Pinpointing Morphology and Projection of Excitatory Neurons in Mouse Visual Cortex

The excitatory neurons in the visual cortex are of great significance for us in understanding brain functions. However, the diverse neuron types and their morphological properties have not been fully deciphered. In this paper, we applied the brain-wide positioning system (BPS) to image the entire brain of two Thy1-eYFP H-line male mice at 0.2 μm × 0.2 μm × 1 μm voxel resolution. A total of 103 neurons were reconstructed in layers 5 and 6 of the visual cortex with single-axon-level resolution. Based on the complete topology of neurons and the inherent positioning function of the imaging method, we classified the observed neurons into six types according to their apical dendrites and somata location: star pyramidal cells in layer 5 (L5-sp), slender-tufted pyramidal cells in layer 5 (L5-st), tufted pyramidal cells in layer 5 (L5-tt), spiny stellate-like cells in layer 6 (L6-ss), star pyramidal cells in layer 6 (L6-sp), and slender-tufted pyramidal cells in layer 6 (L6-st). By examining the axonal projection patterns of individual neurons, they can be categorized into three modes: ipsilateral circuit connection neurons, callosal projection neurons and corticofugal projection neurons. Correlating the two types of classifications, we have found that there are at least two projection modes comprised in the former defined neuron types except for L5-tt. On the other hand, each projection mode may consist of four dendritic types defined in this study. The axon projection mode only partially correlates with the apical dendrite feature. This work has demonstrated a paradigm for resolving the visual cortex through single-neuron-level quantification and has shown potential to be extended to reveal the connectome of other defined sensory and motor systems.