The Circadian Neuropeptide PDF has Sexually Dimorphic Effects on Activity Rhythms.

The circadian system regulates the timing of multiple molecular, physiological, metabolic, and behavioral phenomena. In Drosophila as in other species, most of the research on how the timekeeping system in the brain controls timing of behavioral outputs has been conducted in males, or sex was not included as a biological variable. The main circadian pacemaker neurons in Drosophila release the neuropeptide Pigment Dispersing Factor (PDF), which functions as a key synchronizing factor in the network with complex effects on other clock neurons. Lack of Pdf or its receptor, PdfR, results in most flies displaying arrhythmicity in activity-rest cycles under constant conditions. However, our results show that female circadian rhythms are less affected by mutations in both Pdf and PdfR . Crispr-Cas9 mutagenesis of Pdf specifically in the ventral lateral neurons (LN v s) also has a greater effect on male rhythms. We tested the influence of the M-cells over the circadian network and show that speeding up the molecular clock specifically in the M-cells leads to sexually dimorphic phenotypes, with a more pronounced effect on male rhythmic behavior. Our results suggest that the female circadian system is more resilient to manipulations of the PDF pathway and that circadian timekeeping is more distributed across the clock neuron network in females.

Orai-mediated calcium entry determines activity of central dopaminergic neurons by regulation of gene expression.

Maturation and fine-tuning of neural circuits frequently require neuromodulatory signals that set the excitability threshold, neuronal connectivity, and synaptic strength. Here, we present a mechanistic study of how neuromodulator-stimulated intracellular Ca2+ signals, through the store-operated Ca2+ channel Orai, regulate intrinsic neuronal properties by control of developmental gene expression in flight-promoting central dopaminergic neurons (fpDANs). The fpDANs receive cholinergic inputs for release of dopamine at a central brain tripartite synapse that sustains flight (Sharma and Hasan, 2020). Cholinergic inputs act on the muscarinic acetylcholine receptor to stimulate intracellular Ca2+ release through the endoplasmic reticulum (ER) localised inositol 1,4,5-trisphosphate receptor followed by ER-store depletion and Orai-mediated store-operated Ca2+ entry (SOCE). Analysis of gene expression in fpDANs followed by genetic, cellular, and molecular studies identified Orai-mediated Ca2+ entry as a key regulator of excitability in fpDANs during circuit maturation. SOCE activates the transcription factor trithorax-like (Trl), which in turn drives expression of a set of genes, including Set2, that encodes a histone 3 lysine 36 methyltransferase (H3K36me3). Set2 function establishes a positive feedback loop, essential for receiving neuromodulatory cholinergic inputs and sustaining SOCE. Chromatin-modifying activity of Set2 changes the epigenetic status of fpDANs and drives expression of key ion channel and signalling genes that determine fpDAN activity. Loss of activity reduces the axonal arborisation of fpDANs within the MB lobe and prevents dopamine release required for the maintenance of long flight.

Dissecting neuron-specific functions of circadian genes using modified cell-specific CRISPR approaches.

Circadian behavioral rhythms in Drosophila melanogaster are regulated by about 75 pairs of brain neurons. They all express the core clock genes but have distinct functions and gene expression profiles. To understand the importance of these distinct molecular programs, neuron-specific gene manipulations are essential. Although RNAi based methods are standard to manipulate gene expression in a cell-specific manner, they are often ineffective, especially in assays involving smaller numbers of neurons or weaker Gal4 drivers. We and others recently exploited a neuron-specific CRISPR-based method to mutagenize genes within circadian neurons. Here, we further explore this approach to mutagenize three well-studied clock genes: the transcription factor gene vrille, the photoreceptor gene Cryptochrome (cry), and the neuropeptide gene Pdf (pigment dispersing factor). The CRISPR-based strategy not only reproduced their known phenotypes but also assigned cry function for different light-mediated phenotypes to discrete, different subsets of clock neurons. We further tested two recently published methods for temporal regulation in adult neurons, inducible Cas9 and the auxin-inducible gene expression system. The results were not identical, but both approaches successfully showed that the adult-specific knockout of the neuropeptide Pdf reproduces the canonical loss-of-function mutant phenotypes. In summary, a CRISPR-based strategy is a highly effective, reliable, and general method to temporally manipulate gene function in specific adult neurons.

Dopamine and GPCR-mediated modulation of DN1 clock neurons gates the circadian timing of sleep.

The metronome-like circadian regulation of sleep timing must still adapt to an uncertain environment. Recent studies in Drosophila indicate that neuromodulation not only plays a key role in clock neuron synchronization but also affects interactions between the clock network and brain sleep centers. We show here that the targets of neuromodulators, G Protein Coupled Receptors (GPCRs), are highly enriched in the fly brain circadian clock network. Single-cell sequencing indicates that they are not only enriched but also differentially expressed and contribute to clock neuron identity. We generated a comprehensive guide library to mutagenize individual GPCRs in specific neurons and verified the strategy by introducing a targeted sequencing approach. Combined with a behavioral screen, the mutagenesis strategy revealed a role of dopamine in sleep regulation by identifying two dopamine receptors and a clock neuron subpopulation that gate the timing of sleep.

IP3-mediated Ca2+ signals regulate larval to pupal transition under nutrient stress through the H3K36 methyltransferase Set2.

Persistent loss of dietary protein usually signals a shutdown of key metabolic pathways. In Drosophila larvae that have reached a 'critical weight' and can pupariate to form viable adults, such a metabolic shutdown would needlessly lead to death. Inositol 1,4,5-trisphosphate-mediated calcium (IP3/Ca2+) release in some interneurons (vGlutVGN6341) allows Drosophila larvae to pupariate on a protein-deficient diet by partially circumventing this shutdown through upregulation of neuropeptide signaling and the expression of ecdysone synthesis genes. Here, we show that IP3/Ca2+ signals in vGlutVGN6341 neurons drive expression of Set2, a gene encoding Drosophila Histone 3 Lysine 36 methyltransferase. Furthermore, Set2 expression is required for larvae to pupariate in the absence of dietary protein. IP3/Ca2+ signal-driven Set2 expression upregulates key Ca2+-signaling genes through a novel positive-feedback loop. Transcriptomic studies, coupled with analysis of existing ChIP-seq datasets, identified genes from larval and pupal stages that normally exhibit robust H3K36 trimethyl marks on their gene bodies and concomitantly undergo stronger downregulation by knockdown of either the intracellular Ca2+ release channel IP3R or Set2. IP3/Ca2+ signals thus regulate gene expression through Set2-mediated H3K36 marks on select neuronal genes for the larval to pupal transition.

Ral function in muscle is required for flight maintenance in Drosophila.

Ral is a small GTPase of the Ras superfamily that is important for a number of cellular functions. Recently, we found that expression of Ral is regulated by store-operated calcium entry (SOCE) in Drosophila neurons. In this study, through genetic and behavioural experiments, we show that Ral function is required in differentiated muscles for flight. Reducing Ral function in muscles, specifically reduced duration of flight bouts but not other motor functions, like climbing. Interestingly, unlike in the nervous system, Ral expression in the muscle is not regulated by SOCE. Moreover, either knockdown or genetic inhibition of SOCE in muscles does not affect flight. These findings demonstrate that a multiplicity of signalling mechanisms very likely regulate Ral expression in different tissues.

A Multicomponent Neuronal Response Encodes the Larval Decision to Pupariate upon Amino Acid Starvation.

Organisms need to coordinate growth with development, particularly in the context of nutrient availability. Thus, multiple ways have evolved to survive extrinsic nutrient deprivation during development. In Drosophila, growth occurs during larval development. Larvae are thus critically dependent on nutritional inputs; but after critical weight, they pupariate even when starved. How nutrient availability is coupled to the internal metabolic state for the decision to pupariate needs better understanding. We had earlier identified glutamatergic interneurons in the ventral ganglion that regulate pupariation on a protein-deficient diet. Here we report that Drosophila third instar larvae (either sex) sense arginine to evaluate their nutrient environment using an amino acid transporter Slimfast. The glutamatergic interneurons integrate external protein availability with internal metabolic state through neuropeptide signals. IP3-mediated calcium release and store-operated calcium entry are essential in these glutamatergic neurons for such integration and alter neuronal function by reducing the expression of multiple ion channels.SIGNIFICANCE STATEMENT Coordinating growth with development, in the context of nutrient availability is a challenge for all organisms in nature. After attainment of "critical weight," insect larvae can pupariate, even in the absence of nutrition. Mechanism(s) that stimulate appropriate cellular responses and allow normal development on a nutritionally deficient diet remain to be understood. Here, we demonstrate that nutritional deprivation, in postcritical weight larvae, is sensed by special sensory neurons through an amino acid transporter that detects loss of environmental arginine. This information is integrated by glutamatergic interneurons with the internal metabolic state through neuropeptide signals. These glutamatergic interneurons require calcium-signaling-regulated expression of a host of neuronal channels to generate complex calcium signals essential for pupariation on a protein-deficient diet.

dSTIM- and Ral/Exocyst-Mediated Synaptic Release from Pupal Dopaminergic Neurons Sustains Drosophila Flight.

Manifestation of appropriate behavior in adult animals requires developmental mechanisms that help in the formation of correctly wired neural circuits. Flight circuit development in Drosophila requires store-operated calcium entry (SOCE) through the STIM/Orai pathway. SOCE-associated flight deficits in adult Drosophila derive extensively from regulation of gene expression in pupal neurons, and one such SOCE-regulated gene encodes the small GTPase Ral. The cellular mechanism by which Ral helps in maturation of the flight circuit was not understood. Here, we show that knockdown of components of a Ral effector, the exocyst complex, in pupal neurons also leads to reduced flight bout durations, and this phenotype derives primarily from dopaminergic neurons. Importantly, synaptic release from pupal dopaminergic neurons is abrogated upon knockdown of dSTIM, Ral, or exocyst components. Ral overexpression restores the diminished synaptic release of dStim knockdown neurons as well as flight deficits associated with dSTIM knockdown in dopaminergic neurons. These results identify Ral-mediated vesicular release as an effector mechanism of neuronal SOCE in pupal dopaminergic neurons with functional consequences on flight behavior.

A pupal transcriptomic screen identifies Ral as a target of store-operated calcium entry in Drosophila neurons.

Transcriptional regulation by Store-operated Calcium Entry (SOCE) is well studied in non-excitable cells. However, the role of SOCE has been poorly documented in neuronal cells with more complicated calcium dynamics. Previous reports demonstrated a requirement for SOCE in neurons that regulate Drosophila flight bouts. We refine this requirement temporally to the early pupal stage and use RNA-sequencing to identify SOCE mediated gene expression changes in the developing Drosophila pupal nervous system. Down regulation of dStim, the endoplasmic reticular calcium sensor and a principal component of SOCE in the nervous system, altered the expression of 131 genes including Ral, a small GTPase. Disruption of Ral function in neurons impaired flight, whereas ectopic expression of Ral in SOCE-compromised neurons restored flight. Through live imaging of calcium transients from cultured pupal neurons, we confirmed that Ral does not participate in SOCE, but acts downstream of it. These results identify neuronal SOCE as a mechanism that regulates expression of specific genes during development of the pupal nervous system and emphasizes the relevance of SOCE-regulated gene expression to flight circuit maturation.

Drosophila larval to pupal switch under nutrient stress requires IP3R/Ca(2+) signalling in glutamatergic interneurons.

Neuronal circuits are known to integrate nutritional information, but the identity of the circuit components is not completely understood. Amino acids are a class of nutrients that are vital for the growth and function of an organism. Here, we report a neuronal circuit that allows Drosophila larvae to overcome amino acid deprivation and pupariate. We find that nutrient stress is sensed by the class IV multidendritic cholinergic neurons. Through live calcium imaging experiments, we show that these cholinergic stimuli are conveyed to glutamatergic neurons in the ventral ganglion through mAChR. We further show that IP3R-dependent calcium transients in the glutamatergic neurons convey this signal to downstream medial neurosecretory cells (mNSCs). The circuit ultimately converges at the ring gland and regulates expression of ecdysteroid biosynthetic genes. Activity in this circuit is thus likely to be an adaptation that provides a layer of regulation to help surpass nutritional stress during development.

Store-Operated Calcium Entry through Orai Is Required for Transcriptional Maturation of the Flight Circuit in Drosophila.

Store operated calcium entry (SOCE) is thought to primarily regulate calcium homeostasis in neurons. Subsequent to identification of Orai as the SOCE channel in nonexcitable cells, investigation of Orai function in neurons demonstrated a requirement for SOCE in Drosophila flight. Here, by analysis of an Orai mutant and by controlled expression of a dominant-negative Drosophila Orai transgene, we show that Orai-mediated SOCE is required in dopaminergic interneurons of the flight circuit during pupal development. Expression of dominant-negative Orai in dopaminergic neurons of pupae abolished flight. The loss of Orai-mediated SOCE alters transcriptional regulation of dopaminergic neurons, leading to downregulation of the enzyme tyrosine hydroxylase, which is essential for dopamine synthesis, and the dopamine transporter, which is required for dopamine uptake after synaptic release. These studies suggest that modulation of SOCE could serve as a novel mechanism for restoring dopamine levels in dopaminergic neurons. SIGNIFICANCE STATEMENT: The specificity of an animal's response to an environmental stimulus is determined in part by the release of neurotransmitters, which are sensed by responding neurons through cognate receptors on their surface. One way by which neurons respond is through release of calcium from intracellular stores followed by store refilling from extracellular calcium sources. This mechanism is called store-operated calcium entry (SOCE). The function of SOCE in neurons has been debated. Here we describe a new function for SOCE in the regulation of neurotransmitter levels in Drosophila flight neurons. This cell-signaling mechanism is required to maintain optimal levels of a key enzyme for dopamine synthesis and may serve as a mechanism for restoring dopamine levels in relevant pathological conditions.

Loss of IP3 receptor function in neuropeptide secreting neurons leads to obesity in adult Drosophila.

BACKGROUND: Intracellular calcium signaling regulates a variety of cellular and physiological processes. The inositol 1,4,5 trisphosphate receptor (IP3R) is a ligand gated calcium channel present on the membranes of endoplasmic reticular stores. In previous work we have shown that Drosophila mutants for the IP3R (itprku) become unnaturally obese as adults with excessive storage of lipids on a normal diet. While the phenotype manifests in cells of the fat body, genetic studies suggest dysregulation of a neurohormonal axis. RESULTS: We show that knockdown of the IP3R, either in all neurons or in peptidergic neurons alone, mimics known itpr mutant phenotypes. The peptidergic neuron domain includes, but is not restricted to, the medial neurosecretory cells as well as the stomatogastric nervous system. Conversely, expression of an itpr+ cDNA in the same set of peptidergic neurons rescues metabolic defects of itprku mutants. Transcript levels of a gene encoding a gastric lipase CG5932 (magro), which is known to regulate triacylglyceride storage, can be regulated by itpr knockdown and over-expression in peptidergic neurons. Thus, the focus of observed itpr mutant phenotypes of starvation resistance, increased body weight, elevated lipid storage and hyperphagia derive primarily from peptidergic neurons. CONCLUSIONS: The present study shows that itpr function in peptidergic neurons is not only necessary but also sufficient for maintaining normal lipid metabolism in Drosophila. Our results suggest that intracellular calcium signaling in peptidergic neurons affects lipid metabolism by both cell autonomous and non-autonomous mechanisms.