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Ecdysone, an essential insect steroid hormone, promotes larval metamorphosis by coordinating growth and maturation. In  Drosophila melanogaster , prothoracicotropic hormone (PTTH)-releasing neurons are considered to be the primary promoting factor in ecdysone biosynthesis. Recently, studies have reported that the regulatory mechanisms of PTTH release in  Drosophila  larvae are controlled by different neuropeptides, including allatostatin A and corazonin. However, it remains unclear whether neurotransmitters provide input to PTTH neurons and control the metamorphosis in  Drosophila  larvae. Here, we report that the neurotransmitters acetylcholine (ACh) affect larval development by modulating the activity of PTTH neurons. By downregulating the expression of different subunits of nicotinic ACh receptors in PTTH neurons, pupal volume was significantly increased, whereas pupariation timing was relatively unchanged. We also identified that PTTH neurons were excited by ACh application  ex vivo  in a dose-dependent manner  via  ionotropic nicotinic ACh receptors. Moreover, in our Ca 2+  imaging experiments, relatively low doses of OA caused increased Ca 2+  levels in PTTH neurons, whereas higher doses led to decreased Ca 2+  levels. We also demonstrated that a low dose of OA was conveyed through OA β-type receptors. Additionally, our electrophysiological experiments revealed that PTTH neurons produced spontaneous activity  in vivo , which provides the possibility of the bidirectional regulation, coming from neurons upstream of PTTH cells in  Drosophila  larvae. In summary, our findings indicate that several different neurotransmitters are involved in the regulation of larval metamorphosis by altering the activity of PTTH neurons in  Drosophila .

Neurotransmitters Affect Larval Development by Regulating the Activity of Prothoracicotropic Hormone-Releasing Neurons in Drosophila melanogaster