PROJECT SUMMARY Bipolar cells (BCs) are the first interneurons in the retina and are responsible for sampling photoreceptor output and distributing it to ON and OFF streams. Each BC subtype collects and transforms different salient components of the visual scene (luminance, frequency, contrast, etc.), yet the specific mechanisms different subtypes use to process this information into functional channels remain elusive. This proposal focuses on the CBC2 (Type 2 OFF cone bipolar cell). In addition to its conventional role processing photoreceptor output for downstream retinal ganglion cells, the CBC2 is the primary recipient of inhibitory crossover inhibition from AII amacrine cells and provides the majority of glutamatergic feedback onto AIIs, positioning it as an important mediator of bidirectional signaling between ON and OFF steams across the retina. This project will characterize distinctive and novel physiological features of CBC2s to determine their unique contribution to retinal function. My preliminary experiments have identified three particularly interesting features of CBC2s that have not been seen in ON-BCs. I hypothesize that CBC2s, and perhaps OFF-BCs in general, possess unique mechanisms that are likely fundamental to their role in the retina. This proposal will examine each feature through three primary aims. Aim 1 will use electrophysiology, pharmacology, and super-resolution immunofluorescence microscopy to identify and characterize a postsynaptic glutamate receptor in CBC2 dendrites that may augment conventional excitatory input from photoreceptors. Aim 2 will combine patch-clamp electrophysiology with time-resolved membrane capacitance measurements to examine the consequences of microdomain control of vesicle exocytosis on short-term plasticity and the voltage-dependence of exocytosis. Finally, Aim 3 will investigate reciprocal glycinergic feedback between CBC2s and AII amacrine cells and use Ca2+ imaging and computational modeling to create a biophysical model of glutamate release from CBC2s. Combined, these experiments represent the first comprehensive examination of the biophysical properties of the mammalian CBC2, a vital step in determining the role it plays in retinal function. While the von Gersdorff lab has a proven and published record in the realm of retinal biophysics, my proposal will require learning diverse and novel techniques, such as super- resolution microscopy and computational modeling, for which I have sought specialized training from outside my lab. I have carefully assembled a mentorship team to provide me with the technical expertise and dedicated guidance required to achieve my planned scientific aims and further my professional goal of a career in academic research.