Allison Butt - US grants

Project Summary Neurological disorders, such as depression and bipolar disorder, are thought to be caused by compromised neural circuits ? groups of neurons that operate as units to generate a single output. Frequently, drugs that aIter serotonin signaling seem to positively impact mood disorders even though no disruption in serotonin has been clearly identified. Interestingly, serotonin-producing neurons frequently also produce a neuropeptide known as Substance P, but why this occurs is not known. The nematode Caenorhabditis elegans is ideal for studying circuitry with its simple nervous system and well-known anatomy; additionally, C. elegans has an extensively studied egg-laying circuit that has a quantitative behavioral output of egg laying making it an ideal model circuit to study. This circuit is activated by serotonin and a neuropeptide NLP-3 being released from the Hermaphrodite Specific Neurons (HSNs). Previous work has shown that both of these signals are required for proper activation levels; however, it is unclear why two signals are required. While serotonin has been well characterized in the C. elegans egg-laying circuit, the neuropeptide NLP-3 was only recently implicated in egg laying. In my preliminary work I have identified a putative NLP-3 G protein coupled receptor (GPCR), F10D7.1. In this proposal I will test the hypothesis that the HSN signals to the muscles of the egg-laying circuit using NLP-3 and serotonin as partially redundant signals to ensure robust activation of the circuit. My first aim is to vet F10D7.1 as an NLP-3-activated G protein coupled receptor in the C. elegans egg- laying circuit. I will genetically verify that F10D7.1 is coupled to NLP-3 activity. I will assay two loss-of-function F10D7.1 mutants crossed with various genetic backgrounds for egg laying defects. Additionally, I will express F10D7.1 in heterologous cells to determine the binding potential of NLP-3 peptides and their activity levels. My second aim is to identify the cells within the C. elegans egg-laying circuit that express F10D7.1 and receive an NLP-3 signal to activate the egg-laying circuit. I will create a GFP construct driven by a promoter fragment of F10D7.1. After injecting this into C. elegans, I can use this to identify cells that express F10D7.1 and hypothetically are activated by NLP-3. I can then selectively knock down F10D7.1 in these cells and assay for egg retention. My third aim is to determine the effects of NLP-3 signaling on the egg-laying circuit activity using live- animal calcium imagining. Calcium imaging in live C. elegans is an excellent tool developed by my lab to directly observe how a signal affects a circuit's activation. I will express a calcium indicator in the cells that express F10D7.1 found in Aim 2a and quantify how the loss of the receptor changes the activity pattern of the circuit, heightened by the dual knockout of F10D7.1 with serotonin. Overall, this project will address the question of why serotonin and neuropeptides are utilized in the same circuit.