Serotonin and neuropeptides are both released by the HSN command neuron to initiate C. elegans egg laying

Neurons typically release both a small-molecule neurotransmitter and one or more neuropeptides, but how these two types of signal from the same neuron might act together remains largely obscure. For example, serotonergic neurons in mammalian brain express the neuropeptide Substance P, but it is unclear how this co-released neuropeptide might modulate serotonin signaling. We studied this issue in C. elegans, in which all serotonergic neurons express the neuropeptide NLP-3. The serotonergic Hermaphrodite Specific Neurons (HSNs) are command motor neurons within the egg-laying circuit which have been shown to release serotonin to initiate egg-laying behavior. We found that egg-laying defects in animals lacking serotonin were far milder than in animals lacking HSNs, suggesting that HSNs must release other signal(s) in addition to serotonin to stimulate egg laying. While null mutants for nlp-3 had only mild egg-laying defects, animals lacking both serotonin and NLP-3 had severe defects, similar to those of animals lacking HSNs. Optogenetic activation of HSNs induced egg laying in wild-type animals, and in mutant animals lacking either serotonin or NLP-3, but failed to induce egg laying in animals lacking both. We recorded calcium activity in the egg-laying muscles of animals lacking either serotonin, NLP-3, or both. The single mutants, and to a greater extent the double mutant, showed muscle activity that was uncoordinated and unable to expel eggs. Specifically, the vm2 muscles cells, which are direct postsynaptic targets of the HSN, failed to contract simultaneously with other egg-laying muscle cells. Our results show that the HSN neurons use serotonin and the neuropeptide NLP-3 as partially redundant co-transmitters that together stimulate and coordinate activity of the target cells onto which they are released. Author summary Activity of the brain results from neurons communicating with each other using chemical signals. A typical neuron releases two kinds of chemical signals: a small molecule neurotransmitter, such as serotonin, and one or more small proteins, called neuropeptides. For example, neurons in the human brain that release serotonin, a neurotransmitter thought to be involved in depression, also release the neuropeptide Substance P. Neuroscientists have typically studied the effects of neurotransmitters and neuropeptides separately, without considering how these two types of signals from the same neuron might be integrated. Here we analyzed how specific neurons in the model organism C. elegans use both serotonin and a neuropeptide together. The Hermaphrodite Specific Neurons (HSNs) activate a small group of neurons and muscles to generate egg-laying behavior. Killing the HSNs resulted in animals unable to lay eggs, but we found that eliminating either serotonin or the neuropeptide resulted in HSNs that still remained able to activate egg laying. However, eliminating both serotonin and the neuropeptide resulted in HSNs unable to activate coordinated contractions of the egg-laying muscles. Our results show that in a living animal, serotonin acts in concert with a co-released neuropeptide to carry out its functions.