Specification and integration of interneuron subtypes in the neocortex

Cortical microcircuit function relies on the coordinated activity of a large diversity of interneuron (IN) subtypes. These cells originate from discrete areas of the subpallium, namely the medial and caudal ganglionic eminences (MGE and CGE) and the preoptic area (POA). From these sites of origin, INs migrate long distances to reach the developing neocortex and integrate into the cortical microcircuits. Understanding the emergence of their molecular diversity and how they develop in the neocortex will provide important insights on the architecture of the mature circuit in health and disease. In my PhD project, I studied molecular mechanisms controlling the specification, migration and maturation of IN subclasses. In the first study, I aimed at identifying the developmental origin of a specific subclass of cortical IN called neurogliaform cells (NGCs). These cells are recruited by long-range connections, such as interhemispheric and thalamic projections, and are thought to be the effectors of a powerful inhibitory circuit by activating metabotropic GABAB receptors. Using in vivo lineage-tracing in mice, I found that NGCs originate from a pool of cells located in the POA, co-expressing the transcription factor Hmx3 and the serotonin receptor 3A (HTR3A). Through a combination of methods, I found that Hmx3-derived HTR3A+ cortical IN exhibited the molecular, morphological and electrophysiological profile of NGCs. Overall, these results indicate that NGCs are a distinct class of INs with a unique developmental trajectory. In the second study, I focused on mechanisms regulating laminar allocation of superficial neocortical INs. In a previous study, we found that HTR3A controls the migration and laminar positioning of superficial cortical INs, but molecular mechanisms remain unknown. Using a microarray screen on wild-type and Htr3a-KO INs, I identified PlexinA4 (PlxnA4) as a candidate gene possibly acting downstream the Htr3a and specifically upregulated during the phase of cortical plate invasion. Using in vitro and in vivo strategies, I found that PLXNA4 ligand SEMA3A has chemorepulsive effect on PLXNA4+/HTR3A+ superficial INs and that these effects are mediated by the PLXNA4/NRP1 receptor complex. Interestingly, SEMA3A was found to be secreted by deep layer INs, which do not express the HTR3A. Overall, these results suggest a new guidance mechanism for migrating HTR3A+ INs, involving a HTR3A-dependent upregulation of PLXNA4 in superficial cortical INs. These PLXNA4+/HTR3A+ cells will become gradually sensitive to the repulsive ligand SEMA3A, secreted by deep layer INs, and will preferentially settle into superficial ones. In the third study, I aimed at characterizing the role of the potassium/chloride cotransporter KCC2 in INs at early developmental stages. KCC2 plays a main role in driving GABAAR-mediated inhibition in the mature cortex, by tuning intracellular chloride concentration. Interestingly, it is upregulated in INs earlier than in excitatory cells. To determine the role of KCC2 in IN development, I aimed to selectively knock-out this cotransporter in cortical INs using cre-lox approach. Analyses of the somatosensory cortices indicated that IN-specific deletion of KCC2 significantly decreased the density of parvalbumin (PV)-expressing cells in recombined mice and non cell-autonomously affected the morphological maturation of pyramidal neurons. These results suggest an important role for KCC2 in the maturation of PV-expressing INs. Taken together, studies performed in this PhD thesis provide new insight on molecular mechanisms regulating the specification, migration and laminar allocation of cortical INs.