Project Summary/Abstract A fundamental challenge of visual cortical neuroscience is to understand how sensory representations are transformed within and across layers of primary visual cortex (V1). Visual signals are thought to propagate in a feedforward manner from the thalamus through the layered structure of V1, from L4 (L4) to layer 2/3 (L2/3) to layer 5. Here, we will address two major steps of visual processing: what are the mechanisms by which L4 transforms thalamic inputs, and how are representations transformed between L4 and L2/3? Recent studies in mouse V1 suggest that L4 amplifies thalamocortical inputs, but the mechanisms by which such amplification may occur, such as via local recurrent circuitry, have not been tested. Also, models from Hubel and Wiesel proposed that the formation of complex receptive fields in L2/3 arises from the combination of L4 simple cell inputs with similar orientation tuning and phase-offset subfields. Recent experimental work has shown preferential connectivity between L4 and L2/3 neurons that are co-tuned for different visual features, providing support for a potential mechanism by which selectivity is inherited. However, bridging the scales of anatomy and activity measurements to reveal cortical transformations has previously been challenging due to a lack of appropriate causal and functional methods. The goal of this proposal is to employ novel single-neuron resolution in vivo optical approaches for causally perturbing and monitoring neural activity to understand computations within and across layers during visual processing in awake animals. Our lab has developed a method, called influence mapping, for simultaneous two-photon optogenetic photostimulation of targeted individual neurons while imaging the responses of neighboring populations with known visual tuning properties. Aim 1 will use influence mapping in L4 of V1 to test the hypothesis that recurrent connectivity in L4 amplifies visual signals via a “like-excites-like” motif and determine whether such a motif is functionally operational for processing in different visual stimulus regimes (i.e., low vs. high contrast). The studies outlined in Aim 2 will directly test whether L2/3 complex receptive fields are built from L4 simple cell inputs by photostimulating functionally defined L4 neurons while imaging responses in L4 and L2/3. These experiments will advance our understanding of transformations within and between layers in visual cortex. In addition, the new technical approaches developed may serve as a foundation for future studies of laminar cortical mechanisms that underlie visual processing.