Project Summary Neuromodulators including dopamine and serotonin have profound effects on spinal circuits for locomotion. A wealth of pharmacological manipulations has shown that drugs mimicking or blocking these neuromodulators can change the properties of rhythmic motor output in the isolated spinal cord. However, these studies often conflict and cannot capture the normal range of behaviors expressed in vivo. Furthermore, it is entirely unknown whether neuromodulators are released onto different spinal targets across different behaviors. Finally, neuromodulatory neurons are highly branched, making it difficult to disambiguate the spinal vs supraspinal consequences of their action. We will leverage new tools for imaging and manipulating neuromodulator signaling, combined with the transparency and accessibility of the young zebrafish, and a quantitative modeling approach, to understand the effects of dopamine and serotonin on genetically defined components of the spinal locomotor circuit in vivo. First, we will measure the activity of neuromodulatory axons during three distinct behaviors, testing whether dopamine and serotonin axons differentially participate in these behaviors. Next, we will quantify neuromodulator release during these behaviors directly, both in the whole spinal cord and in genetically defined populations of neurons with distinct contributions to locomotion. We will then test the significance of descending neuromodulatory influence on spinal circuits by targeted axotomy that will allow disambiguation of the spinal and supraspinal consequences of neuromodulator release. Finally, using newly developed chemogenetic approaches, we will selectively block neuromodulatory receptors in motor neurons and measure the consequences on the three distinct behaviors in freely moving animals. Throughout the project, we will use experimental data to develop both single-segment and multi-segment computational models of neuromodulatory action, and in turn use these models to make testable predictions about circuits and behavior. Together, these experiments will reveal for the first time when and where dopamine and serotonin are acting in the spinal locomotor circuit, and how their actions influence behavior in vivo.