Project Summary/Abstract The long-term goal of this research is to understand visual processing in the inner plexiform layer of the retina. More immediately, the research serves to provide genetic access to distinct glycinergic amacrine cell types for functional characterization and to understand how they shape the response properties of retinal ganglion cells (RGCs). Amacrine cells (ACs) are the most diverse neurons in the retina, with at least 60 types of ACs identified through single-cell RNA sequencing in the mouse retina, along with the 40-50 AC types described in morphological studies. It is believed that each AC type has a distinct functional role in visual processing, yet the correspondence is only known for fewer than 20 types. Within the 60 identified AC types, approximately 13 are characterized as glycinergic ACs with small dendrites spanning multiple vertical layers. Our understanding of the functions of glycinergic ACs is limited to only about 5 of these types. For most of the remaining glycinergic ACs, we are constrained from assigning precise functions to these genetically and morphologically defined cell types. We propose to use mouse intersectional genetics, combined with functional imaging and electrophysiology recording, to dissect AC circuits in three separate Aims. In Aim 1, we will create Cre/tTA and Cre/Flp intersectional strategies to discover new glycinergic AC types and to minimize the labeling to the fewest possible types. In Aim 2, we will investigate how visual responses are generated and organized across the laminar depth of the dendrites of the newly discovered glycinergic AC types. In Aim 3, we will identify the postsynaptic RGCs of the glycinergic AC types with intersectional ChR2 activation. We will further examine their functional roles with chemogenetic inactivation. We will begin with COMS-AC, a newly identified glycinergic amacrine cell and map its synaptic connectivity with multiple co-stratifying RGCs, followed by testing the hypotheses that it plays a multitasking role in different RGC circuits with selective inactivation. We will use both hypotheses driven and discovery-based approaches to gain insights into the circuit functions of glycinergic ACs. This research will enhance our comprehension of visual processing in the inner retina, and the technologies developed will represent significant advancements compared to existing methods for studying amacrine cells, with potential applicability to other brain circuits. Moreover, this project will offer valuable insights into the pathophysiological neuronal mechanisms underlying retinal diseases, contributing to the development of improved therapeutic strategies.