Functional and structural features of L2/3 pyramidal cells continuously covary with pial depth in mouse visual cortex

Pyramidal cells of neocortical layer 2/3 (L2/3 PyrCs) integrate signals from numerous brain areas and project throughout the neocortex. Within L2/3, PyrCs show functional and structural specializations depending on their pial depth, indicating participation in different functional microcircuits. However, it is unknown whether these depth-dependent differences result from separable L2/3 PyrC subtypes or whether functional and structural features represent a continuum while correlating with pial depth. Here, we assessed the stimulus selectivity, electrophysiological properties, dendritic morphology, and excitatory and inhibitory synaptic connectivity across the depth of L2/3 in the binocular visual cortex (bV1) of female mice. We find that the structure of the apical but not the basal dendritic tree varies with pial depth, which is accompanied by differences in passive but not active electrophysiological properties. PyrCs in lower L2/3 receive increased excitatory and inhibitory input from L4, while upper L2/3 PyrCs receive a larger proportion of intralaminar input. Complementary in vivo calcium imaging revealed a systematic change in visual responsiveness, with deeper L2/3 PyrCs showing more robust responses than superficial PyrCs. Furthermore, deeper L2/3 PyrCs are more strongly driven by contralateral than ipsilateral eye stimulation. In contrast, orientation- and direction-selectivity of L2/3 PyrCs are not dependent on pial depth. Importantly, the transitions of the various properties are gradual, and cluster analysis does not support the classification of L2/3 PyrCs into discrete subtypes. These results show that L2/3 PyrCs' multiple functional and structural properties systematically correlate with their depth within L2/3, forming a continuum rather than representing discrete subtypes.