PROJECT SUMMARY How does neuronal signaling control behavior? Neuroscientists answer this question by mapping neuronal circuits, studying action potential firing, or monitoring the resultant neurochemicals released. Drosophila is a model system used to map neural circuits and study cell firing during innate behaviors, such as feeding. Direct measurements of neuromodulators are required to understand behavioral regulation because release is highly heterogeneous and plastic; i.e. the same firing patterns elicit different neuromodulator release in different circumstances due to complex regulation. To understand how neuromodulation regulates behavior or encodes memories, we need tools to examine multiple neuromodulators throughout a circuit. The goal of this project is to develop new multiplexed tools to study neuromodulator interactions in Drosophila during sugar feeding. The central hypothesis is that dopamine, octopamine, and glutamate neuromodulation will occur only in discrete neuropil, vary with frequency of sugar feeding, and be enhanced when sugar is paired with odors. This research is significant because it will develop tools to map real-time signaling of multiple neuromodulators throughout a circuit and use these tools to show how neuromodulator interactions in a circuit control appetitive memory formation during sugar feeding. In Aim 1, we will develop new nanoelectrodes to co-detect octopamine and dopamine using fast-scan cyclic voltammetry (FSCV) and measure them in the mushroom body (MB) g5 compartment during sugar feeding. Octopamine modulation of dopamine will be tested by selectively activating or suppressing a specific octopaminergic neuron. In Aim 2, the goal is to understand dopamine neuromodulation during sugar feeding across compartments of the MB. A genetically-encoded dopamine sensor (GRABDA) will be selectively expressed in either MB dopamine neurons or the protocerebral anterior medial (PAM) projection neurons and dopamine recorded during sugar feeding and olfactory conditioning. In Aim 3, neuromodulation will be mapped throughout a circuit that regulates sugar reward by measuring dopamine and octopamine in the g5 compartment of the mushroom body and glutamate signaling (iGluSnFR) from a mushroom body output neuron in the superior medial protocerebrum. These experiments will be the first to multiplex FSCV and genetically encoded sensors to understand how dopamine/octopamine regulation in the mushroom body controls output signaling downstream in a circuit. The expected outcomes of this work are mapping of neuromodulator interactions throughout a circuit in adult Drosophila during feeding and olfactory conditioning. The positive impact will be advanced methods and foundational knowledge necessary to perform future studies of how genetics, behavior, or disease alter neuromodulator release. The neuromodulators studied here are implicated in mental health disorders, such as schizophrenia, compulsive eating, or addiction, and this...