PROJECT SUMMARY At the excitatory synapse, a heterogenous molecular layout creates a precise pre- and post-synaptic structure that facilitates different modes of neurotransmission. The spatial segregation of neurotransmission leads to the autonomous function of spontaneous glutamate release. The investigation into the structure/function relationship at the excitatory synapse has facilitated the discovery of disease pathways and targets for psychiatric disease intervention. However, aberrant inhibitory neurotransmission is also implicated in numerous psychiatric illnesses including schizophrenia, depression, and anxiety. Despite the fundamental importance of inhibitory neurotransmission in disease, few studies have investigated the relationship between nanostructure and function at the inhibitory synapse. My preliminary data suggests a segregation of action potential dependent and spontaneous neurotransmission at central inhibitory GABAergic synapses, but how this segregation is achieved is unknown. Few studies have investigated the inhibitory structure/function relationship due to the limited tools that allow for the selective manipulation of different modes of inhibitory neurotransmission. I have generated preliminary data that a novel small molecule drug, Artemisinin, selectively dysregulates inhibitory spontaneous release as it competitively binds in the GABAAR-gephyrin binding pocket; providing a new pharmacological tool that will be utilized throughout this proposal. This project will investigate the GABAergic synapse in two-fold by elucidating the post-synaptic structure that segregates neurotransmission and how this structure leads to an autonomous function of spontaneous GABAergic transmission in homeostatic plasticity. In primary hippocampal culture, Artemisinin will be used as a tool to decrease GABAergic spontaneous release to investigate how this corresponds to nanostructure and signaling pathways. The central hypothesis is GABAergic synapses have a specific post- synaptic gephyrin scaffold and GABAAR nanostructure that facilitates an autonomous role of spontaneous neurotransmission. Aim#1 will investigate how the post-synaptic structure segregates neurotransmission using super resolution and electron microscopy to assess 1a. gephyrin clustering dynamics and 1b. GABAAR subunit localization and clustering. Aim#2 will investigate the functional pathway this nanostructure facilitates by delineating the role of spontaneous GABAergic neurotransmission in homeostatic plasticity. 2a. First downstream gene expression pathways will be assessed, specifically BDNF expression. 2b. Then how spontaneous GABAergic signaling alters calcium dynamics to trigger gene transcription pathways will be delineated. This research will elucidate the relationship between structure and function at the inhibitory synapse, ultimately providing novel insight into the regulation of synaptic strength underlying excitatory/inhibitory balance in health and disease.