Synchronicity in zebrafish locomotive circuit development mediated by electrical pacemaker interneurons

The earliest motor output in vertebrate animals is generated by clusters of early-born motor neurons that occupy distinct regions of the spinal cord, innervating stereotyped muscle groups. Even the simplest movements require coordinated activity across these motor pools, yet motor neurons are not directly interconnected and instead project to the periphery. Instead, emerging motor circuits might be synchronized by pacemaker interneurons. We hypothesize that pacemaker interneurons are required for synchronization of motor neuron activity throughout the spinal cord, coupling motor pools through electrical gap junctions to ultimately drive coordinated motor behavior. With functional imaging in the embryonic zebrafish spinal cord, we show that ipsilateral caudal interneurons possess periodic activity profiles prior to widespread motor circuit activity that transition to synchronized Ca 2+ events in motor neurons throughout the spinal cord. Importantly, we also show that blockade of electrical gap junctions and ablation of pioneer pacemakers leads to desynchronization in developing motor circuits. Further, we use a genetic model of hyperactivity to gain critical insight into the consequences of errors in motor circuit formation and function, finding that Fragile X syndrome (FXS) model mutant zebrafish are hyperexcitable from the earliest phases of spontaneous behavior, show reduced sensitivity to blockade of electrical gap junctions, and have increased expression of the gap junction protein Connexin 36. Taken together, our work highlights the importance of pacemakers in the development of motor circuits and suggests that the origins of hyperactivity in neurodevelopmental disorders may be established during the initiation of motor circuit formation.