Cellular and neuronal circuit mechanisms involved in locomotor activity

Project Summary Mammalian locomotion is an essential behavior for mobility and survival. Locomotion involves coordinated and alternating rhythmic activity between opposing limbs, as well as between antagonistic muscles of the same limb. The locomotor central pattern generator (CPG), a network of spinal interneurons, is thought to produce the locomotor pattern and rhythm and directly activate motor neurons, which in turn activate peripheral muscles resulting in movement. We have recently reported that a set of spinal interneurons, known as ventral spinocerebellar tract (VSCT) neurons, are both necessary and sufficient for locomotor behavior in neonatal and adult mice. VSCT neurons possess two rhythmogenic properties, namely the exhibit an Ih current and they are electrically coupled. Additionally, they possess spinal axon collaterals within the same spinal segment as well as extra-segmentally. VSCTs are also contacted by motor neuron axon collaterals and they are electrically coupled to them, at least during early postnatal development. VSCTs in turn, connect with other spinal interneurons involved in the locomotor central pattern generator, via their axon collaterals. Our published observations provide a strong foundation to dissect further their cellular characteristics and their neuronal circuits that they form with other spinal neurons, including motor neurons. We hypothesize that both cellular characteristics in VSCTs and the spinal neuronal targets contacted by VSCTs’ axon collaterals will be key determinants for locomotor behavior in mice. In Aim 1, we will study the contribution of the HCN channels responsible for the Ih current in VSCTs and determine the role of several connexins that we have identified through our preliminary results in their role as key neurons in locomotor rhythmogenesis. To do so, we will utilize conditional knock out mice, whole-cell patch clamp protocols along with pharmacological studies, immunohistochemistry, and virally-mediated channel knockdown. In Aim 2, we will study the neuronal connectivity of VSCTs with Chx10+, Sim1+ and motor neurons. We will investigate their functional role in these connections utilizing mouse genetics, behavioral assays, physiological approaches and viral-mediated approaches to map out some of the key neuronal circuits involved in locomotor behavior. Furthermore, we will employ a novel “clearing” technique together with 2-photon laser confocal microscopy to image the entire spinal cord of neonatal and adult mice to uncover the relationship between VSCTs and their potential neuronal targets within the spinal cord and across many different spinal segments. In Aim 3, we will establish whether a novel set of transcription factors marking this class of spinal interneurons can be manipulated by chemogenetic and optogenetic approaches and determine their involvement in locomotor-like behavior. In summary, this comprehensive set of experiments will provide a solid foundation to further our understanding of spinal locomotor networks.