Conditional chemoconnectomics (cCCTomics) as a strategy for efficient and conditional targeting of chemical transmission.

Dissection of neural circuitry underlying behaviors is a central theme in neurobiology. We have previously proposed the concept of chemoconnectome (CCT) to cover the entire chemical transmission between neurons and target cells in an organism and created tools for studying it (CCTomics) by targeting all genes related to the CCT in Drosophila. Here we have created lines targeting the CCT in a conditional manner after modifying GFP RNA interference, Flp-out, and CRISPR/Cas9 technologies. All three strategies have been validated to be highly effective, with the best using chromatin-peptide fused Cas9 variants and scaffold optimized sgRNAs. As a proof of principle, we conducted a comprehensive intersection analysis of CCT genes expression profiles in the clock neurons, uncovering 43 CCT genes present in clock neurons. Specific elimination of each from clock neurons revealed that loss of the neuropeptide CNMa in two posterior dorsal clock neurons (DN1ps) or its receptor (CNMaR) caused advanced morning activity, indicating a suppressive role of CNMa-CNMaR on morning anticipation, opposite to the promoting role of PDF-PDFR on morning anticipation. These results demonstrate the effectiveness of conditional CCTomics and its tools created here and establish an antagonistic relationship between CNMa-CNMaR and PDF-PDFR signaling in regulating morning anticipation.

Light and dopamine impact two circadian neurons to promote morning wakefulness.

In both mammals and flies, circadian brain neurons orchestrate physiological oscillations and behaviors like wake and sleep; these neurons can be subdivided by morphology and by gene expression patterns. Recent single-cell sequencing studies identified 17 Drosophila circadian neuron groups. One of these include only two lateral neurons (LNs), which are marked by the expression of the neuropeptide ion transport peptide (ITP). Although these two ITP+ LNs have long been grouped with five other circadian evening activity cells, inhibiting the two neurons alone strongly reduces morning activity; this indicates that they are prominent morning neurons. As dopamine signaling promotes activity in Drosophila like in mammals, we considered that dopamine might influence this morning activity function. Moreover, the ITP+ LNs express higher mRNA levels than other LNs of the type 1-like dopamine receptor Dop1R1. Consistent with the importance of Dop1R1, CRISPR/Cas9 mutagenesis of this receptor only in the two ITP+ LNs renders flies significantly less active in the morning, and ex vivo live imaging shows that dopamine increases cAMP levels in these two neurons; cell-specific mutagenesis of Dop1R1 eliminates this cAMP response to dopamine. Notably, the response is more robust in the morning, reflecting higher morning Dop1R1 mRNA levels in the two neurons. As morning levels are not elevated in constant darkness, this suggests light-dependent upregulation of morning Dop1R1 transcript levels. Taken together with enhanced morning cAMP response to dopamine, the data indicate how light stimulates morning wakefulness in flies, which mimics the important effect of light on morning wakefulness in humans.

Molecular resolution of a behavioral paradox: sleep and arousal are regulated by distinct acetylcholine receptors in different neuronal types in Drosophila.

Sleep and arousal are both important for animals. The neurotransmitter acetylcholine (ACh) has long been found to promote both sleep and arousal in mammals, an apparent paradox which has also been found to exist in flies, causing much confusion in understanding sleep and arousal. Here, we have systematically studied all 13 ACh receptors (AChRs) in Drosophila to understand mechanisms underlying ACh function in sleep and arousal. We found that exogenous stimuli-induced arousal was decreased in nAChRα3 mutants, whereas sleep was decreased in nAChRα2 and nAChRß2 mutants. nAChRα3 functions in dopaminergic neurons to promote exogenous stimuli-induced arousal, whereas nAChRα2 and ß2 function in octopaminergic neurons to promote sleep. Our studies have revealed that a single transmitter can promote endogenous sleep and exogenous stimuli-induced arousal through distinct receptors in different types of downstream neurons.

D-Serine made by serine racemase in Drosophila intestine plays a physiological role in sleep.

Natural D-serine (D-Ser) has been detected in animals more than two decades ago, but little is known about the physiological functions of D-Ser. Here we reveal sleep regulation by endogenous D-Ser. Sleep was decreased in mutants defective in D-Ser synthesis or its receptor the N-methyl-D-aspartic receptor 1 (NMDAR1), but increased in mutants defective in D-Ser degradation. D-Ser but not L-Ser rescued the phenotype of mutants lacking serine racemase (SR), the key enzyme for D-Ser synthesis. Pharmacological and triple gene knockout experiments indicate that D-Ser functions upstream of NMDAR1. Expression of SR was detected in both the nervous system and the intestines. Strikingly, reintroduction of SR into specific intestinal epithelial cells rescued the sleep phenotype of sr mutants. Our results have established a novel physiological function for endogenous D-Ser and a surprising role for intestinal cells.