Multiple neurotransmitter inputs modulate circadian clock neurons in Drosophila

Abstract: Most animals have endogenous circadian clocks, which drive daily rhythms in behavior and physiology to adapt to daily cycles of the environment. Several sets of neurons have been identified as circadian pacemakers (clocks) in the brain of Drosophila melanogaster, but the neuronal circuits and neurotransmitters signalling circadian information are poorly known. The current study aimed at identifying neurons and neurotransmitters that form inputs to circadian clock neurons of Drosophila.Immunochytochemistry and the GAL4-UAS system were used to detect neurotransmitters in neurons possibly contacting the clock neurons. Axonal processes containing acetylcholine, GABA, serotonin (5-HT) and glutamate are in close proximity to dendrites of the major clock neurons, the s-LNvs. To test the action of these transmitters on the s-LNvs, we employed calcium imaging on dissociated GFP-labeled larval s-LNvs. We found that acetylcholine induced excitatory, while 5-HT, glutamate and GABA produced inhibitory responses in intracellular calcium levels of the LNvs. Pharmacological experiments revealed that the receptors for acetylcholine are of ion channel type (nicotinic), while GABA and glutamate act on metabotropic G-protein coupled receptors (designated GABAB-R and DmGluRA). These receptors were identified on the s-LNvs by immunochytochemistry in the intact brain and on dissociated neurons.We investigated functions of the GABAB-R and DmGluRA in the activity rhythms using transgenic flies with altered receptor expressions. Although GABAB-R RNA interference failed to produce altered behavioral phenotypes, the DmGluRA interference indicated that the glutamate signaling to s-LNvs modulates the activity pattern under light-dark conditions. In conclusion, the major circadian clock neurons, the s-LNvs, receive multiple inputs via specific receptors for acetylcholine, GABA, glutamate and 5-HT. Possible roles of these inputs are discussed in relation to circadian rhythms of Drosophila.