The establishment of neural connections requires activity-dependent and independent mechanisms. The well-understood organization of the visual system comprises an accessible model for analyzing the contribution of each of these factors in the formation of connectivity. Retinal projections form a visual map in their target organized in a topographic and eye-specific manner. This specific organization is crucial for vision. The goal of this project is to understand how activity and molecules shape the visual map during development of the visual system in the mouse. We have recently identified a novel role for PlexinA1, a semaphorin receptor usually studied in axon guidance, in the formation of the visual map. Our preliminary results show that PlexinA1 is required for the fasciculation and proper targeting of retinal axons. We propose to use various tracing and anatomical techniques to determine the origin of these abnormalities and to identify which ligand of PlexinA1 is responsible for the defects observed in PlexinA1-KO mice. We will then focus on the role of activity on the formation of visual maps. Before eye opening, retinal waves are critical for the proper refinement of visual maps, both topographic and eye-specific, yet whether calcium-dependent synaptic release is required has not been investigated. Here, we propose to use a conditional deletion of RIM proteins to strongly reduce calcium-dependent synaptic release specifically in different RGCs by mouse genetics and in utero retinal electroporation techniques. This will allow us to study the importance of calcium-dependent synaptic release in a general manner but also at the level of axon-axon interaction and to determine if this effect is cell autonomous or not. In addition, we will use the same genetic mouse model to study the consequences of the reduced calcium-dependent synaptic release on gene expression, and hope to identify possible activity regulated candidates involved in map formation. Finally, we will focus on the role of serotonin on visual map formation. Increased levels of serotonin perturb visual map formation and 5HT1B receptor and SERT transporter are expressed transiently in the visual system during the period of map formation. Based on these results, we will first investigate if serotonin has a role on retinal axon growth and guidance using in vitro assays in combination with pharmacological treatment. Furthermore, we will study the role of serotonin on map refinement. Our working hypothesis is that the specific expression of SERT on ipsilateral RGCs will allow the local modulation of serotonin concentration by clearing serotonin from the ipsilateral synapses. As the 5HT1B receptor is expressed in all RGCs, the 5HT1B receptor will be sujected to differential concentrations of serotonin and thus will modulate differently glutamatergic transmission and/or the response to various guidance cues. We aim to perturb in vivo serotonin signaling by using in utero electroporation of SERT or the 5HT1B receptor, to see if the retinogeniculate axon projection is perturbed. This will allow us to determine the precise role of serotonin on retinal axon guidance and map refinement at the target and thus better understand the possible consequences on fetal development of selective serotonin reuptake inhibitors (SSRIs) used to treat depression in pregnant and breast-feeding women. Finally, we will study the interplay between these different mechanisms, looking for example whether serotonin acts through modulation of synaptic release or whether activity can regulate the expression of molecules known to be involved in visual map formation. Understanding the cellular and molecular mechanisms of visual map formation will provide insight into how vision is established. More generally, this project will help decipher how neural connections are formed and refined to better comprehend the mechanisms at hand in neurodevelopmental diseases such as schizophrenia and autism.

During the development of the cerebral cortex, the radial glia give rise to neurons, which migrate into the upper layers of the cortex. Proliferation, differentiation and migration are processes that must be tightly regulated. Morphogens are extracellular proteins that act at several stages of these processes, but their dynamics and precise molecular pathways are not always clear. In particular, how cells can change their responses to a single morphogen during development is a central and still open question. By studying single-cell RNA-seq data, we discovered that the expression of specific glypicans (Gpc) appears to be associated with different stages of cortical development and different types of signaling pathways, including Sonic Hedgehog (Shh), a morphogen essential for neuronal development. Furthermore, our preliminary data show that manipulation of Gpc gene expression levels in the embryonic brain differentially affects cell distribution in cortical layers. Thus, we hypothesize that each specific Gpc is closely associated with a certain type of cellular response. In this project, we aim to 1/ characterize Gpc-Shh interactions using biophysical and structural approaches and 2/ identify how distinct Gpc differentially regulate specific processes, such as differentiation, neuronal migration and morphogenic responses, in the developing cortex. Overall, the GLYPHH project will identify the molecular code that triggers specific Shh signaling and provide new insights into the roles of specific glypicans in the developing brain. Finally, this study will have an impact on several fields such as glycobiology, neuronal development and cancerology.

Brain reward system plays a crucial role to maximize fitness by adapting behavior to environment contingencies (e.g. to optimize food consumption). Deciphering the cellular and molecular mechanisms by which activation of reward circuits leads to long-lasting behavioral adaptations is a major challenge for neuroscience. This information is highly relevant for medicine and society at large since addiction and eating disorders, two major health problems of modern societies, involve dysfunction of reward systems. Dopamine neurons play a central role in the reward system, by reporting several aspects of reward prediction and value and controlling the function and plasticity of target neurons. However, very little is known about the cellular populations that are actually modified when an animal learns a rewarding task and the possible existence and nature of persistent molecular modifications in these cells. In this project we propose to address this issue by using a simple incentive learning protocol based on food reward and focusing our analyses on striatal projection neurons that are a major target of dopamine. Our general aim is to identify persistent transcriptional/epigenetic traces of operant learning in functionally relevant ensembles of striatal neurons. We will use a combination of powerful mouse models to identify changes in mRNA and DNA modifications following operant conditioning for regular or highly palatable food. We will determine the persistence of these modifications and we will identify in which neuronal populations they take place. We will use an inducible tagging strategy to identify neuronal ensembles specifically activated during conditioning and will characterize the morphological and physiological changes occurring in these activated neurons. We will test their functional role in behavior by blocking their activity with a chemogenetic approach. We will then try and establish a causal relationship between mRNA and epigenetic modifications in these neurons and cellular or behavioral adaptations by specific targeting of candidates genes. This project will shed light on molecular and cellular mechanisms of incentive learning and and memory. It will provide useful information and potential targets in the context of obesity-related eating disorders.

Sleep and emotional functions are tightly linked. Sleep benefits both emotional memory consolidation and emotional regulation. These two aspects of emotional processing are key to the survival of an individual, ensuring the retrieval of emotional memories and appropriate responses to threatening or rewarding information. The mechanisms that underlie emotional processing in sleep, however, are still unclear. Reactivation of waking experiences during sleep has been found to support memory functions, and may similarly underlie emotional consolidation and emotional regulation. Moreover, rapid-eye-movement (REM) sleep has emerged as a promising candidate state to support emotional functions: it benefits emotional memory consolidation, aids the regulation of emotional responses, and is disrupted in emotional disorders. While the neural dynamics of memory reactivation have been extensively studied in non-REM sleep, little is known about information processing during REM sleep and its role in emotional processing. The overarching goal of this proposal is to determine the role of REM sleep for emotional memory consolidation and emotional regulation. We are proposing an innovative, translational approach with parallel experiments in humans (M. Schonauer) and rats (G. Girardeau) along two aims. First, we hypothesize that the valence of a previous learning experience is processed during REM sleep, supporting the consolidation of emotional memories. We will test whether we can detect and quantify reactivation of negative or positive experiences during REM sleep in core structures of the emotional memory network using novel machine learning methods (Aim 1). Further, we expect that emotional regulation is linked to homeostatic neural processes occurring during REM sleep, which could co-occur or compete with memory consolidation. We will record and selectively perturb activity in structures involved in emotional processing during REM sleep to test how this influences emotional responses at the behavioral and physiological levels (Aim 2). To do so, we will record EEG and fMRI in humans while they are exposed to positive, neutral, and aversive stimuli and during subsequent sleep periods. In rats, we will combine in vivo large-scale electrophysiological recordings in the amygdala, hippocampus, and prefrontal cortex with optogenetic manipulations during positive and aversive spatial tasks and sleep. Disturbed sleep and dysfunctional memory processes have been linked to the development and maintenance of most psychiatric disorders, including post-traumatic stress disorder. Our project will significantly further our understanding of memory and emotional regulation during sleep. This is crucial to develop a coherent model of emotional functioning, and, in the long-term, tailor new treatments and interventions targeted at sleep.

Brain plasticity that underlies memory function likely results from a balance between synapse formation, potentiation and removal. The synapse formation and potentiation occur mostly during wakefulness, while synaptic downscaling and removal take place mostly during sleep. Immune signaling molecules present in the healthy brain can interact with neurochemical systems including serotonin to contribute to the regulation of normal sleep. While it has been widely shown that sleep alterations impair learning, there remains much debate over the mechanisms involved. Recent studies revealed key tasks for microglia, the main immune cells in the brain, interactions with neurons during normal physiological conditions, especially in regulating the maturation of neural circuits and shaping their connectivity in an activity- and experience-dependent manner. However, their role in learning and memory remains elusive. Specifying the role of microglia in synaptic consolidation according to the sleep/wake cycle is undoubtedly a particular challenge to research. Consortium members have shown (i) that microglia motility can be modulated by serotonin, whose levels vary with arousal/sleep states, and (ii) that microglia dynamics is different in arousal vs. sleep states and (iii) that mice lacking 5-HT2B receptors, which is the main serotonin receptor expressed by microglia, display sleep and memory deficits. Together, these observations support the hypothesis that microglia and serotonin actively participate to memory consolidation by regulating synaptic plasticity, that microglia interact with synapses in different modes and potentially with different outcome during sleep and wakefulness, and that serotonin could be implicated in this switch. The role of microglia in learning and/or cognitive flexibility could therefore be more prominent during either the wakefulness or the sleep period. Exploring this hypothesis, the consortium will delineate the signaling pathways induced in microglia by serotonin, and show how serotonin participates to microglia dynamics and to synaptic plasticity. By multidisciplinary approaches combining the analysis of EEG (electroencephalogram) and in-vivo imaging, field potential in acute slices, behavior, conditional mutants, optogenetic and chemogenetic (DREADD) approaches, we propose to elucidate how a control of microglia by serotonin is implicated in synaptic and brain plasticity and if this occurs mainly during awake-learning phases, or asleep-consolidation phases and by which intracellular pathway. This proposed preclinical approach includes a totally new pathophysiological axis since no one has ever established a link between immune cells microglia, memory consolidation and sleep. Thus it may open new perspectives for pharmacological treatment of neuropsychiatric disorders, which are associated with microglia activation, and/or sleep alterations such as schizophrenia, autism and depressive disorders.