Neural networks in the spinal cord can generate locomotion in the absence of rhythmic input from higher brain structures or sensory feedback because they contain an intrinsic source of excitation. However, the molecular identity of the spinal interneurons underlying the excitatory drive within the locomotor circuit has remained unclear. Using optogenetics, we show that activation of a molecularly defined class of ipsilateral premotor interneurons elicits locomotion. These interneurons represent the excitatory module of the locomotor networks and are sufficient to produce a coordinated swimming pattern in zebrafish. They correspond to the V2a interneuron class and express the transcription factor Chx10. They produce sufficient excitatory drive within the spinal networks to generate coordinated locomotor activity. Therefore, our results define the V2a interneurons as the excitatory module within the spinal locomotor networks that is sufficient to initiate and maintain locomotor activity.

Neural circuits in the spinal cord transform instructive signals from the brain into well-coordinated locomotor movements by virtue of rhythm-generating components. Although evidence suggests that excitatory interneurons are the essence of locomotor rhythm generation, their molecular identity and the assessment of their necessity have remained unclear. Here we show, using larval zebrafish, that V2a interneurons represent an intrinsic source of excitation necessary for the normal expression of the locomotor rhythm. Acute and selective ablation of these interneurons increases the threshold of induction of swimming activity, decreases the burst frequency, and alters the coordination of the rostro-caudal propagation of activity. Thus, our results argue that V2a interneurons represent a source of excitation that endows the spinal circuit with the capacity to generate locomotion.central pattern generator | premotor interneurons | motor behavior | synaptic transmission T he generation of motor behavior involves decision making, selection, initiation, and execution (1-9). The spinal cord acts as an interface to process descending commands from the brain and sensory inputs from the periphery (5,8,(10)(11)(12)(13)(14)(15)(16). Many insights into the organization and function of circuits underlying motor behavior have been gained from studies of spinal networks controlling locomotor movements (6,13,(17)(18)(19)(20)(21)(22)(23)(24). The basic locomotor activity requires the interplay of ipsilateral excitatory drive and crossed inhibition, which ensures the alternating pattern between the two sides of the spinal cord. Whereas inhibitory interneurons ensure the coordination of motoneurons controlling antagonistic muscles, excitatory interneurons are believed to represent the core components for the generation of the locomotor rhythm.Determining the identity of the interneurons at the origin of excitation necessary for the normal generation of locomotor activity is central for understanding the principles of organization and function of locomotor circuits. However, the molecular identity of these excitatory interneurons has remained unclear. A prominent class of excitatory interneurons is the V2a interneurons, defined by Chx10 expression and derived from the p2 progenitor domain with homologous counterparts across vertebrate species (21,(25)(26)(27). In larval zebrafish and newborn mice, V2a interneurons have been shown to project to motoneurons and to be recruited in a frequency-dependent manner (28-32). These properties are consistent with the possibility that this class of interneurons represents a source of excitation within the spinal locomotor circuit.In newborn mice, however, genetic elimination of V2a interneurons has been reported to have little effect on the rhythmic output of the locomotor circuits induced by pharmacological means (33, 34). The absence of tangible effects on the excitability of the locomotor circuit in the absence of V2a interneurons led to the conclusion that these interneurons are not essential fo...