In the brain, the balance between excitation and modulation plays a crucial role in numerous cognitive processes and pathological impairments of this balance may underlie several neurological disorders. Indeed, dysregulations of two neurotransmitter receptor families, glutamate NMDA (NMDAR) and dopamine (DAR) receptors, are believed to play a direct role in the emergence of psychotic disorders such as schizophrenia. Thus, understanding how these two neurotransmission systems communicate is of growing interest. Intensive investigations have identified intracellular and genomic cascades involved in this dialogue. However, the recent discovery that NMDAR and DAR diffuse and physically interact at the surface of neurons profoundly changed our view of neurotransmitter signaling. A major challenge is now to unveil the contribution of DAR/NMDAR complexes in the regulation of living brain circuits. To this aim, we will combine complementary expertise to investigate the surface crosstalk between NMDAR and DAR both in physiological and psychotic disorder contexts, focusing our attention on D1R, D2R, and NMDAR in hippocampal, prefrontal and striatal circuits. We will assess the roles of this dynamic receptor interplay from a multi-scale perspective. We will first investigate the rules of the DAR/NMDAR surface dialogue at the single molecule level by mapping all interaction sites and monitoring receptor distribution and diffusion using multi-color single molecule imaging in vitro and in brain slices at several developmental stages, both in control and in models of schizophrenia. We will then investigate whether the DAR/NMDAR surface crosstalk regulates glutamatergic transmission along the dendritic tree, its impact on NMDAR-dependent plasticity, and its role in dendritic outgrowth and spine maturation during development using two-photon calcium imaging, electrophysiological recording and competing peptides. Finally, we will investigate the influence of the DAR/NMDAR interplay on physiological and pathological network dynamics and behaviors using multi-electrode array recordings, optogenetic release of dopamine, competing peptides and “data-mining” statistics. In conclusion, we will join international forces to shed the first lights on this unexpected dynamic integration level using nanoscopic, macroscopic and mesoscopic approaches.

Neuroscience research using rodents as an animal model relies on the assumption that results should generalize across species to primates and ultimately to humans. However, many brain areas, including the neocortex, have species specific functional organizations. The mouse lemur (Microcebus murinus), the World’s smallest primate, has the potential to become an ideal animal model bridging the gap between rodents and primates. It has most the advantages of the rodent model (small brain size, quick reproduction, relatively short life cycle), but additionally offers the evolutionary closeness of primates. Therefore, the mouse lemur promises to revolutionize the transferability of experimental results from small sized animal models to human applications. In this project, three labs will combine their expertise in primate behavior, in-vivo optical imaging, and cutting edge histology and molecular biology to explore the functional organization of the mouse lemur cortex, as well its behavioral and cognitive capacity. This collaborative project will lay the groundwork to establish the mouse lemur as a novel neuroscience model system.

Microorganisms are the most abundant living organisms on earth and they thrive in virtually every possible habitat, including in our bodies and those of other organisms. Understanding the causes and consequences of their diversity is therefore essential for controlling microbes in health, agriculture and biotechnology. Studies of microbial diversity are also increasingly important for advancing general ecological and evolutionary theory, because microbes are powerful experimental model systems. We seek to advance our understanding of the mechanisms underlying microbial diversity by focusing on the role of extra-cellular products (EPs). EPs play an interesting but largely unknown role in the evolution of microbial diversity. Given possible effects of EPs on the fitness of cells other than the producer, a growing perception is that EP production is a form of cooperative behavior, suggesting that it has evolved due to benefits conferred on the population of EP producers rather than on individual producers. However, this view rests on models that make simplifying assumptions that need to be rigorously tested. Our international collaborative project combines the expertise of: (1) two biologists, who will use existing and newly developed millifluidic droplet technology to test for collective benefit and its evolutionary consequences in experiments with two bacterial EP systems with different fitness effects and availability to others, (2) a colloid chemist, who will develop novel millifluidic tools that provide a unique opportunity to quantitatively assess the evolutionary consequences of EP production, and (3) a theoretical biophysicist, who will develop computational models that incorporate biological details of both experimental EP systems, and will both generate specific predictions that will be tested experimentally and facilitate the results from these experiments. By generating unique quantitative data on two very different bacterial EP systems and comparing results against predictions from realistic models, we expect that our project will provide conclusive answers and set new standards for the study of microbial interactions.

Living organisms, particularly mammals, constantly shift between different behavioral states according to their external environment. These shifts are largely regulated by specific molecules, termed "neuromodulators" that convey information to a wide range of brain systems. The mechanisms by which these neuromodulators regulate distinct behavioral states remain enigmatic. One important brain neuromodulator is the neuropeptide oxytocin, which recently became well known for its positive, “pro-social” effects on human social behavior. Oxytocin is synthesized and released by a small number of hypothalamic neurons that send their projections to various forebrain regions, to regulate a plethora of social behaviors ranging from aggression to empathy.
Relying on anatomical and physiological distinction between subsets of oxytocin neurons, we hypothesize that these neurons are segregated into functional modules associated with distinct types of social behavior. To test this hypothesis we will focus on one hypothalamic region – the paraventricular nucleus, which is the main source for oxytocin projections throughout the entire brain. We propose a comprehensive multidisciplinary study aiming to identify, analyze and mathematically model functional oxytocin neuronal modules activated during various forms of social behavior.
To pursue this hypothesis we will employ a novel genetic technique named vGAIT (virus-mediated genetic activity-induced tagging), which enables tagging and manipulating oxytocin neurons that were active during a given social behavior, ranging from simple social interaction to a complex “human-like” cooperative behavior. Using this technique in freely moving rats, we will explore the activity and connectivity, as well as the intrinsic electrophysiological and molecular properties of activated oxytocin modules at the single-cell level. Building on this information we will develop a computational model of the oxytocin system and investigate the mechanistic principles underlying its function. The unique combination of cutting-edge techniques and approaches will allow an unprecedented insight into the mode of operation of a central brain neuromodulatory system that controls mammalian social behavior. Furthermore, the expected findings will pave the way for the development of interventions directed towards specific pathologies of social behavior in humans.

In variable environments (either natural or altered by humans) organisms can adjust to new conditions for better success in producing offspring. In many cases, organisms do this through cognitive abilities: their ability to gather information from their surroundings, process that information, and make decisions to improve their lot. We now realize that there is extensive variation in cognitive capacity both between species and within species suggesting that cognitive abilities evolve, yet we still know very little about how cognition evolves. Most organisms have a number of different cognitive abilities (memory, attention, learning, etc…) and comparative research suggests that certain environments select for improvement in some, but not all, of these abilities, but this has not been demonstrated directly. Furthermore, while our understanding of the neural basis of cognition has improved in model species, we still know very little about the ecological importance of most cognitive capacities and their underlying neural structure. Our project will investigate the evolution of cognitive abilities and neural structure in a wild great tits, and thus provide a big step forward in our understanding of cognitive evolution. To do this, we will study 6 populations in the French Pyrenees that differ in ecology (altitude and urbanization), and thus likely favor different cognitive traits. Our team, specialists in cognition, brain imaging, computer science, and evolution will develop new tools (touchscreens) and analytical approaches (automated video analysis, MRI of non-model species) to examine cognitive traits that could present a clear advantage under some, but not all, ecological contexts. We will measure cognitive abilities of birds in these contrasted populations and quantify their fitness to understand what cognitive traits and neural structures are favored in different ecological settings. In doing so, we can measure natural selection on cognitive abilities and neural structure, understand what factors in the environment drive this selection, and ultimately gain a much better understanding of how evolution shapes minds in the wild. This interdisciplinary project provides advances to a number of different fields of research while together contributing to an important stride forward in our understanding of the neural basis of cognition and how cognitive evolves.

In order to understand the brain, tools need to be developed to allow the investigation of interactions between many individual neurons. Noninvasive tools (MRI, EEG) provide structural and functional information, but at low spatial resolution. Invasive recording methods (patch-clamp, multi-electrode array) provide high temporal resolution data from single neurons, but are bulky and lack spatial resolution and throughput. For this reason, considerable efforts have been invested in developing optical detection methods including the utilization of voltage-sensitive dyes (VSDs) and genetically-encoded fluorescent voltage-sensor proteins (VSPs). Despite these advances, VSDs and VSPs are not yet preforming at the needed level of detection.
We therefore propose to develop solid-state nanoscale voltage sensors nanoparticles (vsNPs) that report on action potentials (APs) on the nanoscale. These devices could dramatically change the way we image and study nervous system activities, and how we interface these systems to provide novel therapeutic approaches and prosthetic strategies. The proposed vsNPs are based on the quantum confined Stark effect. They will be designed to be injectable, targetable, and to be self-inserted into the membrane. They will optically record, non-invasively, APs at the single particle level at the nanoscale, at multiple sites and across a large field-of-view. They will display high voltage sensitivity, high brightness, single-particle detection sensitivity, large spectral shift, fast temporal response, minimal photobleaching, large Stokes shifts, large two-photon cross sections, excellent NIR performance, and compatibility with lifetime imaging.
We will pursue the following aims: (i) synthesis of heterostructure vsNPs (Weiss & Oron) and characterization of their performance (Enderlein, Weiss & Oron); (ii) development of functionalization methods based on ligand exchange with transmembrane (amphiphilic) peptides by self-assembly (Weiss) and optimization of vsNPs insertion into the membrane (Weiss and Enderlein); (iii) development of optical vsNPS recording methodologies and benchmarking of vsNPS performance (Enderlein, Oron, & Weiss); (iv) demonstration of vsNPs utility in model neuronal systems (Triller), exploration of their use in studying synapses and neuronal integration of excitatory and inhibitory inputs (Triller).

There is a common perception that larger brains mediate higher ‘intelligence’ or cognitive capacity. Many insects, however, demonstrate that sophisticated social structures and cognition are possible with very small brains. Foraging honeybees, for example, memorize flower signals and display higher-order learning such as categorization, concept learning and ‘counting’. How do bees perform these feats with small nervous systems? Despite the extensive knowledge on the cognitive abilities of bees, to date, no study has attempted to elucidate the neural mechanisms underpinning their higher-order learning. Here we will pool our expertise in cognitive, computational and neuroscience methods to unravel the neural circuits and architecture underpinning such cognitive performances in the honeybee. We will combine behavioral recordings of bees walking stationarily on a locomotion compensator in a virtual environment with access to its brain via multielectrode recordings. Data will be fed into computational models providing testable hypotheses about minimal neural architectures for cognition, and ultimately working towards whole-brain modeling (connectomics).
This project will expand the currently available information on the neurobiology of insect learning, and will provide the first complete computational model for the neuronal mechanisms that underlie cognition in a miniature nervous system. Our expected results have far-reaching implications for psychophysics, neuroscience and behavioral biology as well as the potential to revolutionize the field of visual processing in animals and robotics.

How does a nervous system adapt to the body plan of a growing animal from birth to adult? This question is fundamental to biology, yet is currently impossible to answer without turning to small model organisms, which provide an extraordinary level of access to the nervous system at the molecular, anatomical, and physiological levels. We will explore how a nematode C. elegans manages to drive undulatory locomotion at all life stages, with different motor circuit components, using powerful new tools in serial-section electron microscopy, optical neurophysiology in freely behaving animals, super resolution light microscopy, and biophysical modeling. This research program should provide a unique data set that illuminates developmental dynamics from neural activities to animal behaviors.

Although locomotion may seem effortless, it relies on spinal circuits producing complex patterns of muscle contractions. Spinal cord central pattern generator circuits (CPGs) produce motor output autonomously as a result of descending commands from the brain and modulations caused by internal physiological cues. The cerebrospinal fluid (CSF) constitutes a fascinating interface through which chemical cues produced in the brain can reach neurons within the entire central nervous system. Located along the central canal, CSF-contacting neurons (CSFns) project a ciliary tuft into the CSF and an axon into the spinal cord. These mysterious cells were identified in over 200 species of vertebrates by Kolmer and Agduhr who hypothesized that they could act as a proprioceptive sensory neuron. The difficulties of labeling and accessing these cells have prevented the characterization of their sensory physiology or specification. However newly developed tools and experimental approaches in zebrafish provide us with a unique opportunity to overcome these technical challenges. The transparency of zebrafish at early stages makes it a powerful system for identifying neurons by their morphology and for investigating and manipulating neuronal activity of specific cells in the intact animal. Using an optogenetic approach, Dr. Wyart and colleagues discovered that CSFns could strongly modulate the activity of spinal CPGs. In this proposed project, the Delmas, Lewis and Wyart labs will combine expression profiling, channel physiology, behavioural analysis and loss-of-function assays to elucidate how CSFns are specified and how they sense chemical and mechanical cues in the CSF. The Lewis lab has pioneered the use of FAC sorting and transcriptome analysis to identify genes expressed in distinct subtypes of spinal neurons. The Delmas lab has unraveled the functions of channels underlying chemo- and mechano-sensation in mammals. The Wyart lab has combined optical and physiological approaches to identify the functions of CSFns. Together we will establish how genetic networks specify these cells and unravel the molecular mechanisms by which CSFns are physiologically activated in vivo and contribute to behaviour.

Although locomotion may seem effortless, it relies on spinal circuits producing complex patterns of muscle contractions. Spinal cord central pattern generator circuits (CPGs) produce motor output autonomously as a result of descending commands from the brain and modulations caused by internal physiological cues. The cerebrospinal fluid (CSF) constitutes a fascinating interface through which chemical cues produced in the brain can reach neurons within the entire central nervous system. Located along the central canal, CSF-contacting neurons (CSFns) project a ciliary tuft into the CSF and an axon into the spinal cord. These mysterious cells were identified in over 200 species of vertebrates by Kolmer and Agduhr who hypothesized that they could act as a proprioceptive sensory neuron. The difficulties of labeling and accessing these cells have prevented the characterization of their sensory physiology or specification. However newly developed tools and experimental approaches in zebrafish provide us with a unique opportunity to overcome these technical challenges. The transparency of zebrafish at early stages makes it a powerful system for identifying neurons by their morphology and for investigating and manipulating neuronal activity of specific cells in the intact animal. Using an optogenetic approach, Dr. Wyart and colleagues discovered that CSFns could strongly modulate the activity of spinal CPGs. In this proposed project, the Delmas, Lewis and Wyart labs will combine expression profiling, channel physiology, behavioural analysis and loss-of-function assays to elucidate how CSFns are specified and how they sense chemical and mechanical cues in the CSF. The Lewis lab has pioneered the use of FAC sorting and transcriptome analysis to identify genes expressed in distinct subtypes of spinal neurons. The Delmas lab has unraveled the functions of channels underlying chemo- and mechano-sensation in mammals. The Wyart lab has combined optical and physiological approaches to identify the functions of CSFns. Together we will establish how genetic networks specify these cells and unravel the molecular mechanisms by which CSFns are physiologically activated in vivo and contribute to behaviour.

Although locomotion may seem effortless, it relies on spinal circuits producing complex patterns of muscle contractions. Spinal cord central pattern generator circuits (CPGs) produce motor output autonomously as a result of descending commands from the brain and modulations caused by internal physiological cues. The cerebrospinal fluid (CSF) constitutes a fascinating interface through which chemical cues produced in the brain can reach neurons within the entire central nervous system. Located along the central canal, CSF-contacting neurons (CSFns) project a ciliary tuft into the CSF and an axon into the spinal cord. These mysterious cells were identified in over 200 species of vertebrates by Kolmer and Agduhr who hypothesized that they could act as a proprioceptive sensory neuron. The difficulties of labeling and accessing these cells have prevented the characterization of their sensory physiology or specification. However newly developed tools and experimental approaches in zebrafish provide us with a unique opportunity to overcome these technical challenges. The transparency of zebrafish at early stages makes it a powerful system for identifying neurons by their morphology and for investigating and manipulating neuronal activity of specific cells in the intact animal. Using an optogenetic approach, Dr. Wyart and colleagues discovered that CSFns could strongly modulate the activity of spinal CPGs. In this proposed project, the Delmas, Lewis and Wyart labs will combine expression profiling, channel physiology, behavioural analysis and loss-of-function assays to elucidate how CSFns are specified and how they sense chemical and mechanical cues in the CSF. The Lewis lab has pioneered the use of FAC sorting and transcriptome analysis to identify genes expressed in distinct subtypes of spinal neurons. The Delmas lab has unraveled the functions of channels underlying chemo- and mechano-sensation in mammals. The Wyart lab has combined optical and physiological approaches to identify the functions of CSFns. Together we will establish how genetic networks specify these cells and unravel the molecular mechanisms by which CSFns are physiologically activated in vivo and contribute to behaviour.

Although locomotion may seem effortless, it relies on spinal circuits producing complex patterns of muscle contractions. Spinal cord central pattern generator circuits (CPGs) produce motor output autonomously as a result of descending commands from the brain and modulations caused by internal physiological cues. The cerebrospinal fluid (CSF) constitutes a fascinating interface through which chemical cues produced in the brain can reach neurons within the entire central nervous system. Located along the central canal, CSF-contacting neurons (CSFns) project a ciliary tuft into the CSF and an axon into the spinal cord. These mysterious cells were identified in over 200 species of vertebrates by Kolmer and Agduhr who hypothesized that they could act as a proprioceptive sensory neuron. The difficulties of labeling and accessing these cells have prevented the characterization of their sensory physiology or specification. However newly developed tools and experimental approaches in zebrafish provide us with a unique opportunity to overcome these technical challenges. The transparency of zebrafish at early stages makes it a powerful system for identifying neurons by their morphology and for investigating and manipulating neuronal activity of specific cells in the intact animal. Using an optogenetic approach, Dr. Wyart and colleagues discovered that CSFns could strongly modulate the activity of spinal CPGs. In this proposed project, the Delmas, Lewis and Wyart labs will combine expression profiling, channel physiology, behavioural analysis and loss-of-function assays to elucidate how CSFns are specified and how they sense chemical and mechanical cues in the CSF. The Lewis lab has pioneered the use of FAC sorting and transcriptome analysis to identify genes expressed in distinct subtypes of spinal neurons. The Delmas lab has unraveled the functions of channels underlying chemo- and mechano-sensation in mammals. The Wyart lab has combined optical and physiological approaches to identify the functions of CSFns. Together we will establish how genetic networks specify these cells and unravel the molecular mechanisms by which CSFns are physiologically activated in vivo and contribute to behaviour.

One essential element very often missing to explain cell-fate control concerns the spatiotemporal regulation of translation. How does translation at synapses regulate the growth and connection strength of individual synapses during learning process? How does localized mRNA translation contribute to maintaining the pluripotency of stem cells? To address these fundamental biological questions, we propose a multi-disciplinary approach combining tools and concepts from synthetic biology, biophysics and neurobiology. Our strategy is to rationally design functionally controllable RNA-protein (RNP) complexes to spatiotemporally control mRNA translation in living cells. One of the main objectives will be to build synthetic RNPs that can traffic, translate or degrade a mRNA molecule. Our design will integrate magnetic nanoparticles and biological molecules to combine both the functional properties of native RNPs and the capability of being manipulated in space and time using magnetic forces. We hypothesize that local translation in cells often play critical roles in cell-fate decisions, such as the establishment of polarity and regulation of synapse formation and plasticity. However, currently available techniques are limited with their capacity in regulating local translation with spatiotemporal precision. Our new engineering approaches to develop synthetic RNPs in target cells will compensate pre-existing techniques by adding precise spatiotemporal controls. With synthetic RNP-mediated spatiotemporal translational regulatory systems, we will ask fundamental biological questions that are not accessible using current cell biology and ‘omics’ approaches, including: (1) What is the specific role of local translation at a synapse in terms of synaptic plasticity and memory formation and consolidation? (2) Can we control cellular behaviors of stem cells or differentiated cells by providing spatiotemporal gene expression dimensions? The new apporach will allow us to examine the effect of localized mRNA translation in controlling cell-fate behavior for two general cell states including pluripotency of stem cells and differentiated state of neurons. Such approaches will open new directions to better understand how local translation contributes to cell-fate conversion.

In the left hemisphere of the human brain, damage to the ventrolateral frontal (VLF) cortex, which includes a specialized region known as Broca’s area and adjacent precentral motor cortex, leads to speech problems. At the same time, there is an older system playing some as yet unspecified role in vocalization and speech in the dorsomedial frontal (DMF) region, which includes the supplementary motor area, presupplementary motor cortex, and the adjacent cingulate motor areas. Little is known of how these two systems interact anatomically and functionally. In primates that lack speech, such as chimpanzees and macaque monkeys, these regions may be involved in voluntary vocal production. Critically, there is nothing known of how these two systems may or may not have changed during primate brain evolution with increasing selection for language and speech functions in the human lineage. The aim of this interdisciplinary group of scientists is to adopt an evolutionary framework to study the role of these two brain regions in vocalization/speech, their cytoarchitectonic characteristics, and their anatomical and functional connectivity. The multimodal approach proposed here is particularly innovative because it combines comparative cytoarchitectonic analysis in three critical primate species to establish comparable areas. Further, invasive anatomical connection tracing studies that can only be carried out in the macaque monkey will provide totally unambiguous connection data that can then be employed to test predictions about functional and anatomical connectivity in the human and chimpanzee brains using non-invasive procedures including diffusion tensor imaging and resting state functional magnetic resonance imaging. Finally, functional imaging techniques will be used to characterize the VLF and DMF networks in relation to vocal production. This is the first systematic comparative study undertaken in the three most critical primate species for understanding DMF and VLF regions. Each research team brings a unique set of scientific skills and pragmatic strengths that provides for a novel, interdisciplinary effort to advance our understanding of the origins of vocal production and eventually human speech.

Complex, multicellular organisms are made up of many cell types, each performing different functions. Understanding the evolution of cells' social interactions is hard: multicellularity evolved long ago and dissecting individual interactions is difficult. We therefore evolve social behavior in two microbes, E. coli and budding yeast. Comparing a prokaryote and a eukaryote will suggest hypotheses for how social interactions between cells evolved in plants and animals.
We will use sucrose utilization as a model to study social evolution. Sucrose hydrolysis releases one molecule of glucose and one of fructose, highly preferred carbon sources for many microbes. In yeast, the enzymes that hydrolyze sucrose are extracellular public goods, whereas in E. coli they are plasmid-encoded, private goods.
We will use genetic engineering, experimental evolution, theory, and simulation to investigate three aspects of social evolution: cooperation, the origin of multicellularity, and the division of labor. We have four aims:
1) Engineer and evolve differentiation within multicellular yeast aggregates to test the idea that undifferentiated, multicellular aggregates evolved before the division of labor.
2) Study the role of plasmid transfer in bacterial cooperation. Does the production of secreted public goods by plasmids select for the faster transfer of plasmids to neighboring cells?
3) Ask how prior history and initial conditions determine the trajectory of evolution. We will engineer yeast and bacteria so they can evolve from similar starting points. We will vary whether sucrose-harvesting systems are public or private, whether they are transmissible or chromosomal, and whether cells can differentiate.
4) We will compare our experiments with three different methods to model social evolution: analytical theory, numerical simulation, and evolving digital organisms within computers.
The proposed work will benefit from a) combining engineering and evolution as experimental approaches, b) comparing different approaches to model these problems, c) strong interaction between theory and experiment, d) the interplay between expertise in yeast and bacterial cell biology, and e) regular meetings to exchange results, critiques and ideas that will influence future research.

We propose to elucidate the basis for positional information by hormones during plant morphogenesis. While it is known that cell fate decisions require simultaneous input from multiple hormones, to-date a precise understanding of how these signals are coordinated and act together to drive morphogenesis does not exist. Our limited mechanistic understanding is largely due to the difficulty to quantify the distribution of these small molecules in space and time. To explore this fundamental question, we will exploit recent advances in synthetic biology to engineer an RNA-based biosensor platform applicable to a broad range of small molecules and in particular to hormones. Using live-imaging technologies, we will use the sensors to obtain quantitative dynamic 3D maps of hormone distributions and relate these maps to the spatio-temporal distribution of cell identities, both during normal morphogenesis and upon perturbations of hormone levels. This analysis will be done on the shoot apical meristem, one of the best-characterized developmental systems in higher plants. In this context, mathematical approaches will be essential to analyze and establish a predictive model for how multiple hormones influence cell fate in a spatio-temporal manner.

We address a key question in developmental biology: how does an organism reach its final size and shape, in the face of stochastic variation at the cellular level? Previous research has focused on mutants and conditions that affect the global size and shape of organs, enabling the discovery of a large number of regulators. However, most analyses have considered only average cell behaviors, overlooking local heterogeneity and stochastic variation. In this context, how the final size and shape of an organism are determined remains poorly understood.
Here we adopt an orthogonal strategy by taking advantage of the observation that many organisms have a remarkably consistent shape, yet at the cellular level, cell growth and shape can be highly variable. Our goal is to resolve this apparent contradiction between the cellular and organismal levels. We propose that cell-cell communication mechanisms coordinate cell stochastic variability so as to yield consistent organs. Since growth remains locally heterogeneous, these mechanisms may, counter-intuitively, maintain or even enhance local heterogeneity.
We will test this hypothesis in Arabidopsis thaliana, as it produces a large number of almost identical flowers with stereotyped floral organs. We choose to work on the sepal, which is accessible for live imaging and mechanical measurements. Using a combination of experimental and theoretical approaches, we will identify mutants with variable sepal size and shape. Because of their role in coordination, the corresponding genes are likely to include the hormonal/nutrient pathways and mechanical sensing and response. As plant growth is driven by the internal turgor pressure and restrained by cell walls, we will assess the activity of these growth regulators by quantifying the mechanics of cells in these mutants and in wild type to causally link gene activity and shape. We will use data-driven approach and information theory to extract the underlying patterns of correlations between cells, notably to qualify the most plausible coordinating mechanisms that regulate cell stochasticity. These hypotheses will be tested by combining a mechanical model of sepal growth that incorporates identified signals and local experimental perturbations, such as cell ablation, force application, and genetic mosaics.

A challenging issue for bacterial pathogens is the need to modulate host cells behavior to allow their own successful propagation. Intracellular pathogens secrete a large number of proteins (“effectors”) into their host cells to either trigger entry, escape cellular defense, modulate cellular responses or interact with intracellular components that are critical for infection. We hypothesize that pathogens also secrete small RNAs that interact with host cellular mRNAs and non-coding RNAs, thus modulating cellular activity for their own benefit. Discovery of such RNAs among the vast amount of cellular RNAs is highly challenging due to lack of proper techniques; however, the emergence of ultra-highthroughput sequencing and computational inference (Sorek’s expertise), in combination with single-cell fluorescence Imaging techniques (Palmer) and extensive expertise in pathogenesis and cell biology (Cossart) will allow us to address this challenge. Our aim is to identify secreted RNAs using the model intracellular pathogen Listeria monocytogenes, and understand their roles in pathogenicity. Although scientifically 'risky', if successful these studies could lead to a major breakthrough in the fields of bacterial pathogenicity and RNA biology.

Persistent structural and functional modifications of excitatory synapses are suggested to represent the cellular substrate of learning and memory. Dendritic spine morphology, like synaptic function, is dynamically regulated by neuronal activity. Changes in both spine morphology and synaptic efficacy are primarily influenced by actin remodeling. However, how dynamic actin filaments are anchored within dendritic spines and control their morphology and synaptic function remains unknown.
Our working hypothesis is that anchoring of actin filaments in dendritic spines represents a critical determinant of their dynamic and plastic behavior. Anchoring to the plasma membrane (‘peripheral anchor’) likely contributes to shape spine head and hinder the local diffusion of transmembrane proteins whereas anchoring at the base of the spine head (‘central anchor’) is required to generate enlargement forces upon actin polymerization. Elucidating the role of actin anchoring in dendritic spine structure, function and learning-related plasticity therefore represents the scientific aim of this proposal. To this end, we will develop and implement a highly multi-disciplinary approach based on the complementary expertise of the participants and a combination of i) advanced in vitro and in vivo electrophysiology, ii) cutting-edge imaging techniques, molecular tracking and super-resolution microscopy, iii) innovative photo-chemical tools for protein tagging, acute photoinactivation and crosslinking and iv) analysis of network activities and specific cognitive performances in behaving animals.
We will establish the role of peripheral and central anchors of F-actin in dendritic spines. We will combine single cell electrophysiology and molecular imaging techniques to explore the functional consequences of suppressing these anchors either genetically or acutely using protein photo-inactivation (CALI) techniques. Next, we will directly visualize the 3D network of spine actin, its modulation upon long term plasticity induction as well as its alterations upon selectively suppressing peripheral or central anchoring. We will also adapt molecular tagging to reveal the molecular organization of F-actin and its anchors within dendritic spines. We will then further identify the molecular components of peripheral and central anchors of F-actin in spine using new photo-crosslinking strategies to identify the molecular partners of peripheral and central actin anchors. Finally, we will combine our efforts to explore the importance of actin anchoring regulation in learning and memory processes by combining in vivo electrophysiology, photoinactivation and Ca imaging while monitoring cognitive performances measured in behavioral assays.

The main aim of this proposal is to understand the cellular and circuit mechanisms that establish spatial coding cellular ensembles in the mammalian hippocampus. The cognitive spatial map theory of the hippocampus posits that internal representations of space are implemented by a sparse subset of 'place cells' that display location-specific firing during spatial navigation, while other neurons remain silent. Moreover, this spatial code is highly dynamic, such that place cells alter their firing properties when the spatial environment changes. The cellular and circuit mechanisms that establish sparsely distributed and dynamic spatial coding schemes in the hippocampus are poorly understood. Longstanding theories posit that the hippocampal inhibitory circuitry plays a central role in the formation and segregation of spatially informative cellular ensembles. If so, the anatomical diversity of local GABAergic interneurons may further promote dynamic organization of place cell assemblies in the population by regulating active dendritic input processing at the subcellular level, which can define the output behavior of principal cells to synaptic excitation in a given environment. Understanding multimodal dynamics in the hippocampus will, therefore, have important consequences for our understanding of how cortical circuits are organized to process and store information. To directly test these hypotheses we will perform multi-level analyses requiring a multidisciplinary approach that brings together experts in neurophysiology, nonlinear optics, molecular genetics and synthetic chemistry.
Our research plan is composed of five tightly integrated projects. 1) Functional population imaging in hippocampal CA1 of behaving mice to determine the fine-scale spatial organization of hippocampal spatial coding ensembles and their dynamic reorganization during controlled changes in the spatial environment and spatial learning. 2) Assessment of multimodal organization and dynamics in synaptic microcircuits of genetically-identified interneurons. 3) Implementation of novel cellular-resolution optical, photochemical and pharmacogenetic techniques for manipulating activity of identified inhibitory circuits in vivo. 4) Dissecting multimodal population and microcircuit dynamics of spatial coding CA1 hippocampal neuronal ensembles using cellular-resolution manipulations in vivo. 5) Measurements and manipulation of subcellular integration of excitatory and inhibitory synaptic inputs in functionally-identified hippocampal CA1 pyramidal cells during spatial navigation and learning.
This project will yield a detailed understanding of the cellular and circuit mechanisms that establish spatial coding ensembles in the mammalian hippocampus. The complexity of this problem necessitates a multidisciplinary and integrated approach.