The brain responds to experiences in the world through the modification of individual synapses. Changes to synaptic architecture underlie the cellular basis for learning and memory and synaptic dysfunction results in a range of neurodevelopmental and psychiatric disorders including epilepsy, autisms, and schizophrenia. The strength of synaptic connections is governed by the underlying molecular architecture at the post-synaptic density; neurotransmitter receptors, adhesion proteins, signaling molecules and cytoskeletal elements all interact in transient and highly regulated ways to shape neurotransmission. Modular scaffolding proteins play a decisive role in this organization. Recent studies at glutamatergic excitatory synapses have demonstrated that postsynaptic scaffolding proteins are not homogeneously distributed but instead are clustered near pre-synaptic active zones into subsynaptic “nanodomains” within the postsynaptic membrane. This organization is thought to facilitate fast, efficient transmission. The subsynaptic organization of inhibitory synapses remains poorly characterized, although analogous principles likely apply. The functional significance of this nano-scale organization at either excitatory or inhibitory synapses remains unclear due to a lack of tools for inducibly and reversibly disrupting molecular architecture while simultaneously measuring synaptic function. In this proposal I will address this using a novel optogenetic approach we have developed for rapidly (within seconds) and reversibly (within minutes) perturbing the nanoscale architecture of the major inhibitory postsynaptic scaffolding protein Gephyrin. I will utilize the optical dimerization protein CRY2olig, which self- oligomerizes within seconds of exposure to 488 nm light12, attached to an intrabody against gephyrin (CRY2olig- GephIB). This novel optogenetic tool provides an approach to acutely perturb endogenous gephyrin organization in real time. In preliminary experiments we find a robust and persistent decrease to inhibitory synaptic strength in cells expressing CRY2olig-GephIB within 60-120 seconds of photo induced cross-linking. I will use this optogenetic tool in combination with live cell imaging, electrophysiology and super-resolution microscopy to directly test the effect of manipulating subsynaptic scaffolding domains on synaptic transmission. This approach will not only provide novel insight into synaptic function but will also fill a major gap in the optogenetic toolkit for new approaches studying circuit dynamics through rapid and direct manipulation of synaptic strength.