Excitatory projection neurons of the neocortex are thought to play important roles in perceptual and cognitive functions of the brain by directly connecting diverse cortical and subcortical areas. However, many aspects of the anatomical organization of these inter-areal connections are unknown. Here, we studied long-range axonal projections of excitatory layer 2/3 neurons with cell bodies located in mouse primary somatosensory barrel cortex (wS1). As a population, these neurons densely projected to secondary whisker somatosensory cortex (wS2) and primary/secondary whisker motor cortex (wM1/2), with additional axon in the dysgranular zone surrounding the barrel field, perirhinal temporal association cortex and striatum. In three-dimensional reconstructions of 6 individual wS2-projecting neurons and 9 individual wM1/2-projecting neurons, we found that both classes of neurons had extensive local axon in layers 2/3 and 5 of wS1. Neurons projecting to wS2 did not send axon to wM1/2, whereas a small subset of wM1/2-projecting neurons had relatively weak projections to wS2. A small fraction of projection neurons solely targeted wS2 or wM1/2. However, axon collaterals from wS2-projecting and wM1/2-projecting neurons were typically also found in subsets of various additional areas, including the dysgranular zone, perirhinal temporal association cortex and striatum. Our data suggest extensive diversity in the axonal targets selected by individual nearby cortical long-range projection neurons with somata located in layer 2/3 of wS1.

The brain processes sensory information in a context- and learning-dependent manner for adaptive behavior. Through reward-based learning, relevant sensory stimuli can become linked to execution of specific actions associated with positive outcomes. The neuronal circuits involved in such goal-directed sensory-to-motor transformations remain to be precisely determined. Studying simple learned sensorimotor transformations in head-restrained mice offers the opportunity for detailed measurements of cellular activity during task performance. Here, we trained mice to lick a reward spout in response to a whisker deflection and an auditory tone. Through two-photon calcium imaging of retrogradely labeled neurons, we found that neurons located in primary whisker somatosensory barrel cortex projecting to secondary whisker somatosensory cortex had larger calcium signals than neighboring neurons projecting to primary whisker motor cortex in response to whisker deflection and auditory stimulation, as well as before spontaneous licking. Longitudinal imaging of the same neurons revealed that these projection-specific responses were relatively stable across 3 days. In addition, the activity of neurons projecting to secondary whisker somatosensory cortex was more highly correlated than for neurons projecting to primary whisker motor cortex. The large and correlated activity of neurons projecting to secondary whisker somatosensory cortex might enhance the pathway-specific signaling of important sensory information contributing to task execution. Our data support the hypothesis that communication between primary and secondary somatosensory cortex might be an early critical step in whisker sensory perception. More generally, our data suggest the importance of investigating projection-specific neuronal activity in distinct populations of intermingled excitatory neocortical neurons during task performance.