18Blindness to Yale University. We thank Tim Maugel and the Laboratory for Biological 19 Ultrastructure (Department of Biology, UMD) for assistance with transmission electron 20 microscopy, Alexander Baden for assistance with 3D visualization, and Kacie Furcolo and Adit 21 Sabnis for assistance with SBEM dataset skeletonization. We thank Cole Graydon for 22 comments on the manuscript. 24 Summary 31The mammalian rod bipolar (RB) cell pathway is perhaps the best-studied circuit in the 32 vertebrate retina. Its synaptic interactions with other retinal circuits, however, remain 33 unresolved. Here, we combined anatomical and physiological analyses of the mouse retina to 34 discover that the majority of synaptic inhibition to the AII amacrine cell (AC), the central neuron 35 in the RB pathway, is provided by a single interneuron type: a multistratified, axon-bearing 36 GABAergic AC, with dendrites in both ON and OFF synaptic layers, but with a pure ON 37 (depolarizing) response to light. We used the nNOS-CreER mouse retina to confirm the identity 38 of this interneuron as the wide-field NOS-1 AC. Our study demonstrates generally that novel 39 neural circuits can be identified from targeted connectomic analyses and specifically that the 40 NOS-1 AC mediates long-range inhibition during night vision and is a major element of the RB 41 pathway. 42 43 74 remain. Most significantly, we do not know the identity of the spiking, GABAergic AC that drives 75 a receptive field surround in the AII during rod-mediated vision via synaptic inhibition of the AII 76 itself (Bloomfield and Xin, 2000); understanding mechanisms contributing to surround inhibition 77 4 is important because such inhibition tunes AII responses to spatial features of the visual 78 stimulus. 79Here, we identified the major inhibitory input to the AII by combining anatomical, genetic, 80 and electrophysiological analyses in a three-step process. One, we reconstructed ACs that 81 provided synaptic input to AIIs in a volume of mouse retina imaged by scanning block-face 82 electron microscopy [SBEM; (Denk and Horstmann, 2004)]. Two, we evaluated published 83 descriptions of reporter mouse lines to identify genetically-accessible ACs that had the 84 anatomical characteristics of the cells reconstructed from SBEM images. And three, we used 85 electrophysiological recordings and genetics-based circuit analysis to demonstrate that a 86 candidate AC, which provided the great majority of the inhibitory synaptic input to AIIs, exhibited 87 a light response predicted by its anatomy and made GABAergic synapses onto AIIs. This 88 spiking, GABAergic AC is a multistratified, ON AC denoted NOS-1 AC and identified in the 89 nNOS-CreER mouse (Zhu et al., 2014). We conclude that the NOS-1 AC is an integral 90 component of the RB pathway and a significant source of long-range inhibition during night 91 vision. More generally, our study demonstrates the utility of targeted, small-scale "connectomic" 92 analysis for identification of novel neural circuits. 94 Results 95AIIs in the ...