ABSTRACTSurvival for vertebrate animals is dependent on the ability to successfully find food, locate a mate, and avoid predation. Each of these behaviors requires fine motor control, which is set by a combination of kinematic properties. For example, the frequency and amplitude (vigor; strength) of motor output combine to determine features of locomotion such as distance traveled and speed. Although there is a good understanding of how different populations of excitatory spinal interneurons establish locomotor frequency, there is not a mechanistic understanding for how locomotor vigor is established. Recent evidence indicates that locomotor vigor is regulated in part by subsets of identified excitatory spinal interneurons (INs), such as the V2a neuronal population in adult zebrafish. Here we provide evidence that the majority of V3 interneurons (V3-INs), which are a developmentally and genetically defined population of ventromedial glutamatergic spinal neurons, are active during fictive swimming. Further, that targeted ablation of V3-INs reduces the proportion of active MNs during fictive swimming, but ablation does not affect the locomotor frequencies produced. These data are consistent with a role of V3-INs in providing excitatory drive to spinal motor neurons during swimming in larval zebrafish, which suggests that locomotor vigor (but not locomotor frequency) may be regulated, in part, by V3-INs.SIGNIFICANCE STATEMENTCurrently, there is a fundamental lack of knowledge about the cellular and spinal network properties that produce locomotor vigor in vertebrates. Here we show, directly for the first time, that V3 interneurons in zebrafish larvae are active duringin vivofictive locomotion, and that targeted ablation of the spinal V3 interneuron population reduces the probability of motoneuron firing during fictive swimming. In contrast to V2a interneurons, ablation of V3 interneurons does not affect locomotor frequency, the fictive neural correlate of speed, which clarifies their role in motor control rather than rhythm generation. Thus, we propose that the V3 interneuron subpopulation is a source of excitation in the vertebrate locomotor neural circuitry that regulates locomotor vigor independently of speed.