Project Summary Altered serotonin signaling is linked to brain disorders like depression and schizophrenia. A current hypothesis is that serotonin 'modulates' the action of other neural signals with critical roles in depression. However, the vast complexity of the human brain makes it challenging to test this idea, and there is no neural circuit in any organism in which we understand in mechanistic detail how serotonin modulates other signals. This project seeks to fill this gap in knowledge by determining how serotonin modulates other signals in a powerful model system: the well-studied neural circuit that controls egg-laying in the C. elegans nematode. A pair of HSN neurons release serotonin to make the vulval type-1 and type-2 muscle cells (vm1s and vm2s) and the ventral cord type C (VCs) neurons more excitable. Simultaneous contraction of the vm1s and vm2s is required for egg laying. The VCs release acetylcholine to depolarize the vm2s; however, so far, no signal that could depolarize the vm1s and thus be potentiated by serotonin has been identified. I discovered that a pair of poorly-studied neurons called PVW produce branches that end in presynaptic termini over the vm1s. My preliminary data also shows that the PVW branches have strong calcium transients during egg laying, and that silencing PVW's activity mildly inhibits egg laying. Furthermore, weaker PVW calcium transients are observed when the vm1s produce small twitches that do not result in egg laying. My central hypothesis is that the PVW branches provide the unknown excitatory signal that triggers vm1 contractions and that serotonin potentiates this effect to result in egg laying. My first aim is to determine if the PVW branches form chemical and/or electrical synapses onto the vm1s. I will use fluorescent presynaptic protein markers to examine if the PVW varicosities contain small-clear vesicles, dense- core vesicles, and/or gap junctions. I will also analyze existing electron micrographs to reconstruct the ultrastructure and synaptic content of the PVW branch onto the vm1s. Next, I will use trans-synaptic labeling methods to determine if the PVW varicosities contact the vm1s. My second aim is to use a genetically-encoded fluorescent calcium indicator to record PVW neural activity within freely-behaving animals to determine if PVW is active when the vm1s are activated, and to study if the HSN or VC neurons modulate PVW's activity during egg laying. My third aim is to manipulate PVW activity with optogenetic and chemogenetic methods to test if the PVW neurons provide a serotonin-potentiated signal that excites the vm1s to induce egg laying. I will silence or induce PVW and VC activities to determine if both neurons are necessary and/or sufficient to induce egg laying in the presence and absence of serotonin. Lastly, I will use RNAi to disrupt presynaptic gene function in PVW to investigate if PVW uses neurotransmitter, neuropeptide, or gap junction signaling to induce egg laying and...