Circuit Hubs

The Marie Curie Actions circuit-hubs FP7-PEOPLE-2007-2-1-IEF

Proposal Part B /34

STARTPAGE

PEOPLE

MARIE CURIE ACTIONS

Intra-European Fellowships (IEF)

Call: FP7-PEOPLE-2007-2-1-IEF

PART B

“Circuit-hubs”


Proposal Part B /34

Table of Contents

B1 SCIENTIFIC AND TECHNOLOGICAL QUALITY ...................................................................................3

• Scientific and technological Quality ...........................................................................................3

• Research methodology................................................................................................................5

• Originality/innovative nature of the project, & relationship to the appropriate 'state of the art' 7

• Timeliness and relevance of the project......................................................................................8

• Host scientific expertise in the field............................................................................................9

• Quality of the group/scientists in charge...................................................................................10

B2 TRAINING ...................................................................................................................................11

• Clarity and quality of the research training objectives for the researcher ................................11

• Relevance and quality of additional scientific training/complementary skills offered.............12

• Host expertise in training experienced researchers in the field and capacity to provide

mentoring/tutoring .........................................................................................................................13

B3 RESEARCHER .............................................................................................................................13

• Research experience..................................................................................................................13

• Research results.........................................................................................................................16

• Independent thinking and leadership qualities..........................................................................17

• Match between the fellow's profile and project ........................................................................17

• Potential for reaching a position of professional maturity........................................................17

• Potential to acquire new knowledge .........................................................................................18

B4 IMPLEMENTATION ......................................................................................................................18

• Quality of infrastructures/facilities and international collaborations of host............................18

• Practical arrangements for the implementation and management of the project ......................19

• Feasibility and credibility of the project, including work plan .................................................20

• Practical and administrative arrangements and support for the hosting of the fellow..............23

B5 IMPACT ........................................................................................................................................23

• Potential of acquiring competencies .........................................................................................23

• Contribution to career development..........................................................................................24

• Contribution to European excellence and European competitiveness ......................................24

REFERENCES .......................................................................................................................................25

B6 ETHICAL ISSUES ............................................................................................................................28

ANNEX : AUTORIZATION FOR ANIMAL EXPERIMENTATION ..................................................................31


Proposal Part B /34

B1 SCIENTIFIC AND TECHNOLOGICAL QUALITY

• Scientific and technological Quality State of the art - Brain function depends on the coordinated and synchronized activity of large

populations of neurons. For example environment exploration or sleep are associated to rhythmic

populations discharges (Destexhe et al. 2003) while pathological disorders including the epilepsies

involve excessive synchrony between neurons. Remarkably, oscillatory synchronous activities are

the signature of the developing brain since it has been observed in a wide range of peripheral (Galli

and Maffei, 1988; Meister et al., 1991; O'Donovan, 1989; Syed et al., 2004) and central structures

(Ben Ari, 2001; Ben Ari et al., 1989; Ben Ari, 2002; Garaschuk et al., 2000; Kandler and Katz,

1998; Owens and Kriegstein, 1998; Palva et al., 2000; Yuste et al., 1992). Moreover, most of the

activity in developing cortical structures is concentrated in these rhythmic population bursts (Ben

Ari, 2002; Dupont et al., 2005; Garaschuk et al., 2000; Kandler and Katz, 1998; Katz and Shatz,

1996; Peinado, 2000; Weissman et al., 2004; Yuste et al., 1992). Since such coordinated activities

in large population of neurons play a major role in the construction of neuronal circuits, it is

essential to understand the mechanisms controlling the onset of synchronisation in immature

networks.

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 are “super-connected” cells that provide developing networks with fast transmission of information. This hypothesis is largely supported by theoretical models

and experimental observations. Models of complex systems and networks, like scale-free networks,

small-world networks and the Self-Organized Criticality (SOC) paradigm (Almaas, 2007; Song et

al., 2005, Barabasi Albert 1999; Jensen, 1998; Vespignani and Zapperi, 1998) are based on the

existence of “circuit-hubs”, i.e. units with spread and/or much larger number of connections, which

strongly influence the state of the network. Many real-world networks present scale-free properties

(Almaas, 2007; Song et al., 2005). Interestingly, such networks were shown to grow following a

“preferential attachment" rule: units with a higher degree of connectivity receive more new links

than less connected units. This theoretical model is very appealing for living neuronal networks in

development. Moreover, neuronal activity in living neuronal networks was described in scale free

framework in different experimental preparations (in vivo, in cultures, slices) and species (human,

invertebrates and rodents) (Beggs and Plenz, 2003; Beggs and Plenz, 2004; Linkenkaer et al., 2001;

Freeman, 2004; Bedard et al., 2006; Mazzoni et al., 2007).

Technological quality - We propose to describe connectivity patterns in developing cortical

networks using a multidisciplinary (Neurophysiology/Physics) and interdisciplinary approach

(Two-Photon Network Imaging/Electrophysiology/Morphology). Our research proposal is

multidisciplinary since it aims at characterizing developing neuronal networks based on a

hypothesis derived from theoretical models describing complex dynamical systems (originally

developed in Physics). Our experimental approach is interdisciplinary since it combines multibeam

two-photon calcium imaging of network dynamics to online computational analysis and targeted

electrophysiological recordings, a unique method recently developed by the host team. The

technological quality of the approach is testified by the recent publications of the host team in high

profile journals (Cossart et al., 2005, Crepel et al., 2007, Tyzio et al., 2006, Goldin et al. 2007, see

below). The feasibility of this project is based 1) on the peculiar education of Dr. Bonifazi who has

a strong mathematical background (Degree in Physics) complemented by a robust research

experience on living neuronal networks (PhD in Neurobiology); 2) on the supervision of Dr.

Cossart expert of imaging/electrophysiology; 3) on the additional support for morphological studies

provided in the host institution by Dr. Represa and 4) the experience/leading role of the host

institution in neurosciences.

Research objectives - A solid scientific framework- Since synchronous coordinated activities

play a major role in the construction of neuronal circuits, it is essential to understand the


Proposal Part B /34

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 multidisciplinary 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.


Proposal Part B /34

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


Proposal Part B /34

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.

Aim #1: description of the functional topography of hippocampal circuits. The methodological approach will consist in designing the appropriate software tools to predict

online the connectivity patterns of a network of hundreds of cells in a living brain slice from the

analysis of their individual calcium dynamics obtained with fast two-photon microscopy.

Data analysis - In order to accomplish this aim, the applicant based on the previous work carried by

D. Aronov, will develop first new software for analysis in order to perform a quick online detection

of calcium spikes during network oscillations. This preliminary analysis will allow to reconstruct

online the temporal dynamics of the network and to identify “hub cells” that (1) show high

correlation with network 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 based on

cross-correlation, principal component analysis, graph partitioning algorithms or other multivariate

data methods. The ultimate goal is to be able to localize during an experiment neurons driving

population activity with any a priori assumption (hubs). Also, Dr. Bonifazi will profit from several

starting collaborations with physicists and mathematicians (Drs. Hentschel and Boccaletti).

Aim#2: morpho-physiological and chemical description of “circuit-hubs”. Electrophysiological recordings - The applicant will carry on experiments to identify and perform

patch-clamp recordings of candidate cell-hubs during spontaneous GDPs. Cells will be recorded

both in cell-attached and whole cell configurations (current-clamp mode) in order to measure their

spontaneous firing properties, active membrane properties and synaptic influx. The activity within

this cells will also be stimulated (membrane potential steps and holding voltage changes) and

compared to the state of the network. The only disadvantage of this approach is that patch recording

is rather invasive since it can affect the natural membrane potential state of the cell. However it is

the only method that enables reliable morpho-physiological characterization of specific neurons.

The candidate will be initially assisted in imaging and electrophysiology by Drs. Cossart and

Goldin in order to let him progressively acquire independent expertise.

Morphological characterization - Patched cells will be filled with biocytin in order to reconstruct

their morphology. Immunohistochemical characterization of the imaged cells will be performed in

collaboration with the group of Dr. A. Represa (group leader at INMED). We have successfully

performed immunohistological processing of imaged and patch-clamp recorded slices using

calretinin antibodies. This series of experiments is likely to narrow the class of neurons driving

network activity to specific morpho-functional phenotypes.


Proposal Part B /34

Aim # 3: function of “circuit hubs” and simulation of network dynamics Previous network studies suggested that in certain conditions a single cell is capable of driving an

oscillation (Cobb et al., 1995, De la Prida et al., 2006, Brecht et al. 2004). Whether the activity of a

single cell can have deep effect on the network state is definitely a central issue in neuronal network

studies. We hypothesize that online detected “circuit-hubs” are capable of influencing network

dynamics i.e. the spatial and temporal structure of coactive cells within a GDP.

Therefore, first we will study network oscillations during hub stimulation. This hypothesis will be

tested reconstructing and characterizing the network activity and GDPs occurrence while different

activity patterns were evoked in “circuit-hubs” in current clamp recordings or while different

membrane potentials were imposed in voltage clamp mode.

Secondly, we will develop a model of the network using the gathered experimental parameters. The

model will be essentially based (1) on the morphology of the cell-hubs and their capability to

establish long range connections, (2) on the experimental reconstruction of network dynamics with

single cell resolution. All the information provided by calcium signals about different firing

properties, pair-wise correlations in relation to spatial distance will be included in the model.

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


Proposal Part B /34

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.

• Timeliness and relevance of the project

Timeliness - The project essentially requires the possibility to monitor the network dynamics with

single-cell resolution and is based on the assumption that neuronal networks are organized as scale-

free or small-world networks. Recently, the experimental strategy and the theoretical approach have

been demonstrated to be appropriate strong tools to describe neuronal circuits.

In particular, a dual experimental strategy is required to study the activity of large cell populations,

which combines calcium imaging to reconstruct the overall network activity in hippocampal slices,

and patch clamp recordings from individual neurons followed by morphological identification post-

hoc. This pioneering approach has been recently designed by Rosa Cossart during her postdoctoral

work in Prof. Yuste’s laboratory and produced already very interesting results (Goldin et al. 2007;

Crepel et al, 2007).

Additionally just in the last few years it has been shown that (1) many, perhaps most, real-world

networks studied so-far present a scale-free or small-world topology and (2) that spontaneous

neuronal activity shows a power-law distribution, a signature of scale-free organization.

Therefore, the project lies at the edge of the last experimental and theoretical studies and is based on

the considerable progress made by imaging techniques. It may pave the way for a new

interdisciplinary approach for the study of the nervous system.

Finally, as we will develop later (B5), brain research in the context of our ageing societies is at

stake on the Health policy agenda.

Relevance and impact for the EU - Coordinated and synchronized activity in large population of

neurons play a major role in the construction of neuronal circuits. Inappropriate driving of this

activity might bring to severe diseases (e.g. epilepsy) and it is essential to understand its dynamic.


Proposal Part B /34

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).

• Host scientific expertise in the field The host Institute, “Institut de Neurobiologie de la Méditerranée” (INMED, Luminy, Marseille),

belongs to the Université de la Méditerranée. INMED is directed by Prof. Y. BEN-ARI and

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, which gathers 35

researchers, is now an appreciated centre of training as reflected by the growing number of

candidates including from the US and European countries and the venue for long stays of highly

recognized researchers (Drs Mc Naughton, Segal, Holmes and Baram). Additionally, every year

INMED organizes the INMED-TINS conference which attract the best scientists in the field from

all over the world and it is an exceptional opportunity for researchers to exchange their ideas.

Competences and techniques available include patch clamp recordings in vivo and in vitro,

dynamic two-photon microscopy in vitro, mathematical analysis of oscillations, in utero

transfection of siRNA, morphology and molecular biology. Several preparations have been

developed in INMED: the interconnected intact hippocampi or cortices, the basal ganglia slice,

human cortical slices removed surgically from epileptic brains, fast dynamic imaging of immature

oscillations and in vivo patch recordings from local superfused neocortex or hippocampus. All

INMED teams share these common facilities, equipments and funding.

PhD students - their number is steadily increasing although we are limiting them to 1 per

permanent or established researcher. A strong effort is made to include MDs (residents for PHDs) to

foster collaborations with hospitals.

School for residents - Hippocrate. This school is now operating for 2 years with increasing

success. It enables the venue of 25 residents for 2 weeks to INMED

National Medical projects - INMED has an extended collaboration with several centers most

importantly: in Marseille with La Timone Hospital for the identification of mutations involved in

mental retardation, and early onset epilepsies and migration disorders and Paris (Necker and

foundation Rotschild for the use of human slices).

International collaborations – There are many steady collaborations developed by the different

INMED teams and between them: Buzsaki G. (Rutgers University, USA), Holmes G.L. (Harvard-

Dartmouth, USA), Kaminska A. (St Vincent Paul, Paris, France), Garcia L. (Universite de

Bordeaux 2) and D’Alessandro G. (Southampton, UK). Additionally, we stress two institutional

collaborations: 1) Jo Lo Turco (University of Connecticut, USA) on in utero disorders and 2) Prof

Remy Quirion (Mc Gill -Douglas Hospital, Montreal, Canada) with an official collaboration for 5

years being signed to foster exchanges and collaboration on human brain disorders


Proposal Part B /34

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)

• Quality of the group/scientists in charge Group composition - The group involved in this project is composed of two postdoctoral fellows

(Dr. Goldin and Dr. Bonifazi), a graduate student (C. Allène) and a young female principal

investigator (Dr. Cossart). The main research interest of the team is to study the dynamics of

network activities at the scale of functional microcircuits.

Expertise - The group focuses on developmental aspects with a combined imaging and

electrophysiological approach. Therefore the group has extensive experience in electrophysiology

and calcium imaging and will provide the applicant with a specified training in patch-clamp

recordings and two-photon imaging systems. The pioneering approach to study network dynamics

based on two-photon imaging and patch-clamp recordings has been designed by Rosa Cossart. A

new imaging system has been successfully implemented in the group based on multibeam two-

photon microscopy. It enables the significant increase of temporal resolution. Rosa Cossart has also

a strong experience in the study of interneurones in the hippocampus and cortex. Besides, the group

benefits from several collaborations within the INMED (Drs. Represa and Khazipov) and outside

(Dr. Boccaletti, Hentschel, Aronov).

Supervision quality – Dr. Rosa Cossart is a very young researcher with an exceptional CV who is

often invited to present the last results of her research at most prestigious conferences (i.e.

Physiological Society Meeting. UCL, London, July 2006; Gordon Research Conferences,

“inhibition in the CNS”, July 2007). In addition, she has received two awards as young scientist

(2006 ANR Jeune Chercheuse; 2005 Médaille de Bronze du CNRS). Although at an early stage of

her career as a team leader (since June 2003), Rosa Cossart has successfully trained 4 students with

a biology or mathematics background (mathematics: L. Sasportas (ECP); biology: D. Dumitriu, R.

Ammari, C. Allène) and a postdoctoral fellow (Dr. Goldin). Her supervision was positively

evaluated since R. Ammari and C. Allène obtained funding for their PHDs and since Dr. Goldin

published her work in a high profile journal (Goldin et al. 2007). She is currently supervising the

graduate work of C. Allène. She also has important responsibilities in INMED since she is in charge

of the recruitment of new researchers as well as is a member of the Scientific Committee that

decides for the orientations of research. She has thus demonstrated her capacity for such supervision

and is very much implicated in the host institution.


Proposal Part B /34

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). Most relevant publications of the group:

• Goldin M, Epsztein J, Represa A, Crépel V, Ben-Ari Y, Cossart R. Specific processing of afferent theta

inputs via synaptic kainite receptors in O-LM neurons. Journal Neuroscience, in press

• Crepel V, Aronov D, Jorquera I, Represa A, Ben-Ari Y, Cossart R. A parturition-associated

nonsynaptic coherent activity pattern in the developing hippocampus. Neuron. 2007 Apr 5;54(1):105-20.

• Tyzio R, Cossart R, Khalilov I, Minlebaev M, Hubner CA, Represa A, Ben-Ari Y, Khazipov R.

Maternal oxytocin triggers a transient inhibitory switch in GABA signalling in the fetal brain during

delivery. Science. 2006 Dec 15;314(5806):1788-92.

• Cossart R, Petanjek Z, Dumitriu D, Hirsch JC, Ben-Ari Y, Esclapez M, Bernard C. Interneurons

targeting similar layers receive synaptic inputs with similar kinetics. Hippocampus. 2006;16(4):408-20.

• Cossart R, Ikegaya Y, Yuste R. Calcium imaging of cortical networks dynamics. Cell Calcium. 2005

May;37(5):451-7. Review.

• Cossart R, Bernard C, Ben-Ari Y. Multiple facets of GABAergic neurons and synapses: multiple fates

of GABA signalling in epilepsies. Trends Neurosci. 2005 Feb;28(2):108-15. Review.

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


Proposal Part B /34

provided by my studies in Physics, my interests in neurobiology have always been focused on

complex theoretical problems, like those involving information processing. During my PhD I

worked on a simplified model of network, dissociated neuronal cultures which possibly develop

random connections and I have focused my attention on information processing. As a next step in

my studies, during my first year of postDoc, I decided to investigate information processing in

neuronal networks with well-defined architecture, i.e. acute cortical slices. I carried on these studies

using Multi-Electrode Arrays which allow to monitor the electrical activity of the network with

optimal temporal resolution and to clearly identify the electrical activity of few dozens of units.

As I clearly realised that a complementary approach to the study of networks requires the possibility

to monitor the activity of the circuits with single cell resolution in order to have potential access to

the morphological properties of the cells. Hence my strong will to terminate my postdoc education

with this project

Importance of the training - This experience will represent a test of validation for an

interdisciplinary (Neurophysiology/Physics) and multidisciplinary approach (Two-Photon Network

Imaging/Electrophysiology/Morphology) to investigate fundamental neurobiological issues.

Dealing with heterogeneous disciplines and methodologies during the project (e.g.,

immunohistochemistry and morphology) will be certainly important in terms of source of

inspiration for my future research projects.

Therefore I am convinced that working in the team of Dr. Cossart, who pioneered this approach

combining fast multi-beam two-photon calcium imaging and patch-clamping, is an essential step in

my carrier. In the proposed research project I focus on a fundamental question regarding

developmental neurobiology. For the first time in my research career I will be working in a

laboratory focusing more on physiological questions of neurobiology (development of neural

circuits) where the large majority of the researchers have biological background. This is a missing

essential step of my scientific training. I will also be trained as an electrophysiologist and as an

imaging expert. During the first period of the project, I will work in close collaboration with Dr.

Goldin, a postdoctoral fellow that received a successful strong training in both aspects in the host

group.

• Relevance and quality of additional scientific training/complementary skills offered

Besides the interdisciplinary and multidisciplinary nature of the project, I will also acquire the

following complementary skills.

Supervision - In INMED I am the only researcher with a strong mathematical background.

Therefore, since three teams are using imaging tools to reconstruct network dynamics, I will be

responsible for training people in mathematical analysis and developing data analysis software. In

addition, as already happened in the last few months, groups working on different subjects could

require directly my collaboration for mathematical analysis or modelling. This will enrich my

scientifical experiences, my ability to supervise people and my future possible collaborations.

Publications - I will be first author of the publications issued from this project. I will therefore

design figures and write first drafts of the papers. Also, given the inter/multi-disciplinary approach

it will be definitely possible to write several publications focusing on the methodology, on

intermediate/final outcomes and on the theoretical models dealing with network connectivity

underlying the all project. I will be personally in charge for writing these publications and will

profit from the financial support offered by this fellowship to present my results in international

events on that subject.

Building a network of young investigators - Thus, in order to be able to establish new contacts

and new collaborations with experts of the different fields involved in the project, using the

possibility offered by the fellowship I will present the results of the research at international

conferences (SFN, FENS, IBRO). Travel funding is essential since I will need to discuss my results

with experts outside the INMED. My goal is to establish a network gathering young experts related

to the inter/multi-disciplinary nature of the project. Within this context, in INMED it is possible to


Proposal Part B /34

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 /34

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.

PAOLO BONIFAZI - Curriculum Vitae

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)


Proposal Part B /34

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)

Background: General Physics, Biophysics, Mathematics, Electronics, Computational

neuroscience, Neuroengineering, Networks and Complex systems.

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

SCHOOLS AND CONFERENCES 1. • School and Workshop on Structure and Function of Complex Networks. ICTP from 16 - 28

May 2005, Trieste.

2. • 2nd European School on Neuroengineering "Massimo Grattarola", Genova 9-12 June 2004.

Oral presentation: “Rate and temporal coding in neuronal cultures from hippocampal rat

neurons”, Paolo Bonifazi, Maria Elisabetta Ruaro, and Vincent Torre.

3. • Young Neuroscientist Meeting, 24th may 2004, Trieste. Oral presentation: “Parallel

processing in natural neuronal networks: technological issues and neurobiological questions“,

Paolo Bonifazi, Maria Elisabetta Ruaro, Vincent Torre.


Proposal Part B /34

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


Proposal Part B /34

cortex is modified as a consequence of repetitive stimulation (Bonifazi P. and Robinson H.P.C.,

in preparation).

• Independent thinking and leadership qualities

Independent thinking- My challenge to cover an exploratory and interdisciplinary educational

path, unusual compared to classic academic studies, somehow by itself testifies my independent

thinking. In fact, starting from my theoretical background, I tried to become more and more familiar

with experimental tools for neuronal networks studies, to acquire more and more knowledge in

neurobiology and to understand the most relevant issues, regardless of the difficulties that breaching

scientific segmentation can lead to. My independent thinking has therefore certainly determined my

peculiar and hybrid education which I consider innovative, original and appropriate for linking experimental and theoretical emerging strategies in neurobiology. My postdoctoral

training in INMED represents a logical step in this context since it will allow me to acquire new

knowledge, not only in developmental neurobiology, but also in basic physiology and brain

diseased in one of the reference centre in Europe. Finally, the mode of organization of the INMED

favours exchanges and interactions between groups (with weekly data club meetings, journal clubs),

with clinicians and biotech companies. These interactions will strongly enrich my biological culture

as well as opening new perspectives for intersectorial contacts.

Leadership- Concerning supervision and training of people, three independent teams of the

INMED are using imaging tools to reconstruct network dynamics and since I joined the INMED

(April 2007), I was named responsible for training students, postdocs and P.I.s to data analysis and

for developing user-friendly software. I have organized weekly meetings where people studying

network dynamics in the Institute share their difficulties and present novel approaches. Certainly

this ‘leading’ role for training people will help me to strengthen my capability to supervise other

researchers and to finally obtain my independent research position.

Future development-This fellowship and the global experience acquired during its duration will

help me to proceed on my next logic step, i.e. trying to start my own independent research position

for living neuronal networks studies possibly in a European-Mediterranean country. I will greatly

benefit from all the collaborations established by the Institute and the host team. Additionally, in

order to be able to establish new contacts and new collaborations with experts of the different fields

involved in the project, using the specific funding of the fellowship I will present the results of the

research at international conferences. Travel funding is essential since I will need to discuss my

results with experts outside the INMED. My goal is to establish a network gathering young experts

related to the inter/multi-disciplinary nature of the project. This will definitely be useful to develop

my own independent carrier.

• Match between the fellow's profile and project The project is definitely interdisciplinary and multidisciplinary since it involves electrophysiology,

optical imaging, morphology, immunochemistry, online analysis and reconstruction of network

dynamics. My previous experience on multi-electrode recording, my original research on neuronal

networks and my strong mathematical background provide the appropriate expertise for driving the

project. Additionally, at the present moment, although three independent teams of the INMED are

using imaging tools to reconstruct network dynamics, there is not any researcher with deep interest

and proofed experience in the study of network dynamics.

Therefore there is no doubt that my skills are very appropriate for the project and the host institution

will greatly benefit from them since I have the appropriate education and experience to link

experimental neurobiology to theoretical models underlying network dynamics.

• Potential for reaching a position of professional maturity This fellowship would represent a fundamental step in my carrier since it would allow me to add an

essential complementary experience in neurosciences, completing my cursus on that regard. First, I

will learn a new and powerful experimental technique for studying neuronal networks, e.g. fast


Proposal Part B /34

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.

• Potential to acquire new knowledge The cursus of my education demonstrate that I could easily 1) work in different teams (Dr. H.P.C.

Robinson, Prof. V. Torre, Prof Fromherz) with successful results, 2) merge different methodologies

and disciplines (Physics, Neurobiology), 3) move and live in different countries (Germany,

England, France). Therefore I proved that I am highly capable of adaptability and I am very

motivated for mobility. Indeed, during my undergraduate studies I spent one year working on

interface between neurons and electronics. At that time I had no experience and knowledge in

neurobiology and I had to learn to culture neurons and to perform intracellular recordings. Next, I

decided to strengthen this peculiar personalised cursus in moving from my undergraduate studies

(Degree in Physics) to Neurobiology (PhD) to develop my own path in research. At the moment my

actual experience as a postdoctoral fellow at INMED studying networks during development

represents a further complementary competence in my educational, oriented towards

neurobiological issues. Certainly working on an interdisciplinary (Neurophysiology/Physics) and

multidisciplinary (Two-Photon Network Imaging/Electrophysiology/Morphology) project will

allow me to publish several papers dealing with not only new methodologies but also with the

development of cortical networks. Additionally, since our potential results will concern a broad

audience of scientists, the outcomes of our research might be published in non-specialized top

scientific journals.

B4 IMPLEMENTATION

• Quality of infrastructures/facilities and international collaborations of host The host team is part of a research Institute called “Institut de Neurobiologie de la Mediterranee”

(INMED, INSERM U29, Université de Luminy, Marseille), that is directed by Y. Ben-Ari and

located in Marseille on the campus of Luminy (France). It is a large institute with over 5000 square

meters that gathers 35 researchers and 7 technicians. INMED has long-lasting interest in the

functional maturation of the developing brain, as well as cellular and molecular mechanisms

involved in pathological conditions such as ischemia and epilepsy. INMED is divided in 9

identified teams that continuously interact with each other as testified by the number of

collaborative projects and publications (see for example Tyzio et. Al. Science 2006). Regarding

infrastructures, the Institute has exceptional facilities which include 12 set ups for in vitro patch-

clamp recordings (cultures, slices and intact brain structures), microscopes, molecular and cellular

biological tools, surgery and culture rooms. For imaging experiments, the institute possesses three


Proposal Part B /34

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.

• Practical arrangements for the implementation and management of the project

Supervision - Additionally to day by day interaction between supervisor and candidate, Dr.

Bonifazi will periodically summarize and update the results of his research 1) to Dr. Cossart’s team

every about 3 weeks (group data club) and 2) to all INMED researchers every about 3 months

(institute data club). Therefore he will receive strong and continuous feedbacks during the all

duration of the fellowship. If external collaborations will be required, in addition to the funding of

the fellowship, Dr. Cossart and INMED will support the cost of related travels.

Concerning the project, Dr. Cossart’s group has extensive experience in electrophysiology and

calcium imaging and will provide the applicant with a specified training in patch-clamp recordings

and two-photon imaging systems. Furthermore, Dr. Cossart has a double competence since she did

her undergraduate studies in Physics and Mathematics. A new imaging system has been

successfully implemented in the group based on multibeam two-photon microscopy. Rosa Cossart

has also a strong experience in the study of interneurones in the hippocampus and cortex.

Additionally to Dr. Cossart, Dr. Represa will work in close collaboration with the applicant

concerning morphological and immunohistochemical studies. Besides, the group benefits from

several collaborations within the INMED (Dr. Khazipov) and with physicist and mathematicians

outside (Dr. Boccaletti, CNR, Sesto Fiorentino, Italy; Dr. Hentschel, Emory University, Atlanta,

USA).

Financial Support - Thanks to this fellowship the applicant will be able to have all the necessary

support for carrying on properly this ambitious interdisciplinary project and to achieve it (without

this funding, the applicant won’t be able to conduct the on-going project until the end). This

fellowship represents the necessary economical support for Dr. Bonifazi (applicant) to devote all his


Proposal Part B /34

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.

• Feasibility and credibility of the project, including work plan Integration in the host team and institution- Paolo Bonifazi will develop his interdisciplinary

project in close collaboration to Dr. Cossart (supervisor; two-photon imaging, electrophysiology

and data analysis), Dr. Goldin (postDoc in Cossart’s team; two-photon imaging and

electrophysiology) and Dr. Represa (team leader; morphology and immunohistochemistry). The

team has extensive experience in electrophysiology and calcium imaging and will provide training

in patch-clamp recordings and multibeam two-photon microscopy. All experimental set-ups and

facilities needed are available at INMED and no particular purchase of equipment that could delay

the actual start of the experimental work will be necessary.

IP issues - Concerning the intellectual property that may arise from the research project, all the

necessary steps will be taken in order to protect, valorize, transfer/license, any exploitable results

arising from this project, in accordance with the rules of the University on that matter (UnivMed IP

Charter) and with the help of the University valorization affiliate, Protisvalor.

Workplan - To identify neurons driving network oscillations, one needs 1) to monitor network

activity with single-cell resolution 2) perform quick and accurate on-line reconstruction of network

dynamics 3) patch neurons potentially playing the role of “network-hubs” 4) perform post-hoc

morphological identification. This pioneering approach has been designed by Rosa Cossart and

implemented in the last years at INMED (Crepel et al., 2007; Goldin et al., 2007). The publications

of the host team testify the validity and feasibility of this research strategy. Software for analysis of

calcium signals and network dynamics have already been developed in collaboration with Dirty

Aronov (Aronov, 2003; Aronov et al., 2003; Aronov and Vicor, 2004; Crepel et al., 2007; Cossart et

al., 2003; Goldberg et al., 2003) and will represent the starting point for Dr. Bonifazi to develop

more advanced mathematical tools to characterize the network activity. Also, Dr. Bonifazi will

profit from several starting collaborations with theoreticians (Drs. Hentschel and Boccaletti).

Concerning the time schedule of the project, as specified in B1 (research methodology) the overall

project has three main aims or work-packages: 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. The detailed work plan and the

feasibility of these aims, supported by preliminary results, is described below and summarized in

table 1. Each aim has an associated final milestone. For each aim, the candidate will write several

deliverables which will summarize periodically the progress of the research and allowing the

overall follow-up and supervision of this project.

Aim 1: description of the functional topography of hippocampal circuits (Months 0-6) During this period Paolo Bonifazi, based on the previous work carried by D. Aronov, will develop

new software for analysis in order to perform a quick online detection of calcium spikes during

network oscillations. This preliminary online analysis will allow to reconstruct the temporal

dynamics of the network and to identify “hub cells” that (1) show high correlation with network


Proposal Part B /34

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

Figure 1. Online

identification of

candidate “network

hub”. (Top left) Raster plot of

network activity. For

each neuron, dots

represent the onset of

firing based on calcium

spikes (see red circles in

bottom left panel).

(middle left) Coordinated

network events (GDPs)

revealed by synchronous

onset of firing in a large

fraction of imaged cells.

(bottom left)

Automatically detected

calcium spikes in the

fluorescence traces. Red

and green circles

represent respectively

onset and offset of firing.

Time resolution:

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.

Aim 2: Morpho-physiological and chemical description of “circuit-hubs” (Months 6 - 17) Once aim 1 will be accomplished, all the experiments to identify and patch candidate cell-hubs

during spontaneous GDPs will be carried on. While being completely responsible for the online

analysis, the applicant will be assisted in imaging and electrophysiology by Drs. Cossart and Goldin

in order to let him progressively acquire complementary expertise. Patched cells will be filled with

biocytin in order to reconstruct and study their morphology (fig. 2). Immunohistochemical

characterization of the cells will be performed in collaboration with the group of Dr. A. Represa

(group leader at INMED). Cell-hub evoked and spontaneous firing properties will also be measured

0

50

100

150

200

0

5

10

15

20

0 150 300 450 600-40

-20

0

20

-4 -2 0 2 40

5

10

15

20

25

cro

ss c

orr

ela

tion

time lag (# frames)

time (s)

0 150 300 450 600

0 150 300 450 600

∆F/F

(%

)onse

t (%

)ce

ll (#

)

0

50

100

150

200


Proposal Part B /34

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.

Aim 3: function of “circuit hubs” and simulation of network dynamics The overall aim of this part of the project is to understand how circuit-hubs drive the network

activity. Therefore, while all the experiments will be carried on, a detailed analysis of the network

dynamic during hub stimulation will be performed. This analysis should first reveal how different

“protocols of stimulation” of the hub affect the network activity (e.g. depolarization,

hyperpolarization, periodic firing, etc.) in order to provide continuous feedback for the experiments.

Secondly, the analysis carried on during this period will add more and more information about the

network dynamics. Therefore, this analysis will represent the base to develop a model of the

network using all the gathered experimental parameters. The model, capable of describing how

network hubs drive the network activity, will be essentially based (1) on the morphology of the cell-

hubs and their capability to establish long range connections, (2) on the experimental reconstruction

of network dynamics with single cell resolution. All the information provided by calcium signals

about different firing properties, pair-wise correlations in relation to spatial distance will be

included in the model. The time course of this part of the project and the correspondent deliverable

is schematized in table 1.

Milestone 3: how circuit hubs

drive the network activity

Deliverables: Driving network

activity with circuit-hubs

(DNAH), due at month 11 and

17; Modeling network dynamics

(MND), due at month 22. Figure 3.

Increasing the firing rate of a “hub

neuron” reduces the occurrence of

synchronous network events. The percentage of active cells in the

network (black line) evidence peak of

synchronous activity (GDPs, marked with asterisk). The occurrence of GDP is reduced when the neuron

(blue line) is depolarized by current injection (horizontal thick black line) and its firing rate increases.

0 150 300 450 6000

10

20

30

40

50

60

70

time (s)

ac

tiv

e c

ells

(%

)

40 mV


Proposal Part B /34

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

OLRND

MP

D IHCC

DNA

H MN

D

Month 1 Month 12 Month 24

Aim 1

Aim 2

Aim 3

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.

B5 IMPACT

• Potential of acquiring competencies This project will first allow the researcher to strongly complement and complete his education:

1. He will learn a new and powerful experimental technique for studying neuronal networks, e.g.

fast multibeam two-photon calcium imaging. This would be a complementary approach for

studying networks in-vitro compared to multi-electrode arrays, a technique that he has already

worked on for 5 years.


Proposal Part B /34

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.

• Contribution to career development As previously said, this fellowship and the global experience acquired during its duration will help

the candidate to proceed on his next logic step, i.e. trying to start his own independent research

position on living neuronal networks studies possibly in a European-Mediterranean country.

Considering the peculiar and hybrid education of the applicant, this fellowship would represent a

unique opportunity for him. In fact the experience in INMED will represent a logical step in his

carrier allowing him to acquire new knowledge, not only in developmental neurobiology, but also in

basic physiology and brain diseases.

He will be responsible to coordinate an interdisciplinary project involving disciplines and technique

with whom he will have to become familiar.

Publishing results of this wide project will introduce his name to scientist working on different

fields with logical positive consequences for his future carrier.

Therefore, the opportunity given by this fellowship will possibly represent the last step for him

before being able to develop his own novel/original research team capable of carrying on

original investigations on living neuronal networks balanced by mathematical,

neurobiological and experimental knowledge.

• Contribution to European excellence and European competitiveness

Several reasons support the idea that the methodological approach is and of great value for the

European Community and will strengthen the European Research Area (ERA).

1. It is a real multidisciplinary project which aims to bridge the gap between experimental life-

sciences to theoretical-mathematical sciences; certainly the validation of our proposed


Proposal Part B /34

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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Proposal Part B /34

B6 ETHICAL ISSUES

ETHICAL ISSUES TABLE

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 animals?

X B1- research 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


Proposal Part B /34

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.

Details on the Use of animals in ‘Circuit-Hubs’ project Experiments will be performed on slices of developing rat hippocampus (P3-P5). There is no

alternative to the use of the rats to perform our proposed research and we estimated that about 50

rats will be enough for experiments for this two-year project.

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).

We also confirm that we will follow all relevant EU legislations, in particular:

- Directive 86/609/EEC on the approximation of laws, regulations and administrative provisions of

the Member States regarding the protection of animals used for experimental and other scientific

purposes.

- Protocol on Protection and welfare of animals (Protocol to the Amsterdam Treaty, 2/10/1997)

- Directive 2000/54/EC on the protection of workers from risks related to exposure to biological

agents at work

- The 2000 Report of the AVMA panel on euthanasia.

- The Recommendations for euthanasia of experimental animals: Part 2-Working Party Report

(Laboratory Animals (1997) - 31, 1-32).

- And as explained above, we are trying to implement whenever possible 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) in order to search for alternative methods, reduction of

the number of animals and the refinement of experiments will be fully applied.

Finally, we comply with the French Legislation:

1) The European directive 86/609/CEE of November 26th 1986, unifying CEE regulations

concerning the protection of animals used for scientific purposes, incorporated into French law by

the decrees N° 87-848 of October 19th 1987 and N° 2001-464 published on May 29th 2001.

Three texts of application of the above French law dated April 19th 1988 were published in the

Official Journal of the French Republic April 27th

1988 concerning: a. the supply of animals to

authorized animal facilities for experimental of scientific research; b. The rules for the authorization

for individuals to undertake experiments using animals; etc. the licensing, construction and

functioning of animal facilities for experimental scientific research.


Proposal Part B /34

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)


Proposal Part B /34

ANNEX : AUTORIZATION FOR ANIMAL EXPERIMENTATION

« arrete prefectoral portant agrement d’un etlabissement d’experimentation animale »


Proposal Part B /34


Proposal Part B /34

ENDPAGE

PEOPLE

MARIE CURIE ACTIONS

Marie Curie Intra-European Fellowships (IEF)

Call: FP7-PEOPLE-2007-2-1-IEF

PART B

“Circuit-hubs”

Circuit Hubs

Documents

Transcript of Circuit Hubs