Project Summary/Abstract Establishing how features of the visual world are represented in the activity of cortical circuits, and how these representations are constructed during development remain fundamental challenges for visual neuroscience and are central to understanding the neural basis of visual perception. A critical function of circuits in primary visual cortex is the integration of the inputs from the two eyes to create a single binocularly aligned columnar representation of stimulus features such as the orientation of edges and their direction of motion. This alignment is evident at the scale of columns, and at the scale of individual neurons: visual stimuli presented to either eye yield highly similar responses at both scales, giving rise to our coherent binocular perception of the world. Our studies during the previous period of support have revealed a surprising new and critical role for visual experience in the development of binocularly aligned columnar representations in ferret visual cortex. Prior to natural eye opening, visual stimulation thru either eye evokes highly structured columnar maps of orientation preference, but there are conspicuous mismatches in the spatial organization of these maps that reflect misalignment in the orientation preference of the inputs that individual neurons receive from the two eyes. Shared binocular experience immediately following eye opening is necessary to drive changes in the orientation preference of individual neurons that achieve a single binocularly unified representation. The goal of this proposal is to employ new technologies that we have developed that make it possible to visualize the functional organization of excitatory synaptic inputs to individual neurons in order to probe the mechanisms responsible for this alignment process. We start in Aim 1 by examining the synaptic basis for binocular integration in the mature visual cortex, combining 2-photon in vivo imaging of calcium signals with post-hoc electron microscopy to elucidate how the numbers, strength, and the spatial arrangement of functionally characterized synapses contribute to the binocular response properties of individual layer 2/3 neurons. In Aim 2, we examine the functional synaptic architecture of binocular integration early in development and use chronic imaging techniques to probe changes induced by the onset of visual experience. In Aim 3 we combine in vivo imaging with expansion microscopy to explore how 2 major sources of inputs to layer 2/3—feedforward inputs from layer 4, and horizontal connections from other layer 2/3 neurons--contribute to the mature pattern of binocular integration and to the development of binocularly aligned representations. Taken together, we believe these experiments will yield a wealth of new insights into the synaptic basis for binocular integration and the experience-dependent mechanisms that achieve aligned representations in visual cortex.