PROJECT SUMMARY The proposed research will use state of the art electron microscopic (EM) connectomics combined with specific pathway tracing to analyze the convergent inputs involving cortical and thalamic circuitry in mice. Labeling will be via different types of engineered ascorbate peroxidase (APX) that will tag either cytoplasm for some injections or mitochondria for others. Volumes of cortex and convergent inputs to be analyzed are: thalamic VPm & POm input to S1; thalamic LGN & Pul input to V1; S1 & POm input to M1; thalamic POm & VA/VL inputs to M1; and layer 5 inputs from different cortical areas to a thalamic nucleus (those to be initially analyzed are V1 & S1 convergent input to LD and V1 & V2, to Pul). We shall also analyze in each of the cortical areas under connectomic analysis inputs from layer 4 to layers 2/3 and local lateral connections within layers 2/3. The detailed connectomics analysis will allow us to determine the different connection patterns of the various labeled convergent afferents, including the extent to which they converge onto single cells. Regarding the analysis of cortical layer 5 convergent inputs to a thalamic nucleus, we expect this to address an open question of great import regarding thalamic functioning. Limited studies so far suggest that there is little or no functional convergence of different information streams onto individual thalamic relay cells. Thus retinal input to LGN cells involves little or no convergence, and even when there is little convergence (usually ≤3 retinal axons), the inputs are of the same type (e.g., X or Y retinal axons) with virtually no mixing of types. This suggests a simple relay function for thalamus. However, these analyses have been mostly limited to first order thalamic nuclei (i.e., those receiving driving input from subcortical sources, like the retina). Recent study of higher order nuclei (i.e., those receiving driving input from cortical layer 5) suggests that these might have convergent input from different cortical areas. We will test this, and if we find such convergent input onto single relay cells, this will transform our understanding of thalamic function, because it will demonstrate that, at least for some higher order nuclei, significant alteration in the nature of incoming information occurs before relay to cortex. In addition to these analyses allowing a better understanding of cortical and thalamic circuitry and their interactions, most of the pathways to be studied have independently been analyzed physiologically for characteristics of driver vs modulator properties. We thus expect to find significant correlations between these physiological data and the proposed connectomics analyses, which we believe will allow future connectomic studies to be interpreted on a functional basis heretofore unavailable.