The Marie Curie Actions 2-1-IEF

circuit-hubs

FP7-PEOPLE-2007-

STARTPAGE

PEOPLE MARIE CURIE ACTIONS Intra-European Fellowships (IEF) Call: FP7-PEOPLE-2007-2-1-IEF

PART B

“Circuit-hubs”

Proposal Part B

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FP7-PEOPLE-2007-

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

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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
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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 morphophysiological description of “circuit-hubs” (3) analysing the function of “circuit hubs”. In the following lines we will describe the methodological approaches implemented to meet these objectives. Objectives against the state of the art - The first coherent activity patterns in developing cortical networks have been described almost twenty years ago but the mechanisms leading to neuronal synchronization are still unknown. One possible explanation relies on the complexity of cortical networks (variety of morpho-physiological cell types, of communication devices between neurons, etc), a property even emphasized at early developmental stages. Indeed, immature cortical networks gather not only different types of neurons but also cells at different developmental stages. Furthermore, developing networks are by definition highly dynamic and plastic. So far two hypotheses have been proposed to drive the onset of early network oscillations: (1) a “network theory”: synchrony is generated by the build-up to a threshold of random local perturbations in a highly recurrent synaptic network (de la Prida et al., 1999; la Prida et al., 2006, Bolea et al., 2006, Marchionni 2007), and/or (2) a “cell theory” where rhythmicity is provided by the autonomous activity of a group of pacemaker neurons (Sipila et al., 2005, Sipila et al., 2006, Zheng et al., 2006, Strata et al. 1997). Therefore, the understanding of the mechanisms leading to population coherence in developing cortical structures requires having simultaneously and dynamically access to two levels of observation: the network and the cell. The considerable progress made by imaging techniques now offers this possibility. Our goal is to localize and characterise “network hubs” based on the analysis of spontaneous network dynamics without any a priori assumption. Expected results - We expect by the end of the project to reach the following results: (1) By reconstructing the temporal dynamics of the network we expect to identify “hub cells” capable to drive its activity, i.e. showing high correlation with global network firing and anticipating synchronous events (GDPs). (2) By performing targeted electrophysiological recordings of spontaneous and evoked activity in hub neurons we expect to characterize their functional properties and confirm that they drive the oscillatory activity of the network. We hope to be able to understand the cellular mechanisms by which these cells influence the network state. (3) Based on morphological reconstruction of biocytin-filled targeted hub-cells we expect that, although still immature, hub neurons display spread and wide morphological features. This observation would support the theoretical model underlying this project. (4) Immunohistochemical characterization of these cells should narrow the functional family of hub neurons to specific cell types. (5) Demonstration that inter- and multi- disciplinary strategies bridging mathematic and physics to neurobiology are strongly appropriate in brain research. Expected outcome beyond the scope of the proposal – By reaching the above mentioned point 4, we will open a wide field of investigation that goes far beyond questions of developmental neurobiology. Indeed, if high-connectivity neurons are of a specific cell class and drive network synchrony, the next logical step following the outcomes of this project would be to perform experiments on genetically engineered mice where the specific neurochemical marker for these neurons would be tagged with GFP. That way, it will be possible to perform a full description of these cells, study their role in mature networks and eventually test whether they also contribute to other types of oscillations such as epileptiform activities. Finally our approach to study the functional topography of networks will be of obvious interest for biologists interested in complex biological systems even outside the field of Neurobiology.

Proposal Part B

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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
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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.
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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 intermultidisciplinary 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
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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 SelfOrganized 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 scalefree 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 posthoc. 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.
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The Marie Curie Actions 2-1-IEF

circuit-hubs

FP7-PEOPLE-2007-

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. (HarvardDartmouth, 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
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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 twophoton 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.
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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
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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
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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
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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 (TwoPhoton 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) 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)

Academic Degree:

RESEARCH EXPERIENCE From 04/2007 : PostDoc in the group of Dr. Cossart at INMED, INSERM U29, Parc Scientifique de Luminy, Marseille (France)
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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). 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). Trainee in the lab. of prof. Fromherz at the Department of Membrane and Neurophysics of the Max Planck Institute for Biochemistry, Martinsried (Germany) General Physics, Biophysics, Mathematics, Electronics, Computational neuroscience, Neuroengineering, Networks and Complex systems.

03/2002 – 01/2006:

01/2000 – 01/2001:

Background:

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) 11901193 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.

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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

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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
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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 patchclamp recordings (cultures, slices and intact brain structures), microscopes, molecular and cellular biological tools, surgery and culture rooms. For imaging experiments, the institute possesses three
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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 multibeam 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
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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
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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 150 20 hub”. 100 (Top left) Raster plot of 15 network activity. For 50 each neuron, dots 10 represent the onset of 0 0 150 300 450 600 firing based on calcium 5 20 spikes (see red circles in bottom left panel). 15 0 -4 -2 0 2 4 (middle left) Coordinated 10 time lag (# frames) network events (GDPs) 5 revealed by synchronous onset of firing in a large 0 fraction of imaged cells. 0 150 300 450 600 (bottom left) 20 Automatically detected calcium spikes in the 0 fluorescence traces. Red and green circles -20 represent respectively onset and offset of firing. -40 0 150 300 450 600 Time resolution: time (s) 100ms/frame. (Top right) For each pair of neurons the cross-correlation (CC) of the onset time series is calculated. The average CC vs. the average time lag of maximum CC is shown for each neuron (dot). Averages were calculated over all possible pairs. Red marked neuron is a candidate network hub. Its calcium trace is shown in bottom left panel. (Bottom right) Two-photon calcium fluorescence image of the analyzed CA3 region from a rat hippocampal slice loaded with a calcium indicator (Fura2-AM) at P6.
200 25
cell (#)

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
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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 morphofunctional 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.

active cells (% )

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 cellhubs 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. 70 Milestone 3: how circuit hubs 40 mV drive the network activity 60 Deliverables: Driving network 50 activity with circuit-hubs (DNAH), due at month 11 and 40 17; Modeling network dynamics (MND), due at month 22. 30
Figure 3. Increasing the firing rate of a “hub 10 neuron” reduces the occurrence of synchronous network events. 0 0 150 300 450 600 The percentage of active cells in the time (s) 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.
20

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Depending on the outcomes of the project, all deliverables, written at the end of the specified periods, are potentially scientific reports, articles or communications to scientific conferences and if they can lead to any exploitable results of interest for the industry/market, appropriate studies will be carried on by Protisvalor to evaluate the IPR and potential patentable results that may arise. The last two month of the fellowship will be left as spare time to summarize the results of the all project and write a final interdisciplinary paper likely accepted in a very prestigious multidisciplinary international journal.
• Practical and administrative arrangements and support for the hosting of the fellow

The candidate has already started a postdoctoral experience at INMED since April 2007. He has already found an accommodation and, as an Italian citizen, he benefits from the Schengen area legislation. Since the fellow is single he has no need at the moment to find schools or childcare facilities. However, the Fellow will be put in contact with the Local Mobility Centre (link to the ERA-Net Mobility Centres) which is located in Marseille to ease his stay in France. Additionally, for any additional burocratical and administrative support, four secretaries working at INMED, which already provide support to all intra- and extra-European students and researcher working at INMED, will help him. For practical and administrative arrangements, the laboratory can also rely on the back up from the European department (Head: C. Damon) at the University in terms of paper work. Furthermore, to help his stay in France, he will receive a salary with the complete social and welfare cover (instead of fees) and additional employee advantages (cheque restaurant, etc.). As the applicant spent already one year in Munich (Germany) and one year in Cambridge (England), he is used to live abroad. About the language teaching, he will have the possibility to attend the courses which are periodically organized at the Scientific Campus of Luminy. Anyway since INMED is a very international institute, the language for working is English. Month 1 Month 12 Month 24 OLRND MP D IHCC DNA H MN D Table 1. Schedule of training management Online reconstruction of network dynamics (OLRND) Morpho-physiological description (MPD) Immunohistochemical characterization of hubs (IHCC) Driving network activity with circuit-hubs (DNAH) Modeling network dynamics (MND) Expected deliverables Aim 1 Aim 2 Aim 3

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.

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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 lifesciences to theoretical-mathematical sciences; certainly the validation of our proposed
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2.

3.

4. 5.

6.

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. 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. 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. 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. 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). 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.

REFERENCES Adelsberger, H., O. Garaschuk, and A. Konnerth. "Cortical calcium waves in resting newborn mice." Nat.Neurosci. 8.8 (2005): 988-90. Almaas E. Biological impacts and context of network theory. J Exp Biol. 2007 May;210(Pt 9):1548-58. Aronov D. Fast algorithm for the metric-space analysis of simultaneous responses of multiple single neurons. J Neurosci Methods. 2003 Apr 15;124(2):175-9 Aronov D, Reich DS, Mechler F, Victor JD. Neural coding of spatial phase in V1 of the macaque monkey. J Neurophysiol. 2003 Jun;89(6):3304-27. Epub 2003 Jan 29. Aronov D, Victor JD. Non-Euclidean properties of spike train metric spaces. Phys Rev E Stat Nonlin Soft Matter Phys. 2004 Jun;69(6 Pt 1):061905. Epub 2004 Jun 2. Bak P, Tang C, Wiesenfeld K (1988) Self-organized criticality. Physical Review A: Atomic, Molecular, and Optical Physics 38: 364-374. Ban J, Bonifazi P, Pinato P, Broccard F, Studer L, Torre V and Ruaro ME: “ES-derived neurons form functional networks in vitro”, Stem Cells. 2006 Nov 16. Barabasi AL, Albert R. Emergence of scaling in random networks. Science. 1999 Oct 15;286(5439):509-12. Barbin, G., et al. "Involvement of GABAA receptors in the outgrowth of cultured hippocampal neurons." Neuroscience Letters 152.1-2 (1993): 150-54. Bedard C, Kroger H, Destexhe A (2006) Does the 1/f frequency scaling of brain signals reflect selforganized critical states? Phys Rev Lett 97: 118102 1-4. Beggs JM, Plenz D (2003) Neuronal avalanches in neocortical circuits. J Neurosci 23: 11167-11177. Beggs JM, Plenz D (2004) Neuronal avalanches are diverse and precise activity patterns that are stable for many hours in cortical slice cultures. J Neurosci 24: 5216-5229. Ben Ari, Y. "Developing networks play a similar melody." Trends in Neurosciences 24.6 (2001): 353-60.

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Ben Ari, Y. "Excitatory actions of gaba during development: the nature of the nurture." Nat.Rev.Neurosci. 3.9 (2002): 728-39. Ben Ari, Y., et al. "Giant synaptic potentials in immature rat CA3 hippocampal neurones." J.Physiol 416 (1989): 303-25. Ben-Ari, Y., et al. "GABAA, NMDA and AMPA receptors: a developmentally regulated 'menage a trois'." Trends.Neurosci. 20 (1997): 523-29. Bolea S, Sanchez-Andres JV, Huang X, Wu JY. Initiation and propagation of neuronal coactivation in the developing hippocampus. J Neurophysiol. 2006 Jan;95(1):552-61. Epub 2005 Sep 21 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 Bonifazi P1, Ruaro ME1, 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) Brecht M, Schneider M, Sakmann B, Margrie TW. Whisker movements evoked by stimulation of single pyramidal cells in rat motor cortex Nature. 2004 Feb 19;427(6976):704-10 Cang, J., et al. "Optical imaging of the intrinsic signal as a measure of cortical plasticity in the mouse." Vis.Neurosci. 22.5 (2005): 685-91. Cang, J., et al. "Ephrin-as guide the formation of functional maps in the visual cortex." Neuron 48.4 (2005): 577-89. Cobb, S. R., et al. "Synchronization of neuronal activity in hippocampus by individual GABAergic interneurons." Nature 378 (1995): 75-78. Cossart R, Tyzio R, Dinocourt C, Esclapez M, Hirsch JC, Ben-Ari Y, Bernard C. Presynaptic kainate receptors that enhance the release of GABA on CA1 hippocampal interneurons. Neuron. 2001 Feb;29(2):497-508 Cossart, R., D. Aronov, and R. Yuste. "Attractor dynamics of network UP states in the neocortex." Nature 423.6937 (2003): 283-88. Cossart, R., et al. "Dendritic but not somatic GABAergic inhibition is decreased in experimental epilepsy." Nat.Neurosci. 4.1 (2001): 52-62. Cossart, R., et al. "Quantal Release of Glutamate Generates Pure Kainate and Mixed AMPA/Kainate EPSCs in Hippocampal Neurons." Neuron 35.1 (2002): 147-59. Cossart, R., et al. "GluR5 kainate receptor activation of interneurons increases tonic GABAergic inhibition in pyramidal cells." Nat.Neurosci. 1 (1998): 470-78. Cossart, R., Y. Ikegaya, and R. Yuste. "Calcium imaging of cortical networks dynamics." Cell Calcium 37.5 (2005): 451-57. Crepel, V., et al. "A parturition-associated nonsynaptic coherent activity pattern in the developing hippocampus." Neuron 54.1 (2007): 105-20. De la Prida LM, Huberfeld G, Cohen I, Miles R. Threshold behavior in the initiation of hippocampal population bursts. Neuron. 2006 Jan 5;49(1):131-42 De la Prida LM, Sanchez-Andres JV. Nonlinear frequency-dependent synchronization in the developing hippocampus. J Neurophysiol. 1999 Jul;82(1):202-8 Destexhe, A., M. Rudolph, and D. Pare. "The high-conductance state of neocortical neurons in vivo." Nat.Rev.Neurosci. 4.9 (2003): 739-51. Dupont, E., et al. "Rapid developmental switch in the mechanisms driving early cortical columnar networks." Nature (2005). Freeman WJ (2004) Origin, structure, and role of background EEG activity. Part 2. Analytic phase. Clin Neurophysiol 115: 2089-2107. Galli, L. and L. Maffei. "Spontaneous impulse activity of rat retinal ganglion cells in prenatal life." Science 242.4875 (1988): 90-91. Garaschuk, O., et al. "Large-scale oscillatory calcium waves in the immature cortex." Nat.Neurosci. 3.5 (2000): 452-59. Goldberg JH, Tamas G, Aronov D, Yuste R. Calcium microdomains in aspiny dendrites. Neuron. 2003 Nov 13;40(4):807-21. 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 Hennou, S., et al. "Early sequential formation of functional GABA(A) and glutamatergic synapses on CA1 interneurons of the rat foetal hippocampus." European Journal of Neuroscience 16.2 (2002): 197-208. Proposal Part B Page 26/34

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Hopfield JJ. Neural networks and physical systems with emergent collective computational abilities. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2554-8 Jensen HJ (1998) Self-Organized Criticality – Emergent Complex Behavior in Physical and Biological Systems. Cambridge: Cambridge University Press. Kandler, K. and D. C. Gillespie. "Developmental refinement of inhibitory sound-localization circuits." Trends in Neurosciences 28.6 (2005): 290-96. Kandler, K. and L. C. Katz. "Coordination of neuronal activity in developing visual cortex by gap junctionmediated biochemical communication." J.Neurosci. 18.4 (1998): 1419-27. Khazipov, R., et al. "Early development of neuronal activity in the primate hippocampus in utero." J.Neurosci. 21.24 (2001): 9770-81. Khazipov, R., et al. "Developmental changes in GABAergic actions and seizure susceptibility in the rat hippocampus." European Journal of Neuroscience 19.3 (2004): 590-600. Leinekugel X, Khazipov R, Cannon R, Hirase H, Ben-Ari Y, Buzsaki G. Correlated bursts of activity in the neonatal hippocampus in vivo. Science. 2002 Jun 14;296(5575):2049-52. Linkenkaer-Hansen K, Nikouline VV, Palva JM, Ilmoniemi RJ (2001) Long range temporal correlations and scaling behavior in human brain oscillations. J Neurosci 21: 1370-1377 Marchionni I, Omrani A, Cherubini E. In the developing rat hippocampus a tonic GABAA-mediated conductance selectively enhances the glutamatergic drive of principal cells. J Physiol. 2007 Jun 1;581(Pt 2):515-28. Epub 2007 Feb 22 Mazzoni A, Broccard F, Garcia E, Bonifazi P, Ruaro ME and Torre V: “ On the dynamics of the spontaneous activity in neuronal networks “, PLoS ONE. 2007 May 9. Meister, M., et al. "Synchronous bursts of action potentials in ganglion cells of the developing mammalian retina." Science 252.5008 (1991): 939-43. O'Donovan, M. J. "Motor activity in the isolated spinal cord of the chick embryo: synaptic drive and firing pattern of single motoneurons." J.Neurosci. 9.3 (1989): 943-58. Owens, D. F. and A. R. Kriegstein. "Patterns of intracellular calcium fluctuation in precursor cells of the neocortical ventricular zone." J.Neurosci. 18.14 (1998): 5374-88. Palva, J. M., et al. "Fast network oscillations in the newborn rat hippocampus in vitro." J.Neurosci. 20.3 (2000): 1170-78. Peinado, A. "Traveling slow waves of neural activity: a novel form of network activity in developing neocortex." J.Neurosci. 20.2 (2000): RC54. Ruaro ME2, Bonifazi P2, and Torre V, “Towards the neurocomputer: Image processing and pattern recognition with neuronal cultures”, IEEE Transactions on Biomedical Engineering, vol. 52, pp. 371383, 2005 (2 Paolo Bonifazi and Maria Elisabetta Ruaro equally contributed) Schneidman E, Berry MJ 2nd, Segev R, Bialek W. Weak pairwise correlations imply strongly correlated network states in a neural population. Nature. 2006 Apr 20;440(7087):1007-12. Epub 2006 Apr 9. Sipila, S. T., et al. "Depolarizing GABA acts on intrinsically bursting pyramidal neurons to drive giant depolarizing potentials in the immature hippocampus." J.Neurosci. 25.22 (2005): 5280-89. Sipila, S. T., et al. "Intrinsic bursting of immature CA3 pyramidal neurons and consequent giant depolarizing potentials are driven by a persistent Na+ current and terminated by a slow Ca2+ -activated K+ current." European Journal of Neuroscience 23.9 (2006): 2330-38. Strata, F., et al. "A pacemaker current in dye-coupled hilar interneurons contributes to the generation of giant GABAergic potentials in developing hippocampus." J.Neurosci. 17 (1997): 1435-46. Syed, M. M., et al. "Stage-dependent dynamics and modulation of spontaneous waves in the developing rabbit retina." J.Physiol 560.Pt 2 (2004): 533-49. Tyzio, R., et al. "Maternal oxytocin triggers a transient inhibitory switch in GABA signaling in the fetal brain during delivery." Science (2006). Tyzio, R., et al. "The establishment of GABAergic and glutamatergic synapses on CA1 pyramidal neurons is sequential and correlates with the development of the apical dendrite." J.Neurosci. 19.23 (1999): 10372-82. Vespignani A, Zapperi Z (1998) How self organized criticality works: a unified mean field picture. Physical Review E: Statistical, Nonlinear, and Soft Matter Physics 57: 6345-6363 Weissman, T. A., et al. "Calcium waves propagate through radial glial cells and modulate proliferation in the developing neocortex." Neuron 43.5 (2004): 647-61. Yuste, R., A. Peinado, and L. C. Katz. "Neuronal domains in developing neocortex." Science 257.5070 (1992): 665-69. Proposal Part B Page 27/34

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B6 ETHICAL ISSUES

ETHICAL ISSUES TABLE
YES? 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? • 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 PAGE

X

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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.

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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° 99961 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)

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ANNEX : AUTORIZATION FOR ANIMAL EXPERIMENTATION

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

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ENDPAGE

PEOPLE MARIE CURIE ACTIONS

Marie Curie Intra-European Fellowships (IEF) Call: FP7-PEOPLE-2007-2-1-IEF

PART B

“Circuit-hubs”

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