In many parts of the nervous system, interneurons, which mediate local interactions within a circuit, are more diverse than projection neurons, which transmit information between subsequent circuits in a pathway. Due in part to this diversity, the functions of many interneurons are unknown and general operating principles of interneuron circuits remain to be identified. The diversity of interneurons may be greatest in the retina, where approximately 40 distinct types of amacrine cells (ACs) form specific patterns of connections with bipolar cells, which transmit photoreceptor signals from the outer to the inner retina, and retinal ganglion cells, which transmit retinal information to the brain. Most AC types release GABA or glycine, and many release excitatory neurotransmitters or neuromodulators as well (i.e. dual transmitter neurons), further enhancing the diversity of their signals. Here, we will analyze the contributions of specific AC types to motion processing in the retina and to characteristic behaviors elicited by different forms of visual motion. In doing so, we will test a set of general principles (i.e. functional modularity), which we hypothesize govern the operation of AC circuits. We recently identified VGluT3-expressing ACs (VG3-ACs) as local motion detectors in the retina, and showed that VG3-ACs provide excitatory input to object motion sensitive ganglion cells. The selectivity of this circuit relies on fast inhibitory inputs that cancel responses to global motion stimuli. Which AC type(s) provide this input is currently unknown. Preliminary results show that two genetically identified wide-field AC types form inhibitory connections with object motion sensitive ganglion cells. In Aim 1, we will test whether either or both AC types inhibit additional tiers of the excitatory axis of this circuit (i.e. bipolar cells, VG3-ACs). We will then use mice in which these ACs are transiently or stably silenced, or are removed from mature retinas, to probe the functional contribution of their input to motion processing in the object motion sensitive circuit. In addition, we will assess their influence on orienting responses of mice to local motion stimuli. Optogenetic experiments suggest that VG3-ACs provide excitatory input to additional ganglion cell types, with distinct motion preferences. Whether this input occurs during vision, and how VG3-ACs contribute to motion processing in these circuits and influence characteristic behaviors elicited by different forms of visual motion is unclear. In Aim 2, we will test the functional significance and anatomical basis of excitatory input from VG3-ACs to different motion sensitive ganglion cells and assess changes in behavioral responses to visual motion in mice in which VG3-ACs are transiently or stably silenced, or are removed from mature retinas. Intriguingly, preliminary results indicate that VG3-ACs provide selective inhibitory input to a ganglion cell that is suppressed by motion. We will analyze the patterns and function of these connections and test the contribution of this target-specific use of dual transmitters to suppressive responses of these ganglion cells.