Second messengers including cGMP, cAMP and calcium are signaling molecules shared by many pathways. Although modulated by a plethoric number of intra and extracellular signals, these molecules drive selectively their various downstream effectors. The mechanisms of such a specificity are poorly understood. The subcellular control of second messengers emerged as a flexible strategy to provide specific and coordinated regulation of cellular events. We aim at demonstrating that spatially restricted signals are critical to achieve specific shape transformations of growing processes exposed to extracellular cues in the developing nervous system. Signals at the tip of leading processes during axon pathfinding (using retinal ganglion cells as a model) and in the centrosomal region during neuronal migration (focusing on cortical interneuron) will be analyzed. We will identify the local events occurring in two signaling hubs: the lipid rafts at the plasma membrane and the primary cilium.

Ciliopathies are human pathologies related to abnormal functioning of the primary cilium located at the surface of most cells of the body. They are characterized by developmental abnormalities and generally affect the brain. They are not necessarily associated with visible structural defects when using brain imaging techniques, and can induce functional defects such as behavioral or mental disability. An essential function of the primary cilium in all vertebrates is to control the activation, and conversely the repression, of transcriptional targets of Sonic Hedgehog ( Shh). The binding of Shh to its receptor Patched causes the accumulation of Smoothened (Smo), the unique transducer of Shh, in the primary cilium and the activation of transcriptional targets by Gli factors. This function of Shh is essential during early development of the nervous system. It is also involved in the formation of cancers. Its role at intermediate stages of brain development is uncertain, especially during cell migration. Shh continues to be expressed in the brain during the phase of cell migration. Then it functions as a diffusible cue able to guide the migration of axonal growth cones and of migrating cells. It can play either and attractive or a repulsive role. This activity is independent of Gli factors and does not seem to involve the primary cilium. Both gene transcription and chemotaxis activities of Shh are often presented as mutually exclusive. It is established that they act on different cellular processes, whose mutual independence is not clear. The objective of MIGRACIL is to study the cellular mechanisms by which Shh controls the migration of a population of cortical neurons, the future inhibitory interneurons during embryonic development, and to distinguish between mechanisms controlled by the transcriptional activity and by the chemotactic activity of Shh. Recent studies in which the first partner contributed, showed that interneurons born outside the cortex assemble a primary cilium during their migration to the cortex. These neurons join the cortex after re-orienting their trajectory, a phase sensitive to Shh and which involves primary cilium activity. We now wish to understand the respective roles of Shh and the primary cilium in the final targeting of interneurons to the cortex . To characterize the role of transcriptional and non- transcriptional pathways activated by Shh during migration in cortical interneurons, we intend to analyze by imaging techniques and cell biology approaches the in vivo and in vitro migratory behavior and the response to Shh of interneurons that suffer i / an ablation of ciliary genes, which impairs the normal function of the primary cilium , ii / an ablation of genes involved in the canonical Shh signaling pathway and iii / an ablation of Smo, the mandatory transducer of Shh in and outside the primary cilium. After settling in the cortex, interneurons establish inhibitory circuits to control the activity of the other cortical neurons. Abnormal positioning of interneurons leads to functional defects in both excitatory and inhibitory circuits, and cognitive alterations. We will study the activity of these circuits by electrophysiological and behavioral techniques in conditionnal mutant mice with ciliary genes or Smo invalidation in interneurons. Finally, we will study the histological organization and interneuron distribution in the cortex of fetuses with ciliopathies in order to identify structural abnormalities associated with mutations in ciliary genes and in the SHH pathway. These studies should allow us to increase the understanding of the mechanisms by which Shh controls the migration and development of inhibitory circuits in the cerebral cortex, and should lead to develop new hypotheses about the pathophysiological origin of cortical abnormalities associated with ciliopathies in humans .

Contextual fear memory is a conserved, still mysterious, process allowing vital response to dangerous situations. Microglia, which are the resident macrophages of the brain, are known to contribute to synaptic remodeling, by eliminating or promoting the appearance of synapses, and it has recently been shown that they also exert an inhibitory control over the activity of neurons. They can therefore potentially contribute to memory processes, but upstream signals regulating their actions on neurons and synapses are unknown. The neuromodulator serotonin is a good candidate, given its role in plasticity and our identification of functional serotonin receptors in microglia, the 5-HT2BR subtype. We have shown that serotonin, when applied locally to acute brain slices, induces an attraction of microglial processes, which depends on these 5-HT2B receptors. Recently, we have also shown that in the absence of this receptor since birth, microglia are more reactive, and in case of peripheral inflammation, the induced neuroinflammation is stronger and takes longer to resolve. Thus, serotonin has both acute and long-term effects on microglia, both on their morphology and function. Finally, our unpublished results indicate that when this receptor is inactivated in microglia, from adulthood onwards in order to avoid developmental effects, mice can learn to memorise an association between a context and a danger, but this contextual fear memory is not reinforced, not consolidated, beyond 1.5 hours. Morevoer, in these mice, the increase in dendritic spines density which is normally induced 48h after contextual fear conditioning, is not observed. Our working hypothesis is thus that microglia and serotonin work together to allow contextual fear memory consolidation, the microglia being regulated, engaged, by 5-HT in the hippocampus to act on neurons. Combining sophisticated genetic and viral models (conditional KOs and GFP expression in microglia, biosensors to detect changes in serotonin levels in vivo in behaving animals, optogenetics to stimulate serotonergic neurons, viral infections to locate dendritic spines in hippocampal neurons), cutting-eddge in vivo imaging approaches (fiber photometry, GRIN lenses and multiphoton imaging in the hippocampus on awake animals), and transcriptomic and translatomic approaches, we will investigate the provocative hypothesis that microglia, under the timely control of 5-HT, contributes to contextual fear memory consolidation. One of our goals will be to understand the mechanisms by which microglia, under the control of serotonin, modulates the stability of synapses and thereby contributes to the consolidation of fear memory. Finally, along these experiments, we will develop a microglial activation reporter system in order to be able to identify and tag, specifically, the microglial cells which are actively engaged upon fear conditioning. This would allow to follow and study them specifically, and to compare them to other microglial cells that have not been activated by fear conditioning. We aim at producing a versatile mouse system that may have many more applications for studying activated microglial cells in other contexts, like stress, addiction, inflammation. Our results, by documenting the involvement of microglia and serotonin in the consolidation of contextual fear memory, will suggest new therapeutic targets to treat emotional memory disorders, for example post-traumatic stress disorders, in which contextual emotional memory is abnormally generalized. Moreover, they could open to fascinating hypotheses about the potential modulation of this memory and thus of our behaviour by situations such as infection, stress, and changes in microbiota, which are known to affect microglia.