Differences in the morphology of spinal V2a neurons reflect their recruitment order during swimming in larval zebrafish

Networks of neurons in spinal cord generate locomotion. However, little is known about potential differences in network architecture that underlie the production of varying speeds of movement. In larval zebrafish, as swimming speed increases, Chx10-positive V2a excitatory premotor interneurons are activated from ventral to dorsal in a topographic pattern that parallels axial motoneuron recruitment. Here, we examined whether differences in the morphology and synaptic output of V2a neurons reflect their recruitment order during swimming. To do so, we used in vivo single-cell labeling approaches to quantify the dorsoventral distribution of V2a axonal projections and synapses. Two different classes of V2a neurons are described, cells with ascending and descending axons and cells that are only descending. Among the purely descending V2a cells, more dorsal cells project longer distances than ventral ones. Proximally, all V2a neurons have axonal distributions that suggest potential connections to cells at and below their own soma positions. At more distal locations, V2a axons project dorsally, which creates a cumulative intersegmental bias to dorsally located spinal neurons. Assessments of the synapse distribution of V2a cells, reported by synaptophysin expression, support the morphological observations and also demonstrate that dorsal V2a cells have higher synapse densities proximally. Our results suggest that V2a cells with more potential output to spinal neurons are systematically engaged during increases in swimming frequency. The findings help explain patterns of axial motoneuron recruitment and set up clear predictions for future physiological studies examining the nature of spinal excitatory network connectivity as it relates to movement intensity.