The thalamus relays sensori-motor information to the cortex and is an integral part of cortical executive functions. The precise distribution of thalamic projections to the cortex is poorly characterized, particularly in mouse. We employed a systematic, high-throughput viral approach to visualize thalamocortical axons with high sensitivity. We then developed algorithms to directly compare injection and projection information across animals. By tiling the mouse thalamus with 254 overlapping injections, we constructed a comprehensive map of thalamocortical projections. We determined the projection origins of specific cortical subregions and verified that the characterized projections formed functional synapses using optogenetic approaches. As an important application, we determined the optimal stereotaxic coordinates for targeting specific cortical subregions and expanded these analyses to localize cortical layer-preferential projections. This data set will serve as a foundation for functional investigations of thalamocortical circuits. Our approach and algorithms also provide an example for analyzing the projection patterns of other brain regions.
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