Cell class-specific long-range axonal projections of neurons in mouse whisker-related somatosensory cortices

Abstract

The extensive long-range axonal projections of diverse classes of neocortical excitatory neurons are thought to contribute importantly to the highly integrative brain-wide interactions underlying the processing of sensory, cognitive and motor signals. Here, we investigated the long-range axonal output of various classes of genetically-defined projection neurons with cell bodies located in the whisker-related somatosensory cortices of the mouse through brain-wide light-sheet imaging of fluorescently-labeled axons segmented by specifically-trained convolutional networks quantified within the Allen Mouse Brain Atlas Common Coordinate Framework. We injected Cre-dependent virus to express GFP or tdTomato in the posterior primary somatosensory barrel cortex and the posterior supplemental somatosensory cortex, which contain the representations of the large posterior mystacial whiskers. We investigated the six following transgenic mouse lines which preferentially express Cre in different glutamatergic neocortical cell classes: Rasgrf2-dCre for layer 2/3 intratelencephalic projection neurons, Scnn1a-Cre for layer 4 intratelencephalic projection neurons, Tlx3-Cre for layer 5 intratelencephalic projection neurons, Sim1-Cre for layer 5 pyramidal tract projection neurons, Rbp4-Cre for layer 5 projection neurons and Ntsr1-Cre for layer 6 corticothalamic neurons. We found long-range axonal projections in many diverse downstream brain areas with largely similar output from primary and secondary cortices, but with genetically-defined cell classes showing distinct innervation patterns, with Rbp4-Cre mice showing the broadest innervation targets, subsets of which were innervated in the other mouse lines. To test whether the revealed axonal projections might underpin functional circuits, we compared the spatial organization of the axonal innervation with functional connectivity maps obtained from optogenetic stimulation of sensory cortex and wide-field imaging of the activity propagation to frontal cortices. Both methods indicated that neurons located more laterally in somatosensory cortex topographically signaled to more anteriorly located regions in motor cortex. The current methodology therefore appears to quantify brain-wide axonal innervation patterns supporting brain-wide signaling, and, together with further technological advances, this will help provide increasingly detailed connectivity information of the mouse brain, essential for understanding the complex neuronal circuitry underlying even simple goal-directed behaviors.