Monosynaptic pathway from rat vibrissa motor cortex to facial motor neurons revealed by lentivirus-based axonal tracing

The mammalian motor cortex typically innervates motor neurons indirectly via oligosynaptic pathways. However, evolution of skilled digit movements in humans, apes, and some monkey species is associated with the emergence of abundant monosynaptic cortical projections onto spinal motor neurons innervating distal limb muscles. Rats perform skilled movements with their whiskers, and we examined the possibility that the rat vibrissa motor cortex (VMC) projects monosynaptically onto facial motor neurons controlling the whisker movements. First, single injections of lentiviruses to VMC sites identified by intracortical microstimulations were used to label a distinct subpopulation of VMC axons or presynaptic terminals by expression of enhanced green fluorescent protein (GFP) or GFP-tagged synaptophysin, respectively. Four weeks after the injections, GFP and synaptophysin-GFP labeling of axons and putative presynaptic terminals was detected in the lateral portion of the facial nucleus (FN), in close proximity to motor neurons identified morphologically and by axonal back-labeling from the whisker follicles. The VMC projections were detected bilaterally, with threefold larger density of labeling in the contralateral FN. Next, multiple VMC injections were used to label a large portion of VMC axons, resulting in overall denser but still laterally restricted FN labeling. Ultrastructural analysis of the GFP-labeled VM Caxons confirmed the existence of synaptic contacts onto dendrites and somata of FN motor neurons. These findings provide anatomical demonstration of monosynaptic VMC-to-FN pathway in the rat and show that lentivirus-based expression of GFP and GFP-tagged presynaptic proteins can be used as a high-resolution neuroanatomical tracing method.