo Bonifazi and Maria Elisabetta Ruaro equally contributed to the paper
but the policy of the journal did not accept co-authorship)
5. Bonifazi P., Fromherz P.. Silicon Chip for Electronic Communication between Nerve Cells by
Noninvasive Interfacing and Analog-Digital Processing. Advanced Materials 14 (2002) 1190-
1193
In Preparation:
1. Bonifazi P., Broccard F., Ruaro M.E. and Torre V.. Population coding in networks of rat
hippocampal neurons. In preparation.
2. Bonifazi P. and Robinson H.P.C.. Dynamic of gamma oscillations evoked in mouse
somatosensory cortex in vitro. In preparation.
PATENT
1. PCT/IT03/00317, date 23.05.2003, " Method and device for image processing and learning with
neuronal cultures". S.I.S.S.A. Scuola Internazionale Superiore di Studi Avanzati; Maria
Elisabetta Ruaro, Paolo Bonifazi and Vincent Torre
4. • 1st European School on Neuroengineering "Massimo Grattarola", Venice 16-20 June 2003.
Oral presentation: “Towards the neurocomputer: Image processing and learning with neuronal
cultures”, Paolo Bonifazi, Maria Elisabetta Ruaro, and Vincent Torre.
5. • Workshop on Nonlinear Signal and Image Processing, June 8-11, 2003 GRADO – Italy.
Oral presentation: “Towards the neurocomputer: Image processing and learning with neuronal
cultures”, Paolo Bonifazi, Maria Elisabetta Ruaro, and Vincent Torre.
SKILLS
Computation: Data analysis and modelling with Matlab; Computer programming in Basic,
Pascal and Fortran 77 languages; Use of Windows and DOS operating systems;
Use of Office, Origin and Labview.
Neurophyisiology: Double intracellular recording with sharp electrodes; extracellular recording
with planar electrode arrays; isolation of single neuron from snail, preparation
of hippocampal and cortical slices from rats/mice.
Foreign languages: Good knowledge of English (written and oral). Good knowledge of Spanish
(oral and written). Basic knowledge of German. I am beginning to study
French.
• Research results
Undergraduate studies. The result of the research carried on during this period brought to the
development of a chip for direct electronic communication between two disconnected nerve
cells via a semiconductor chip (a neuroelectronic prosthesis; Bonifazi P. and Fromherz P., 2002).
Graduate studies. My two major findings during this period indicate that very different networks in
terms of connectivity patterns and cell types share remarkably similar properties regarding their
spontaneous and evoked dynamics.
I studied a simplified network, at least in terms of connectivity pattern, since I used dissociated
neuronal cultures which more likely develop random connections. I focused on evoked network
dynamics. My goal was to understand how information is encoded in the neuronal firing, i.e. how
different stimuli (INPUT) are translated into an evoked train of action potentials in postsynaptic
neurons (OUTPUT). To this aim I used multi-electrode arrays (MEAs) which allow the reliable
delivery of reproducible stimuli and to record the response of a large sample of neurons. My first
major research finding is that small groups of neurons (a few dozens) composing a neuronal
ensemble are much more reliable computing elements compared to single neurons. This
extends the findings previously reported in more complex and integrated brain preparations
indicating that even a simplified network architecture such as neuronal cultures, is capable of
processing information through the activation of neuronal ensembles (Bonifazi et al., 2005;
Ruaro et al., 2005; see above patent PCT/IT03/00317). Using the methodology which I developed
to quantify the ability of neuronal cultures to process information based on information theory and
classification analysis, I contributed to demonstrate for the first time that cultures of neurons
derived from embryonic stem cells generate functional networks capable of information
processing with spontaneous and evoked dynamics very similar to hippocampal cultures (Ban
et al., 2007).
Additionally, I contributed to show that spontaneous activity in two very different networks in
terms of wiring and cell types (intact leech ganglia and dissociated cultures of rat
hippocampal neurons) share several features and have very common dynamics (Mazzoni et al.,
2007).
First postdoctoral year. During this period I have pursued my network studies in a more
physiological brain preparation since I investigated synchronisation and spatiotemporal dynamics of
gamma oscillations in acute cortical slices. For the first time, using MEAs I was able to monitor and
reliably evoke gamma oscillations. I have shown that synchrony across different areas of the
multibeam two-photon calcium imaging. This would be a complementary approach for studying
networks in-vitro compared to multi-electrode arrays, a technique that I have already worked on for
5 years. Secondly, rather than studying information processing which is a more theoretical issue (as
I did in graduate and postgraduate experiences) I will focus on neural circuits during development, a
central issue of neurobiology.
As a further proof of the potential of this fellowship for reaching a position, Dr. Cossart is
successfully concluding the supervision of Dr. Goldin (Marie Curie fellow Frame Program 6) with
excellent results, as said before. In fact, Dr. Goldin was already able to present her results in several
international conferences including FENS (2006) and Gordon Conference (2007) and publish part
of her results obtained during the fellowship’s duration in one of the most prestigious journals in the
field (Goldin et al., Journal of Neuroscience 2007). Finally, she already received serious job offers
to develop her own independent research carrier in Israel (her home country). Therefore, similarly I
will very likely be able to publish the outcomes of my research carried on during the fellowship in
prestigious journals, and using the career exploratory allowance and the funding of the host
institution I will attend workshops and present his results in international conferences. Therefore I
will be able to get international contacts both at INMED (with permanent researchers and
international visiting scientists) and at international conferences.
B4 IMPLEMENTATION
two-photon microscopes and a confocal system, including two recently acquired TrimScope
multibeam scanning systems coupled to a pulsed IR LASER (Chameleon, Cohérent) and associated
to double patch-clamp amplifiers. The host team headed by Rosa Cossart implemented both
multibeam systems and has an exclusive access to one of them. The group facilities include a multi-
beam two-photon LASER scanning system (Trimscope-LaVision Biotec, Bielefeld, Germany)
coupled to an Olympus Microscope (Hamburg, Germany). At the moment this set-up is the unique
exemplar worldwide applied in neurophysiology and was developed by Dr. Cossart in collaboration
with Trimscope-LaVision Biotec. The system is based on a patented beamsplitter that splits up the
incoming femtosecond LASER beam (provided by a Ti:Sapphire LASER source, Chameleon,
Coherent, Santa-Clara, USA), into 64 beamlets, which are scanned simultaneously (scan rate 2KHz)
in the slice. This results in 64 times higher image acquisition rates compared to conventional
multiphoton scanning microscopes. Technical assistance is provided by an optics engineer (Dr.
Michel) and a lab technician specialized in histology (I. Jorquera) which dedicate part of their time
to the host team.
The host Institute is involved in several international collaborations and many steady collaborations
have been developed by the different INMED teams. Out of them, we stress here two institutional
collaborations: 1) University of Connecticut (USA), Pr. Lo Turco on in utero disorders; 2)
Montreal Mc Gill -Douglas Hospital (Prof Remy Quirion) with an official collaboration for 5 years
being signed to foster exchanges and collaboration on human brain disorders. On her side, Dr.
Cossart has a consolidate collaboration with D. Aronov (MIT, USA), co-author and responsible for
data analysis of her pioneering papers on network dynamics monitored with calcium imaging.
Moreover, in order to have a theoretical support to her studies on networks, she has started
collaborations with theoreticians (Drs. Hentschel and Boccaletti). She has a funded collaboration
with KIST laboratory (Pr. Shin, Seoul, Corea, Egide Program) which provides genetically
engineered mice.
Therefore, in order to carry on the research described in the project, all the necessary facilities and
infrastructures are available in the host institution. Additionally, the applicant will have his own
desk and computer in a room shared with one or maximum two postgraduate researchers.
efforts to demonstrate the solidity and scientific relevance of the project whose results are
potentially of real great impact on the scientific community. Indeed, Dr. Bonifazi has a postdoctoral
contract at INMED since April 2007 which will last just till the end of the year. In only few months
he was already able to provide strong evidence (see below, “Feasibility and credibility of the
project”) that it is possible: 1) to reliably reconstruct on-line the network dynamic and identify
network hubs, 2) to strongly influence the state of the network driving the activity of s ingle
network hub. Therefore this fellowship will be necessary to carry on the all project and spread the
results through publications and conferences. Additionally to the funding available at INMED, the
fellowship will allow the candidate to have funding for participating to workshops and conferences.
These experiences will allow him to get feedback about the progressive results of the project and to
establish useful collaboration for the project and for his future. Specifically the interdisciplinary
nature of the project will encourage the candidate to develop external contacts and connections.
firing and (2) anticipate synchronous networks events. Such online analysis has to be fast enough
(~10 minutes) to allow targeted patch clamp recordings of candidate network “hubs“. Additionally,
online analysis has to be as reliable as possible in order to avoid the detection of false-positive
events which would corrupt the reconstruction of spatio-temporal activity patterns. A satisfactory
compromise between duration and quality of the analysis must be obtained. Given these constraints,
from a mathematical point of view the researcher will be required to develop new original and/or
standard strategies of signal processing and data analysis. In this way, the applicant will develop the
required mathematical tools for characterizing the network dynamics. Eventually he will take
advantage of the collaboration established between Drs. Cossart, Hentschel and Boccaletti, experts
of networks and non linear dynamical systems.
Fig. 1 summarizes the preliminary analysis developed by Dr. Bonifazi to identify hub cells. The
time course of this part of the project and the correspondent deliverable is schematized in table 1.
Milestone 1: online identification of network hub
Deliverable 1: Online reconstruction of network dynamics (OLRND), due at month 6
200
Figure 1. Online
25 identification of
150 candidate “network
20 hub”.
cell (#)
0
fluorescence traces. Red
-20 and green circles
represent respectively
-40 onset and offset of firing.
0 150 300 450 600
Time resolution:
time (s) 100ms/frame. (Top right)
For each pair of neurons the cross-correlation (CC) of the onset time series is calculated. The average CC
vs. the average time lag of maximum CC is shown for each neuron (dot). Averages were calculated over all
possible pairs. Red marked neuron is a candidate network hub. Its calcium trace is shown in bottom left
panel. (Bottom right) Two-photon calcium fluorescence image of the analyzed CA3 region from a rat
hippocampal slice loaded with a calcium indicator (Fura2-AM) at P6.
while imaging the network activity. Offline careful analysis of these recordings will be done for the
all duration of this part of the project (see below aim 3).
This series of experiments will likely narrow the class of
neurons driving network activity to specific morpho-
functional phenotypes. The most risky part of the all
project is represented by the immunohistochemical
characterization but its eventual failure won’t drastically
affect the results of the global project. The time course
of this part of the project and the correspondent
deliverable is schematized in table 1.
Milestone 2: morpho-physiological and chemical
description of “circuit-hubs”
Deliverables: Morpho-physiological description of
hubs (MPD), due at month 11 and month 17;
Immunohistochemical characterization of hubs
(IHCC), due at month 11 and month 17
Figure 2
Photomicrographs of the candidate “neuron hub” selected in fig. 1 and filled with biocytin.
Depending on the outcomes of the project, all deliverables, written at the end of the specified
periods, are potentially scientific reports, articles or communications to scientific conferences and if
they can lead to any exploitable results of interest for the industry/market, appropriate studies will
be carried on by Protisvalor to evaluate the IPR and potential patentable results that may arise.
The last two month of the fellowship will be left as spare time to summarize the results of the all
project and write a final interdisciplinary paper likely accepted in a very prestigious
multidisciplinary international journal.
• Practical and administrative arrangements and support for the hosting of the fellow
The candidate has already started a postdoctoral experience at INMED since April 2007. He has
already found an accommodation and, as an Italian citizen, he benefits from the Schengen area
legislation. Since the fellow is single he has no need at the moment to find schools or childcare
facilities. However, the Fellow will be put in contact with the Local Mobility Centre (link to the
ERA-Net Mobility Centres) which is located in Marseille to ease his stay in France. Additionally,
for any additional burocratical and administrative support, four secretaries working at INMED,
which already provide support to all intra- and extra-European students and researcher working at
INMED, will help him. For practical and administrative arrangements, the laboratory can also rely
on the back up from the European department (Head: C. Damon) at the University in terms of paper
work. Furthermore, to help his stay in France, he will receive a salary with the complete social and
welfare cover (instead of fees) and additional employee advantages (cheque restaurant, etc.). As the
applicant spent already one year in Munich (Germany) and one year in Cambridge (England), he is
used to live abroad. About the language teaching, he will have the possibility to attend the courses
which are periodically organized at the Scientific Campus of Luminy. Anyway since INMED is a
very international institute, the language for working is English.
Month 1 Month 12 Month 24
OLRND Aim 1
MP
D Aim 2
IHCC
DNA
Aim 3
H
MN
D
Table 1. Schedule of training management
Online reconstruction of network dynamics (OLRND)
Morpho-physiological description (MPD)
Immunohistochemical characterization of hubs (IHCC)
Driving network activity with circuit-hubs (DNAH)
Modeling network dynamics (MND)
Expected deliverables
B5 IMPACT
2. Rather than studying information processing which is a more theoretical issue (as he did in
graduate and postgraduate experiences), he will focus on neural circuits during development, a
central issue of neurobiology.
3. For the first time in his carrier, he will work in close contact to biologists and he will learn about
immunohistochemistry and morphology.
4. INMED offers the possibility to work in an interdisciplinary and multidisciplinary environment
(clinicians, educational centre (TousChercheurs), three biotech companies) which is a real
chance in terms of carrier’s perspectives for the fellow.
Secondly, the applicant will further strengthen his ability to bridge different scientific approaches
(multi- and inter-disciplinary aspects of the project). In fact, currently, experimental investigations
which can be carried on neuronal networks allow measuring just a limited number of parameters
and variables which are indeed required to simulate and validate network models. Therefore, thanks
to this experience the candidate will certainly widen and reinforce his ability to project new and
original experiments linking experimental neurobiology to theoretical network models.
Thirdly, concerning supervision and training capacities reinforcement:
1. He will be responsible for training students, postdocs and P.I.s to analyse data and to develop
related software.
2. In close contact to his supervisor Dr. Cossart, he will be responsible to coordinate the project
which partially requires the collaboration of other researchers (Dr. Goldin and Dr. Represa).
These both aspects will definitely help the candidate to reinforce his professional maturity and
independence.
Fourthly, since 9 different teams are present and collaborate at INMED, in addition the applicant
will have the opportunity to get in contact with all of them so that he might establish new
collaborations for his future.
Finally, this fellowship and the potential deep impact of the outcomes will strongly strengthen the
scientific CV of the applicant by publishing relevant papers and by presenting results at
international conferences where he will be able to get feedback about his research line and to
establish new contacts.
multidisciplinary approach will encourage other groups to establish such kind of collaborations.
One of the main objectives of the ERA is indeed to limit the fragmentation of research in
Europe, notably through the establishment of inert & multidisciplinary collaborations.
2. The researcher has already worked in three European Research Institutions world-widely known
for their excellence, i.e. Max-Planck (Martinsried/Munich, Germany), SISSA/ISAS (Trieste,
Italy) and University of Cambridge (Cambridge, England); supporting the mobility of the
researcher within Europe is one of the main issue of the European Research Charter and fits
perfect with the previous research experiences of the applicant and the proposed project.
3. In agreement with previous issue, this fellowship will contribute to strengthen links between
European institutions as the concept of ERA requires: the former labs visited by the applicant
will probably established long-lasting contacts with INMED via the researcher, while the
researcher will benefit from the network of INMED.
4. The research project lies at the frontier of science, since it is a as novel and original approach to
study a problem and is in opposite direction to fragmentation of science.
5. The project is in line with the European objectives for Research and Health, as described in the
FP7 Work Programme, in particular has a strong accent on multidisciplinary, on development of
new tools/equipment for research and medical research and on translation research results into
clinical application and the brain research is a central priority in Europe in the context of ageing
(population and existing burden caused by neurodegenerative diseases).
6. It is an original and cutting-edge project for the EU so it represents the chance for the EU to
support a young researcher with innovative ideas who aims to investigate experimentally a
neurobiological issues (coordination of electrical activity in the nervous system) within a strong
mathematical framework (complex systems). At our knowledge there are no other teams which
will invest such big efforts on similar inter- and multi-disciplinary projects. Therefore this
project will represent a proof of the validity of this approach, while it is pioneering compared to
the state-of-the-art and projects developed elsewhere in the world.
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B6 ETHICAL ISSUES
YES? PAGE
Informed Consent
• Does the proposal involve children?
• Does the proposal involve patients or persons
not able to give consent?
• Does the proposal involve adult healthy
volunteers?
• Does the proposal involve Human Genetic
Material?
• Does the proposal involve Human biological
samples?
• Does the proposal involve Human data
collection?
Research on Human embryo/foetus
• Does the proposal involve Human Embryos?
• Does the proposal involve Human Foetal
Tissue / Cells?
• Does the proposal involve Human Embryonic
Stem Cells?
Privacy
• Does the proposal involve processing of
genetic information or personal data (e.g.
health, sexual lifestyle, ethnicity, political
opinion, religious or philosophical conviction)
• Does the proposal involve tracking the
location or observation of people?
Research on Animals
• Does the proposal involve research on B1- research
animals?
X methodologies (page 5)
• Are those animals transgenic small laboratory
animals?
• Are those animals transgenic farm animals?
• Are those animals cloning farm animals?
• Are those animals non-human primates?
Research Involving Developing Countries
• Use of local resources (genetic, animal, plant
etc)
• Benefit to local community (capacity building
i.e. access to healthcare, education etc)
Dual Use
• Research having potential military / terrorist
application
I CONFIRM THAT NONE OF THE ABOVE ISSUES
APPLY TO MY PROPOSAL
We confirm that the issues of informed consent, data protection issues, research on human embryos,
human embryonic stem cells, use of human biological samples and involvement of third countries
are not applicable in this project. On the other hand, we will face the issue of use of animals.
We are well aware of the “3 Rs” policy of Refinement, Reduction and Replacement towards the
use of animals for scientific procedures (99/167/EC: Council Decision of 25/01/99) and will comply
with them as follows:
- As the host institution has a long standing experience with all animal procedures
necessary for this proposal, we have conducted a careful analysis of the proposed experiment to
evaluate how many animals need to be used so that the experiments will be (i) reliable and (ii) no
animal life will be unnecessarily wasted; the number of animals used will be that needed to
demonstrate a fact (we estimated about 50 rats) (Reduction).
- Dr. Cossart has extensive practice of these types of experiments (in vitro two-photon
calcium imaging) so that all aspects of the experiments will be properly designed and carried out
correctly (Reduction).
- We will keep any distress or suffering of the animal to a minimum. To do so, we will
anesthetize the mice completely with Ketamine before decapitation, brain extraction and slicing.
(Refinement).
- We will house several animals per cages respecting the number of mice per surface
(Refinement).
2) French law N° 2001-486 of June 6th 2001 being the official French publication of the European
Convention concerning the protection of vertebrates used in experiments or for other scientific
purposes (convention adopted in Strasbourg March 18th 1986 and signed by France on September
2nd 1987).
3) French legislation concerning living animal transportation : Arrêté of 19 July 2002 (fixant les
conditions sanitaires pour l’importation et le transit des animaux vivants) and Décret N° 99-
961 of 24 November 1999 (relatif à la protection des animaux en cours de transport). Mice
transport will be performed by accredited transporters.
4) Additionally, experiments will be performed under the guidelines of the French National Ethic
Comity for Sciences and Health report on “Ethic Principles for Animal Experimentations”.
This compliance with French, European international legislations is under the responsibility of host
institution and applicant which already have the following agreements to perform animal
experimentation: “arrete prefectoral portant agrement d’un etlabissement d’experimentation
animale” (A 13 055 19, see in Annex).
Additionally, use of animals for experiments will be carried on under the supervision of Dr. A.
Represa (authorization n. 13.125, April 17th 2002, released by “Direction Departemental des
Service Veterinaire”, Prefecture de Bouches du Rhone)
ENDPAGE
PEOPLE
MARIE CURIE ACTIONS
PART B
“Circuit-hubs”