PROJECT SUMMARY The neurotransmitter dopamine (DA) is well known as a regulator of vertebrate locomotor behaviors, but prior research has largely ignored the contributions of DA-producing neurons in the hypothalamus. Working in the larval zebrafish, we have discovered that a population of DA neurons in the hypothalamic preoptic nucleus, defined by their expression of the tyrosine hydroxylase gene, th2, are critically important for generating most forms of spontaneous and evoked swimming. Functional imaging reveals that these cells exhibit complex sensory and motor encodings, firing intense bursts of activity in acute correlation with movement, auditory cues, or both, and optogenetic manipulation elicits a variety of kinematically distinct swim bouts. When the th2+ neurons are ablated, fish initiate spontaneous swimming dramatically less often. We have identified a group of premotor spinal projection neurons (SPNs) in the mid- and hindbrain as particularly important mediators of the th2+ neurons’ behavioral functions. Activation of the th2+ afferents to this region rapidly elicits sustained bursts of activity in a majority of SPNs, driving the resulting behavior. The SPNs comprise a group of roughly 250 neurons, which are anatomically and functionally invariant between animals, and activity in individual SPNs has been directly linked to particular behaviors. As the targets of th2+ DA neuron activity, these cells present a unique opportunity for understanding the functional architecture of a modulatory network – that is, how DA neurons that project onto distinct functional targets might differ in their physiological properties, and how that organization might influence the behavioral contributions of specific DA cells. We propose that functionally heterogeneous subgroups of preoptic th2+ neurons differentially release DA onto specific SPNs under different sensorimotor conditions, enabling the selective recruitment of premotor ensembles to drive contextually appropriate behaviors. To test this idea, we will first use calcium imaging in traceable neurons to determine whether th2+ afferents to the SPN subgroups that mediate different behaviors – i.e. routine vs. defensive swimming – are selectively activated during the associated bout type. Next, we will directly image DA secretion to determine whether the modulatory signals at different sites vary independently from one another, in a way compatible with selective modulation of particular SPNs. Last, we will use in vivo electrophysiology to precisely determine how the th2+ neurons affect SPN function. The result of our work will be a detailed model linking functional heterogeneity in a modulatory network to the performance of specific behaviors.