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.

Neural control of wing coordination in flies.

At the onset of each flight bout in flies, neural circuits in the CNS must rapidly integrate multimodal sensory stimuli and synchronously engage hinges of the left and right wings for coordinated wing movements. Whereas anatomical and physiological investigations of flight have been conducted on larger flies, molecular genetic studies in Drosophila have helped identify neurons that mediate various levels of flight control. However, neurons that might mediate bilateral coordination of wing movements to precisely synchronize left and right wing engagement at flight onset and maintain their movement in perfect coordination at rapid frequencies during flight maneuvers remain largely unexplored. Wing coordination could be directly modulated via bilateral sensory inputs to motoneurons of steering muscles and/or through central interneurons. Using a Ca(2+)-activity-based GFP reporter, we identified three flight-activated central dopaminergic interneurons in the ventral ganglion, which connect to and activate motoneurons that innervate a pair of direct-steering flight muscles. The activation of these newly identified dopaminergic interneurons is context specific. Whereas bilateral wing engagement for flight requires these neurons, they do not control unilateral wing extension during courtship. Thus, independent central circuits function in the context of different natural behaviors to control the motor circuit for Drosophila wing movement.

Serotonergic neurons of the Drosophila air-puff-stimulated flight circuit.

Monoaminergic modulation of insect flight is well documented. Recently, we demonstrated that synaptic activity is required in serotonergic neurons for Drosophila flight. This requirement is during early pupal development, when the flight circuit is formed, as well as in adults. Using a Ca2+-activity-based GFP reporter, here we show that serotonergic neurons in both prothoracic and mesothoracic segments are activated upon air-puff-stimulated flight. Moreover ectopic activation of the entire serotonergic system by TrpA1, a heat activated cation channel, induces flight, even in the absence of an air-puff stimulus.

A genetic RNAi screen for IP3/Ca²âº coupled GPCRs in Drosophila identifies the PdfR as a regulator of insect flight.

Insect flight is regulated by various sensory inputs and neuromodulatory circuits which function in synchrony to control and fine-tune the final behavioral outcome. The cellular and molecular bases of flight neuromodulatory circuits are not well defined. In Drosophila melanogaster, it is known that neuronal IP3 receptor mediated Ca²âº signaling and store-operated Ca²âº entry (SOCE) are required for air-puff stimulated adult flight. However, G-protein coupled receptors (GPCRs) that activate intracellular Ca²âº signaling in the context of flight are unknown in Drosophila. We performed a genetic RNAi screen to identify GPCRs that regulate flight by activating the IPIP3 receptor. Among the 108 GPCRs screened, we discovered 5 IPIP3/Ca²âº linked GPCRs that are necessary for maintenance of air-puff stimulated flight. Analysis of their temporal requirement established that while some GPCRs are required only during flight circuit development, others are required both in pupal development as well as during adult flight. Interestingly, our study identified the Pigment Dispersing Factor Receptor (PdfR) as a regulator of flight circuit development and as a modulator of acute flight. From the analysis of PdfR expressing neurons relevant for flight and its well-defined roles in other behavioral paradigms, we propose that PdfR signaling functions systemically to integrate multiple sensory inputs and modulate downstream motor behavior.

Altered lipid homeostasis in Drosophila InsP3 receptor mutants leads to obesity and hyperphagia.

Obesity is a complex metabolic disorder that often manifests with a strong genetic component in humans. However, the genetic basis for obesity and the accompanying metabolic syndrome is poorly defined. At a metabolic level, obesity arises from an imbalance between the nutritional intake and energy utilization of an organism. Mechanisms that sense the metabolic state of the individual and convey this information to satiety centers help achieve this balance. Mutations in genes that alter or modify such signaling mechanisms are likely to lead to either obese individuals, who in mammals are at high risk for diabetes and cardiovascular disease, or excessively thin individuals with accompanying health problems. Here we show that Drosophila mutants for an intracellular calcium signaling channel, the inositol 1,4,5-trisphosphate receptor (InsP3R) store excess triglycerides in their fat bodies and become unnaturally obese on a normal diet. Although excess insulin signaling can rescue obesity in InsP3R mutants to some extent, we show that it is not the only cause of the defect. Through mass spectrometric analysis of lipids we find that homeostasis of storage and membrane lipids are altered in InsP3R mutants. Possibly as a compensatory mechanism, InsP3R mutant adults also feed excessively. Thus, reduced InsP3R function alters lipid metabolism and causes hyperphagia in adults. Together, the metabolic and behavioral changes lead to obesity. Our results implicate altered InsP3 signaling as a previously unknown causative factor for metabolic syndrome in humans. Importantly, our studies also suggest preventive dietary interventions.

Synaptic activity in serotonergic neurons is required for air-puff stimulated flight in Drosophila melanogaster.

BACKGROUND: Flight is an integral component of many complex behavioral patterns in insects. The giant fiber circuit has been well studied in several insects including Drosophila. However, components of the insect flight circuit that respond to an air-puff stimulus and comprise the flight central pattern generator are poorly defined. Aminergic neurons have been implicated in locust, moth and Drosophila flight. Here we have investigated the requirement of neuronal activity in serotonergic neurons, during development and in adults, on air-puff induced flight in Drosophila. METHODOLOGY/PRINCIPAL FINDINGS: To target serotonergic neurons specifically, a Drosophila strain that contains regulatory regions from the TRH (Tryptophan Hydroxylase) gene linked to the yeast transcription factor GAL4 was used. By blocking synaptic transmission from serotonergic neurons with a tetanus toxin transgene or by hyperpolarisation with Kir2.1, close to 50% adults became flightless. Temporal expression of a temperature sensitive Dynamin mutant transgene (Shi(ts)) suggests that synaptic function in serotonergic neurons is required both during development and in adults. Depletion of IP(3)R in serotonergic neurons via RNAi did not affect flight. Interestingly, at all stages a partial requirement for synaptic activity in serotonergic neurons was observed. The status of serotonergic neurons was investigated in the central nervous system of larvae and adults expressing tetanus toxin. A small but significant reduction was observed in serotonergic cell number in adult second thoracic segments from flightless tetanus toxin expressing animals. CONCLUSIONS: These studies show that loss of synaptic activity in serotonergic neurons causes a flight deficit. The temporal focus of the flight deficit is during pupal development and in adults. The cause of the flight deficit is likely to be loss of neurons and reduced synaptic function. Based on the partial phenotypes, serotonergic neurons appear to be modulatory, rather than an intrinsic part of the flight circuit.

Inositol 1,4,5-trisphosphate receptor and dSTIM function in Drosophila insulin-producing neurons regulates systemic intracellular calcium homeostasis and flight.

Calcium (Ca(2+)) signaling is known to regulate the development, maintenance and modulation of activity in neuronal circuits that underlie organismal behavior. In Drosophila, intracellular Ca(2+) signaling by the inositol 1,4,5-trisphosphate receptor and the store-operated channel (dOrai) regulates the formation and function of neuronal circuits that control flight. Here, we show that restoring InsP(3)R activity in insulin-producing neurons of flightless InsP(3)R mutants (itpr) during pupal development can rescue systemic flight ability. Expression of the store operated Ca(2+) entry (SOCE) regulator dSTIM in insulin-producing neurons also suppresses compromised flight ability of InsP(3)R mutants suggesting that SOCE can compensate for impaired InsP(3)R function. Despite restricted expression of wild-type InsP(3)R and dSTIM in insulin-producing neurons, a global restoration of SOCE and store Ca(2+) is observed in primary neuronal cultures from the itpr mutant. These results suggest that restoring InsP(3)R-mediated Ca(2+) release and SOCE in a limited subset of neuromodulatory cells can influence systemic behaviors such as flight by regulating intracellular Ca(2+) homeostasis in a large population of neurons through a non-cell-autonomous mechanism.