Project Summary Vision arises from the combination of viewing and visual processing. How animals align viewing strategies with regional processing specializations to accomplish specific behavioral tasks is unclear. We recently discovered that mice use binocular vision to pursue and capture insects. Here, we follow up on this discovery to understand the retinal signals and downstream pathways that mediate binocular vision for predation (Aim 1) and control the gaze to keep targets within the binocular visual field (Aim 2). Mammalian binocular vision relies on the presence of ipsilaterally projecting retinal ganglion cells (RGCs). We recently reported that nine of the 40+ mouse RGC types have ipsilateral projections. Here, we test the hypothesis, that two ipsilaterally projecting RGC types, the sustained ON and sustained OFF alpha (sONα- and sOFFα-) RGCs, guide binocular predation. We have developed intersectional transgenic tools to selectively label, silence, and remove the ipsilaterally projecting sONα- and sOFFα-RGCs (~300 cells, ~0.6% of RGCs). The sONα- and sOFFα-RGCs show increased density and acuity (i.e., reduced receptive field size) in the ventrotemporal retina. In Aim 1, we will combine transgenic and immunohistochemical labeling and high-resolution imaging of whole retinas to understand the organization of the sONα- and sOFFα-RGC acute zone in the ventrotemporal retina. We will analyze how sONα- and sOFFα- RGCs encode local, global, and combined motion individually and as populations with targeted patch clamping and two-photon calcium imaging. Visual stimulus parameters will be based on analyses of our large 3D tracking dataset of mice hunting crickets. Next, we will assess the binocular processing of sONα- and sOFFα-RGC signals by specific neurons in the superior colliculus, the retinorecipient target mediating predation. Finally, we will measure the contributions of ipsilaterally projecting sONα- and sOFFα-RGCs to predation using selective silencing and removal and 3D behavior tracking. In Aim 2, we will test the hypothesis that ON-OFF direction- selective (DS-) RGCs control the gaze to keep prey within the binocular visual field during pursuit and capture. Combining two-photon calcium imaging and immunohistochemistry, we will analyze the topographic maps of DS-RGC direction preferences around the sONα- and sOFFα-RGC acute zone. We will use targeted patch clamp recordings and two-photon calcium imaging to understand how ON-OFF DS-RGCs encode local, global, and combined motion stimuli with ethologically relevant parameters and in vivo electrophysiology to analyze the transformation of their signals by specific neurons in the superior colliculus, which mediates gaze shifts. Finally, we will test the impact of removing direction selectivity from ON-OFF DS-RGCs on gaze control during predation. Our studies will reveal how two conserved RGC subclasses, their regional specializations in the retina (acute zones and topographic direction pref...