Project Summary Astrocytes are important regulators of neural circuit activity. Intracellular astrocyte Ca2+ signaling often correlates with local synaptic activity and behavioral state. Astrocytes express a range of neurotransmitters (NTs) receptors, in many contexts respond to NTs with increases in cellular Ca2+, and disruption of astrocyte calcium signaling can meaningfully impact neurophysiology and animal behavior. However, the mechanisms by which astrocytes sense neurotransmission, signal intracellularly and ultimately modulate circuit function remain poorly defined. Our goal is to identify and functionally characterize endogenous signaling molecules that allow astrocytes to regulate neural circuits. We use Drosophila as a model to study astrocyte biology. Flies astrocytes are similar to their mammalian counterparts by morphological, developmental, molecular and functional criteria, and this system offers a powerful array of tools for characterizing gene function in vivo. In the previous cycle, we identified a simple circuit where octopamine/tyramine (the fly orthologs of norepinephrine, NE) signal directly to astrocytes thought the Octopamine- Tyramine Receptor (Oct-TyrR) GPCR, this activates the TRP channel Water Witch (Wtrw), which stimulates intracellular astrocyte Ca2+ signaling and modifies downstream neuronal activity and behavior. This work (1) established a fly circuit to study how astrocytes respond to neuromodulatory input and modulate circuits, and (2) demonstrated NE/Oct/Tyr- dependent modulation of astrocyte Ca2+ signaling is an evolutionarily conserved astrocytic signaling mechanism. In preliminary work we found bath application of many NTs (i.e. glutamate, acetylcholine, GABA and dopamine) failed to elicit Ca2+ signaling events in astrocytes. However, a brief pre-exposure of astrocytes to Oct/Tyr resulted in potent astrocytic Ca2+ signaling events in response to these NTs within seconds, which depended on Oct-TyrR. This suggests an arousal stimulus (i.e. norepinephrine/Oct/Tyr) gates the ability of astrocytes to sense the NTs Glu, Ach, GABA and DA, and dramatically changes astrocyte function in vivo. In Aim 1 we will determine how PLC and DAG/IP3 signaling downstream of Oct-TyrR regulate astrocyte sensitivity to Glu, GABA and Ach. In Aim 2 we will characterize the role of adenylate cyclase, cAMP and PKA in gating astrocyte sensitivity to dopamine after Oct/Tyr exposure. We will identify the source(s) of Ca2+ signaling activated by these NTs (e.g. is it Wtrw?), and explore how these Oct/Tyr-gated events affect animal behavior. Finally, we will explore how astrocyte Ca2+ signaling events regulate neural activity and behavior—among the most important unsolved questions in our field. In preliminary work we performed a forward genetic screen for genes that when knocked down selectively in astrocytes, suppressed the ability of TRP channel-mediated astrocyte Ca2+ entry to induce seizure behavior. We identified many genes, most o...