Abstract
Association projections from cortical pyramidal neurons connect disparate intrahemispheric cortical areas, which are implicated in higher cortical functions. The underlying developmental processes of these association projections, especially the initial phase before reaching the target areas, remain unknown. To visualize developing axons of individual neurons with association projections in the mouse neocortex, we devised a sparse labeling method that combined in utero electroporation and confocal imaging of flattened and optically cleared cortices. Using the promoter of an established callosal neuron marker gene that was expressed in over 80% of L2/3 neurons in the primary somatosensory cortex (S1) that project to the primary motor cortex (M1), we found that an association projection of a single neuron was the longest among the interstitial collaterals that branched out in L5 from the earlier-extended callosal projection. Collaterals to M1 elongated primarily within the cortical gray matter with little branching before reaching the target. Our results suggest that dual-projection neurons in S1 make a significant fraction of the association projections to M1, supporting the directed guidance mechanism in long-range corticocortical circuit formation over random projections followed by specific pruning.