Summary A robust and well-organized rhythm is a key feature of many neuronal networks, including those that regulate essential behaviors such as circadian rhythmogenesis, breathing and locomotion. Here we show that excitatory V3-derived neurons are necessary for a robust and organized locomotor rhythm during walking. When V3-mediated neurotransmission is selectively blocked by the expression of the Tetanus toxin light chain subunit (TeNT), the regularity and robustness of the locomotor rhythm is severely perturbed. A similar degeneration in the locomotor rhythm occurs when the excitability of V3-derived neurons is reduced acutely by ligand-induced activation of the allatostatin receptor. The V3-derived neurons additionally function to balance the locomotor output between both halves of the spinal cord, thereby ensuring a symmetrical pattern of locomotor activity during walking. We propose that the V3 neurons establish a robust and balanced motor rhythm by distributing excitatory drive between both halves of the spinal cord.

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