PROJECT SUMMARY/ABSTRACT: Proper cortical network development and function is reliant on the generation, maturation, and activity of numerous cell types in addition to their complex cell-cell interactions. Essential to this process is the output of glutamatergic pyramidal neurons (PyNs), which is highly modulated by inhibitory GABAergic interneurons. One subset of interneurons that exerts powerful control over PyN spiking is the chandelier cell (ChC), which forms connections specifically at the site of action potential initiation in PyNs, referred to as the axon initial segment (AIS). Due to the unique connections formed between the terminals of ChC axonal arbors and the AISs of large populations of spiking PyNs, ChCs are physiologically poised to regulate the output of excitatory cortical networks. As a result, it is not surprising that disruptions in ChC biology have been linked to autism spectrum disorder (ASD) and schizophrenia, debilitating mental health disorders resulting from aberrant neuronal network activity. Despite the importance of ChCs, virtually nothing is known about the molecular factors governing their selective innervation at the AIS of neocortical PyNs. By performing a novel, in vivo RNA interference (RNAi) screen against PyN AIS-specific and -enriched adhesion molecules, this study intriguingly revealed an essential role for the axonal cell adhesion molecule L1CAM in ChC/PyN AIS innervation. Specifically, L1CAM knockdown in neocortical PyNs was found to significantly reduce PyN AIS innervation by ChCs, thus identifying L1CAM as the only known molecule to date to regulate ChC/PyN subcellular target recognition. This application aims to elucidate how L1CAM governs ChC/PyN AIS innervation and to identify the presynaptic binding partner(s) of L1CAM on ChC axon terminals. To this end, Aim 1 will use molecular tools to disrupt interactions between L1CAM and the AIS cytoskeleton to determine whether cytoskeleton-mediated L1CAM clustering is necessary for proper ChC/PyN AIS innervation. In addition, molecular replacement strategies will be utilized to investigate L1CAM’s subcellular distribution on the AIS and axon of neocortical PyNs in vivo. Finally, Aim 1 will determine whether PyN L1CAM is required early on for the establishment and/or later on for the maintenance of ChC/PyN innervation using RNAi technology in combination with in utero electroporation (IUE)- and adeno-associated viral-based strategies. Aim 2 focuses on identifying the presynaptic binding partner(s) of L1CAM on ChC axon terminals. Based on mutant L1CAM screening, neuropilin-1 (Nrp1) is hypothesized to serve as the presynaptic partner of L1CAM on ChCs necessary for proper ChC/PyN innervation. To test this, experiments utilizing ChC- targeting IUE, RNAi technology, and conditional N...