Abstract
The fruit fly Drosophila melanogaster possesses approximately 150 brain clock neurons that control circadian behavioral rhythms. Even though individual clock neurons have self-sustaining oscillators, they interact and synchronize with each other through a network. However, little is known regarding the factors responsible for these network interactions. In this study, we investigated the role of CCHamide1 (CCHa1), a neuropeptide expressed in the anterior dorsal neuron 1 (DN1a), in intercellular communication of the clock neurons. We observed that CCHa1 connects the DN1a clock neurons to the ventral lateral clock neurons (LNv) via the CCHa1 receptor, which is a homolog of the gastrin-releasing peptide receptor playing a role in circadian intercellular communications in mammals. CCHa1 knockout or knockdown flies have a generally low activity level with a special reduction of morning activity. In addition, they exhibit advanced morning activity under light-dark cycles and delayed activity under constant dark conditions, which correlates with an advance/delay of PAR domain Protein 1 (PDP1) oscillations in the small-LNv (s-LNv) neurons that control morning activity. The terminals of the s-LNv neurons show rather high levels of Pigment-dispersing factor (PDF) in the evening, when PDF is low in control flies, suggesting that the knockdown of CCHa1 leads to increased PDF release; PDF signals the other clock neurons and evidently increases the amplitude of their PDP1 cycling. A previous study showed that high-amplitude PDP1 cycling increases the siesta of the flies, and indeed, CCHa1 knockout or knockdown flies exhibit a longer siesta than control flies. The DN1a neurons are known to be receptive to PDF signaling from the s-LNv neurons; thus, our results suggest that the DN1a and s-LNv clock neurons are reciprocally coupled via the neuropeptides CCHa1 and PDF, and this interaction fine-tunes the timing of activity and sleep.
Highlights
It has been demonstrated that cyanobacteria, which are simple unicellular organisms, can generate robust circadian rhythms (Golden et al, 1997)
We performed a double staining with the anti-TIM and antiCCHa1 antibodies; TIM and CCHa1 were clearly co-localized in the DN1a neurons (Figures 1A,B) but not in other clock neurons (Supplementary Figure S1)
We investigated whether the expression of CCHa1 in the DN1a neurons is regulated by the circadian clock
Summary
It has been demonstrated that cyanobacteria, which are simple unicellular organisms, can generate robust circadian rhythms (Golden et al, 1997). Multiple circadian pacemakers are required to form a network that coordinates behavioral rhythms. For example, the suprachiasmatic nucleus (SCN) in the anterior hypothalamus is the master clock, and is composed of approximately 20,000 pacemaker neurons (Welsh et al, 2010). Individual SCN oscillators contain autonomous transcriptional-translational feedback loops whereby the mRNA and proteins of clock genes are expressed in a circadian manner. The oscillators are synchronized and reinforced chemically using neurotransmitters such as γ-aminobutyric acid, gastrin-releasing peptide (GRP), and vasoactive intestinal polypeptide (VIP), thereby generating coherent and robust rhythms at the network level (Ramkisoensing and Meijer, 2015). The detailed mechanisms of its connections are not fully understood
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