Excitatory spinal Lhx9-derived interneurons modulate locomotor frequency in mice

Locomotion allows us to move and interact with our surroundings. Spinal networks that control locomotion produce rhythm, left-right and flexor-extensor coordination. Several glutamatergic populations, Shox2 non-V2a, Hb9-derived interneurons and recently, spinocerebellar neurons have been proposed to be involved in the mouse rhythm generating networks. However, these cells make up only a smaller fraction of the excitatory cells in the ventral spinal cord. Here we set out to identify additional populations of excitatory spinal neurons that may be involved in rhythm generation or other functions in the locomotor network. For this, we use RNA-sequencing from glutamatergic, non-glutamatergic, and Shox2 cells in the neonatal mice from both sexes followed by differential gene expression analyses. These analyses identified transcription factors that are highly expressed by glutamatergic spinal neurons and differentially expressed between Shox2 neurons and glutamatergic neurons. From this latter category, we identified the Lhx9-derived neurons that have a restricted spinal expression pattern and do not overlap with Shox2 neurons. They are purely glutamatergic and ipsilaterally projecting. Ablation of the glutamatergic transmission or acute inactivation of the neuronal activity of Lhx9-derived neurons lead to a decrease in the frequency of locomotor-like activity with no change in coordination pattern. Optogenetic activation of Lhx9-derived neurons promotes locomotor-like activity and modulates the frequency of the ongoing drug-induced locomotor activity. Calcium activities of Lhx9-derived neurons in the upper lumbar cord show strong left-right out of phase rhythmicity during locomotor-like activity. Our study identifies a distinct population of spinal excitatory neurons that regulates the frequency of locomotor output with a suggested role in rhythm-generation in the mouse alongside other spinal populations. Significance Statement Ipsilaterally-projecting excitatory interneurons play a crucial role in the generation of locomotor rhythms in the vertebrate spinal cord. While the Shox2, Hb9, and spinocerebellar tract neurons are known components involved in this behavior they represent only a fraction of the entire excitatory population in the spinal cord. In this study, we identify additional excitatory spinal populations that could be essential contributors to the locomotor circuitry and shed light on a new population of ipsilaterally-projecting excitatory neurons expressing the transcription factor Lhx9. Our findings show that this newly identified population has the capacity to modulate the locomotor frequency, suggesting a potential involvement in the rhythm generating network along other spinal neuronal populations.