Motor cortex projections to red and pontine nuclei have distinct roles during movement in the mouse.

Motor control largely depends on the deep layer 5 (L5) pyramidal neurons that project to subcortical structures. However, it is largely unknown if these neurons are functionally segregated with distinct roles in movement performance. Here, we analyzed mouse motor cortex L5 pyramidal neurons projecting to the red and pontine nuclei during movement preparation and execution. Using photometry to analyze the calcium activity of L5 pyramidal neurons projecting to the red nucleus and pons, we reveal that both types of neurons activate with different temporal dynamics. Optogenetic inhibition of either kind of projection differentially affects forelimb movement onset and execution in a lever press task, but only the activity of corticopontine neurons is significantly correlated with trial-by-trial variations in reaction time. The results indicate that cortical neurons projecting to the red and pontine nuclei contribute differently to sensorimotor integration, suggesting that L5 output neurons are functionally compartmentalized generating, in parallel, different downstream information.