InteractiveFly: GeneBrief

Corazonin: Biological Overview | References

BIOLOGICAL OVERVIEW

Stress triggers cellular and systemic reactions in organisms to restore homeostasis. Mammalian gonadotropin-releasing hormone (GnRH) and its insect orthologue, adipokinetic hormone (AKH), are known for their roles in modulating stress-related behaviour. This study shows that corazonin (Crz), a peptide homologous to AKH/GnRH, also alters stress physiology in Drosophila. The Crz receptor (CrzR) is expressed in salivary glands and adipocytes of the liver-like fat body, and CrzR knockdown targeted simultaneously to both these tissues increases the fly's resistance to starvation, desiccation and oxidative stress, reduces feeding, alters expression of transcripts of Drosophila insulin-like peptides (DILPs), and affects gene expression in the fat body. Furthermore, in starved flies, CrzR-knockdown increases circulating and stored carbohydrates. Thus, these findings indicate that elevated systemic Crz signalling during stress coordinates increased food intake and diminished energy stores to regain metabolic homeostasis. This study suggests that an ancient stress-peptide in Urbilateria evolved to give rise to present-day GnRH, AKH and Crz signalling systems (Kubrak, 2016).

Stress can be evoked by a multitude of different environmental factors and animals have evolved an arsenal of mechanisms to respond to such aversive stimuli, both systemically and at the cellular level. At the systems level, hormonal and neuronal pathways are involved both in mediating stress signals and in resetting the homeostasis. Thus, in mammals, corticosteroids as well as several neuropeptides and peptide hormones have been identified in stress response pathways (Kubrak, 2016).

The vinegar fly Drosophila has emerged as a versatile genetic model for analysis of stress responses, both at the cellular and organismal levels. At the organismal level, Drosophila insulin-like peptides (DILPs) and adipokinetic hormone (AKH), an insect orthologue of mammalian gonadotropin-releasing hormone (GnRH), play important roles in various stress responses and affect longevity. Corazonin (Crz) is another Drosophila peptide ancestrally related to AKH/GnRH, which has been proposed as a stress-induced hormone based on various actions revealed in several insect species (Boerjan, 2010; Veenstra, 2009; Zhao, 2010; Johnson, 2005; McClure, 2013; Patel, 2014), but mechanisms of Crz function in stress are not known. The Crz receptor (CrzR; CG10698) is evolutionarily related to that of mammalian gonadotropin-releasing hormone (GnRH), but also those of arthropod AKH and AKH-corazonin-like peptide (ACP) (Li, 2016; Tian, 2016). GnRH is known to mediate metabolic and stress-related effects on reproduction, and thus it may be that an ancestral role of Crz, AKH and GnRH in metabolism and stress has been conserved over evolution in parallel with a diversification of other functional roles (Kubrak, 2016).

Knockdown of Crz in the Crz-producing dorsolateral peptidergic neurons (DLPs) in the Drosophila brain affects metabolism and resistance to starvation stress (Kapan, 2012). As the DLPs are neurosecretory cells with axon terminations in the corpora cardiaca, anterior aorta and intestine, it is likely that Crz primarily functions as circulating hormone acting on peripheral tissues. This is supported by detecting expression of the CrzR in the fat body, salivary glands and heart of adult Drosophila (Sha, 2014; Chintapalli, 2007) (see also FlyAtlas, http://flyatlas.org). Thus, this study investigated the role of systemic Crz signalling under normal conditions and during stress in flies. We used different fat body Gal4 drivers were used to knockdown the CrzR. and the flies were tested in a set of assays for effects on metabolism, stress tolerance, gene expression and neuropeptide levels. These drivers also target expression to the salivary glands, a tissue known to express the CrzR. The findings suggest that systemic Crz signalling predominantly to the fat body and salivary glands regulates starvation, desiccation and oxidative stress resistance, as well as food ingestion. Furthermore, in starving flies CrzR-knockdown leads to increased circulating and stored carbohydrates, and altered expression of several genes in the fat body. Indications were found of feedback from the fat body to endocrine cells of the brain and corpora cardiaca, as targeted CrzR-knockdown alters dilp3, dilp5 and Crz transcript levels and Crz and AKH peptide levels. As a comparison to knockdown of the CrzR with the fat body GAL4 drivers, CrzR-RNAi was targetted more broadly with a CrzR-Gal4 driver, and furthermore the effects of Crz peptide knockdown (Crz-Gal4>UAS-Crz-RNAi) was examined. These experiments produced similar phenotypes in the assays performed, suggesting that a major role of Crz signalling in stress and food intake is via peripheral targets. Thus, systemic Crz signalling, including the fat body as a target, regulates food intake, carbohydrate metabolism and storage, and affects the expression of Upd2 in the fat body, which is a feedback signal from the fat body to the brain. Crz may thus operate in stress responses in association with insulin-like peptides and AKH (Kubrak, 2016).

This study study shows that the CrzR, the insect homologue of GnRH receptors, is expressed in the fat body and salivary glands of adult flies. The fat body thus receives Crz signals from the brain that affects carbohydrate but not lipid metabolism, diminishes resistance to starvation, desiccation and oxidative stress, increases food ingestion, and triggers feedback signals from the fat body to the brain. The possibility cannot be excluded that part of these phenotypes are caused by Crz signalling to the salivary gland. However, the aim of this study was to tease apart the effects of systemic Crz signalling from Crz action in the CNS. Several of the effects of diminishing the CrzR in the periphery are more prominent in starved flies. Thus, it is suggestrf that Crz signalling from neuroendocrine cells of the brain to the fat body (and perhaps salivary glands) is important when flies are under nutritional stress. In mammals, the GnRH producing neuroendocrine cells are located in the hypothalamus and receive nutrient inputs via leptin signalling, and the GnRH system is also affected by stress. The Drosophila neuroendocrine cells releasing Crz are located in a brain area that is a functional equivalent of the hypothalamus, and may be regulated by nutrient-sensing inputs, and stress hormones such as the diuretic hormones DH₃₁ and DH₄₄ that are ancestrally related to calcitonin and corticotropin-releasing factor, respectively (Kubrak, 2016).

It has previously been suggested that Crz is utilized in stress signalling in various insects (Boerjan, 2010; Veenstra, 2009), but only a few studies in Drosophila have actually tested this. An earlier report showed that ablation or inactivation of the Crz-producing DLP neurons in the brain resulted in flies with increased resistance to metabolic, oxidative and desiccation stress, as measured by survival, and also resulted in increased triglyceride levels (Zhao, 2010). The same report also found that the Crz transcript decreased during starvation and osmotic, but not oxidative stress. Furthermore, ablation of Crz neurons resulted in elevated dopamine levels in the circulation and increased locomotor activity in male flies. However, these results need to be interpreted with caution as a later study found that another neuropeptide, short neuropeptide F (sNPF), coexpressed with Crz in DLPs, also affects starvation resistance and other metabolism related phenotypes. Nonetheless, a previous paper (Kapan , 2012) demonstrated that knockdown of Crz in DLPs in the Drosophila brain increased starvation resistance and carbohydrate and TAG levels (Kubrak, 2016).

The current results are derived from selectively diminishing Crz signalling to the fat body and salivary glands by targeted CrzR-knockdown. Thus, direct actions on other targets can be excluded, including the brain and heart. Nevertheless, the effects seen in this study following CrzR-knockdown in the periphery on stress resistance and carbohydrate metabolism are similar to those where DLP neurons were targeted by Crz-RNAi. This study also showed that more global knockdown of CrzR or Crz peptide resulted in stress and feeding phenotypes very similar to those obtained after more selective CrzR-RNAi in fat body/salivary gland. Therefore, it seems that a substantial portion of the systemic effects of Crz are mediated via the fat body. Indeed, clear effects were found of CrzR-knockdown on transcription of a few relevant genes in the fat body. The bmm transcript level decreased. bmm encodes the TAG lipase Brummer, which regulates lipid storage and this gene is therefore important in regulation of energy homeostasis. However, as will be discussed in more detail below, effects were also detected of CrzR-knockdown on the fat body genes Upd2, NLaz and TotA. Upd2 is a leptin-like factor, which is nutrient signal released from the fat body acting on the brain IPCs. Such, a feedback to the brain is supported by changes in transcripts of dilp3 and 5, as well as AKH peptide levels after CrzR-knockdown. This feedback may thus result in complex effects after CrzR-knockdown in the fat body due to both direct and indirect regulation of the adipocytes by Crz as well as DILPs and AKH (Kubrak, 2016).

Earlier studies have shown that Crz displays multiple actions in insects, several of which may be associated with stress responses. Crz was first identified as a cardioactive hormone in cockroaches, but its actions have been extended in Drosophila to roles in reproduction (Taylor, 2012, Bergland, 2012), carbohydrate metabolism (Zhao, 2010; Lee, 2008), modulation of locomotor activity (Zhao, 2010), regulation of ethanol sedation and metabolism, and a role in the clock system. In other insects, Crz induces gregarization-associated colour change in locusts and controls ecdysis behaviour in a moth. Furthermore, it was shown recently that during adult reproductive diapause in Drosophila, when stress resistance is increased, transcripts of both Crz and its receptor are significantly upregulated (Kucerova, 2016). In addition, a possible function of Crz signalling to the salivary glands remains to be determined. Data from FlyAtlas suggest the presence of the CrzR in the Drosophila salivary glands, and it cannot be excluded that CrzR-RNAi experiments with ppl- and to-Gal4 drivers generated effects on salivary gland function that contributed to the phenotypes that were recorded. Adult salivary gland function is not well investigated in Drosophila, but this tissue may contribute to facilitating food ingestion and processing by lubrication and release of digestive enzymes (Kubrak, 2016).

The Drosophila Crz receptor is ancestrally related to the GnRH receptor family, which is known to participate in stress responses in mammals. Also, the CrzR and AKH receptor (AkhR) have been proposed to have a common ancestor, suggesting that Crz and AKH signalling might share some of the ancient functions in regulation of stress and metabolism. AKH predominantly stimulates catabolic processes (mobilization of lipids, carbohydrates and amino acids) while simultaneously inhibiting their biosynthesis. Although both AKH and Crz target the fat body, a comparison of the current results and those of earlier studies analysing Akh and AkhR mutants reveals that these two signalling systems play distinct roles in metabolism and stress responses. Knockdown of Crz or ablation of Crz-producing cells leads to increased levels of stored lipids and carbohydrates, and here this study shows that the effect on carbohydrate metabolism is mediated by Crz signalling to the periphery, and this effect is stronger during stress conditions. One difference between Crz and CrzR-knockdown is the lack of effect on TAG levels after CrzR-knockdown in the periphery. This suggests that Crz regulates lipid metabolism indirectly via another signalling system (Kubrak, 2016).

The fat body is not only a primary metabolic tissue and energy store, but it is also an active endocrine organ. Hence, similar to mammals, Drosophila displays reciprocal humoral interactions between adipocytes/liver and brain neuroendocrine cells. The adult fat body can release hormonal factors to modulate IPCs and systemic insulin signalling, which in turn signals to the fat body. In mammals, systemic insulin signalling is influenced by adipocyte-derived hormonal factors, such as leptin and adiponectin. In Drosophila, a functional leptin homologue, Upd2, produced in the fat body, was shown to regulate IPCs. In addition, DILP6 from the fat body can act on IPCs to decrease DILP2 expression. Fasting induces dilp6 mRNA expression in fat body of Drosophila larvae and adults. In this study, 36 h of starvation failed to affect dilp6 expression but diminished Upd2 in the fat body. Taken together with the altered dilp3 and 5 transcript levels, the results suggest that fat-body-derived humoral signals are affected by Crz activation of the adipocytes (Kubrak, 2016).

This study also assayed a few fat body genes associated with stress signalling in Drosophila. Of these, the mRNA of Turandot A (TotA) was upregulated after CrzR-RNAi in the periphery. TotA is a target gene of Janus kinase/signal transducer and activator of transcription (JAK/STAT) signalling, and is known to play an important role in stress tolerance and immune response. In fed flies, CrzR-knockdown had no effect on the transcript of the antioxidant enzyme manganese-containing superoxide dismutase 2 (Sod2) which is a target of the transcription factor FOXO, but upregulated the lipocalin Neural Lazarillo (encoded by NLaz), which is related to apolipoprotein A (ApoA) in mammals and is part of the stress responsive Jun-N-terminal Kinase (JNK) signalling pathway. Thus, it is concluded that increased Crz action on the fat body upregulates TotA and Nlaz stress signalling (Kubrak, 2016).

In summary, signalling through the CrzR in the periphery during metabolic stress results in increased nutrient intake and reallocated energy stores enabling the fly to reestablish homeostasis. The Crz action triggers transcriptional changes in the adipocytes that include stress genes and one gene involved in metabolism. The alteration of transcript levels of Crz, dilp3 and dilp5 and the decreased AKH peptide levels suggests that Crz signalling to the periphery generates a feedback signal to the brain and corpora cardiaca endocrine cells and thereby gives rise to complex hormonal fine-tuning. The CrzR is considered ancestrally related to the GnRH receptor, which is known to be involved in specific stress responses in mammals. Thus, the role of Crz in stress may be an ancient one, and over evolution Crz, AKH and GnRH signalling systems have acquired additional functions seen both in Drosophila, other invertebrates and in mammals (Kubrak, 2016).

A neuroendocrine pathway modulating osmotic stress in Drosophila

Environmental factors challenge the physiological homeostasis in animals, thereby evoking stress responses. Various mechanisms have evolved to counter stress at the organism level, including regulation by neuropeptides. In recent years, much progress has been made on the mechanisms and neuropeptides that regulate responses to metabolic/nutritional stress, as well as those involved in countering osmotic and ionic stresses. This study identified a peptidergic pathway that links these types of regulatory functions. The neuropeptide Corazonin (Crz), previously implicated in responses to metabolic stress, was uncovered as a neuroendocrine factor that inhibits the release of a diuretic hormone, CAPA, and thereby modulates the tolerance to osmotic and ionic stress. Both knockdown of Crz and acute injections of Crz peptide impact desiccation tolerance and recovery from chill-coma. Mapping of the Crz receptor (CrzR) expression identified three pairs of Capa-expressing neurons (Va neurons) in the ventral nerve cord that mediate these effects of Crz. Crz was shown to act to restore water/ion homeostasis by inhibiting release of CAPA neuropeptides via inhibition of cAMP production in Va neurons. Knockdown of CrzR in Va neurons affects CAPA signaling, and consequently increases tolerance for desiccation, ionic stress and starvation, but delays chill-coma recovery. Optogenetic activation of Va neurons stimulates excretion and simultaneous activation of Crz and CAPA-expressing neurons reduces this response, supporting the inhibitory action of Crz. Thus, Crz inhibits Va neurons to maintain osmotic and ionic homeostasis, which in turn affects stress tolerance. Earlier work demonstrated that systemic Crz signaling restores nutrient levels by promoting food search and feeding. It is additionally proposed that Crz signaling also ensures osmotic homeostasis by inhibiting release of CAPA neuropeptides and suppressing diuresis. Thus, Crz ameliorates stress-associated physiology through systemic modulation of both peptidergic neurosecretory cells and the fat body in Drosophila (Zandawala, 2021).

Environmental conditions continuously challenge the physiological homeostasis in animals, thereby evoking stress that can adversely affect the health and lifespan of an individual. For instance, lack of food and water, extreme temperatures, infection and predation can all evoke stress responses. In order to counter this stress and restore homeostasis, animals have evolved a multitude of physiological and behavioral mechanisms, which involve actions of multiple tissues and/or organs. The core of these mechanisms involves hormones and neuropeptides, which orchestrate the actions of various organs to counteract stress and maintain homeostasis. One well-studied mechanism counteracting water-deficit stress is the mammalian anti-diuretic system that involves hypothalamic osmoreceptors stimulating the sensation of thirst that leads to the release of the anti-diuretic hormone vasopressin, which targets multiple organs, including the kidney, to decrease urine output and conserve water. In insects such as the vinegar fly, Drosophila melanogaster, much progress has been made on the mechanisms and factors regulating metabolic homeostasis, nutritional stress and longevity. Several neuropeptides and peptide hormones have been shown to influence responses to nutrient stress via actions on peripheral tissues such as the liver-like fat body. Specifically, these peptide hormones include Drosophila insulin-like peptides (DILPs), adipokinetic hormone (AKH) and corazonin (Crz). In addition, mechanisms regulating the release of these hormones are being unraveled. Hence, the neural circuits and neuroendocrine pathways regulating metabolic homeostasis and nutritional stress are beginning to be understood. However, the circuits and/or pathways that regulate thermal, osmotic and ionic stresses remain largely unexplored. Thus, this study asked what factors and cellular systems constitute the osmoregulatory axis in Drosophila (and other insects). Since nutrient and osmotic homeostasis are inter-dependent, it was hypothesized that regulation of osmotic stress may involve factors that also regulate nutritional stress. This study identified Crz signaling, a paralog of the AKH/Gonadotropin releasing hormone signaling system, as a candidate regulating these stresses. Based on previous research in Drosophila and other insects, it has been hypothesized that Crz modulates responses to stress, especially nutritional stress. Consistent with this, recent work has shown that neurosecretory cells co-expressing Crz and short neuropeptide F (sNPF) are nutrient sensing and modulate nutrient homeostasis through differential actions of the two co-expressed neuropeptides. Whereas sNPF acts on the insulin-producing cells (IPCs) and AKH-producing cells to stimulate DILP release and inhibit AKH release, respectively, systemic Crz signaling modulates feeding and nutritional stress through actions on the fat body (Zandawala, 2021).

Although the role of Crz in regulating responses to nutritional stress is now established, less is known about its role in cold tolerance and ion/water homeostasis. Hence, to address this, the role of Crz in modulating osmotic and ionic stresses was studied and the cellular systems constituting the Crz signaling axis is furthermore outlined. To this end, this study analyzed the effects of manipulating Crz signaling on desiccation tolerance and chill-coma recovery as these two assays are routinely used to assess responses to osmotic/ionic stress. Both knockdown of Crz and acute injections of Crz peptide impact desiccation tolerance and recovery from chill-coma. Comprehensive mapping of the Crz receptor (CrzR) expression revealed that these effects of Crz are not likely mediated by direct modulation of the osmoregulatory tissues but indirectly via three pairs of neurons (Va neurons) in the ventral nerve cord (VNC), which express diuretic CAPA neuropeptides. Knockdown of the CrzR in Va neurons affects CAPA release and ion/water balance, consequently influencing desiccation tolerance and chill-coma recovery. Crz acts via inhibition of cAMP production in Va neurons to inhibit release of CAPA and thereby reducing water loss during desiccation. These data, taken together with published findings [14] suggest that Crz is released into the hemolymph during nutritional stress and acts on the fat body to mobilize energy for food search to increase food intake. In summary, we propose that Crz acts upstream of CAPA signaling to regulate water and ion balance as well as restore nutrient levels caused by starvation. Thus, in addition to the hormonal actions of Crz on the fat body to maintain metabolic homeostasis and counter nutritional stress (Kubrak, 2021), this peptide also helps maintain osmotic homeostasis (Zandawala, 2021).

This study has found that a peptidergic neuroendocrine pathway in Drosophila, known to restore nutrient deficiency (utilizing Crz), integrates a further peptidergic component (CAPA) to maintain osmotic and ionic homeostasis. The Crz-CAPA signaling thereby also influences tolerance to osmotic and cold stress. An earlier study suggested that Crz is released during nutritional stress to mobilize energy stores from the fat body to fuel food search behavior (Kubrak, 2021). Furthermore, that study suggests that increased Crz signaling compromises resistance to starvation, desiccation and oxidative stress (Kubrak, 2021). This study has confirmed these findings, and also found that Crz inhibits a set of CrzR expressing Va neurons in the abdominal ganglia that produce CAPA peptides, which when released in vivo through optogenetic or thermogenetic control, leads to increased excretion and decreased whole body water content, respectively. Thus, the two peptidergic systems act together to maintain both energy and ion/water homeostasis (Zandawala, 2021).

The Capa gene-derived neuropeptides (CAPA1 and CAPA2) are well established as osmoregulatory factors that act on the Malpighian tubule principal cells (Davies, 2013; Paluzzi, 2012) and perhaps the hindgut, as indicated herein by receptor expression data. Previous ex vivo studies have largely showed that CAPA neuropeptides act as diuretic hormones in Drosophila and other dipterans while anti-diuretic actions have also been reported. This study's in vivo findings are more aligned with the observations supporting a diuretic role of CAPA peptides. Furthermore, this study shows that the CAPA-producing Va neurons are downstream of Crz signaling and it is proposed that under adverse conditions when flies are exposed to dry starvation (desiccation without food) the two signaling systems act in tandem to restore homeostasis. When the fly experiences starvation and nutrients diminish in the fly, nutrient sensors record this deficiency, which triggers release of hormones that act to restore metabolic homeostasis. As indicated above, one such hormone is Crz, known to provide energy for food search and induce feeding. The released Crz also inhibits CAPA release from Va cells during the nutrient shortage and results in diminished excretion (and defecation) during starvation and food search. Additionally, during the revision of this manuscript, a recent paper (Koyama, 2021) reported that CAPA from Va cells also acts on AKH-producing cells in the corpora cardiaca to inhibit AKH release and thereby decreasing lipolysis in adipocytes. Thus, when Crz inhibits the Va neurons, AKH signaling is likely to be elevated and results in increased energy mobilization from the fat body, further facilitating food search. In insects, increased food intake yields water, which requires post-feeding diuresis to be activated to restore water homeostasis. Therefore, the inhibition of CAPA release from Va cells needs to be lifted after successful food search and nutrient intake. This suggests that the Crz action occurs during the starvation/fasting, and thereafter postprandial CAPA action sets in to restore nutrient and water homeostasis. Unfortunately, it was not possible to provide direct evidence for this sequence of events since there is no method sensitive enough to monitor the timing of changes in hemolymph levels of Crz and CAPA in small organisms such as Drosophila. Indirect measurements, like monitoring levels of peptide-immunolabel in neurons of interest are not necessarily accurate, since these levels reflect the 'balance' between peptide release and production and therefore not useful for resolving onset and duration of release with accuracy (Zandawala, 2021).

The evidence for the Crz action on CAPA-producing Va neurons is based on the expression of CrzR in these cells, as well as functional imaging data which shows that Crz inhibits cAMP production in Va neurons (but has no impact on intracellular Ca2+ levels). This led to an investigation of the effects of manipulating Crz signaling and targeted CrzR knockdown in Va neurons on aspects of water and ion homeostasis and cold tolerance, which have been shown earlier to be affected by CAPA signaling.It was observed that global Crz or CrzR knockdown leads to delayed recovery from chill-coma (i.e. jeopardizing cold tolerance) and increased tolerance to ionic stress. Furthermore, experiments that target knockdown of CrzR to Va neurons also affected chill-coma recovery, and tolerance to starvation, desiccation, and ionic stresses. Injecting flies in vivo with Crz in the present study also resulted in effects on chill-coma recovery and survival during desiccation, further strengthening the model in which Crz modulates release of CAPA. However, the effect of CAPA injections on chill-coma recovery shown previously are not directly compatible with the current findings. This may be due to the difficulty in comparing the effects obtained from RNAi manipulations (chronic) and peptide injections (acute). Furthermore, Crz not only affects cold tolerance via its actions on CAPA signaling, it also regulates levels of trehalose, a cryoprotectant, via actions on the fat body [14]. Thus, Crz signaling could modulate cold tolerance via two independent hormonal pathways (Zandawala, 2021).

Previous work in Drosophila showed that not only does global Crz knockdown result in increased resistance to starvation, desiccation and oxidative stress, but also found that CrzR knockdown in the fat body (and salivary glands) led to these same phenotypes. These findings suggest that Crz signaling to the fat body accounts for some of the stress tolerance phenotypes seen following Crz knockdown. Thus, it is possible that the effects observed on desiccation survival following Crz peptide injections in the present study are partly confounded by the actions of this peptide on the fat body and other tissues expressing the CrzR (Zandawala, 2021).

Earlier data show that after CrzR knockdown in the fat body, glucose, trehalose and glycogen levels are elevated in starved flies, but not in normally fed flies. Moreover, starved, but not normally fed flies with Crz knockdown, display increased triacyl glycerides and Crz transcript is upregulated in starved flies with CrzR knockdown in the fat body. This further emphasizes that systemic Crz signaling is critical under nutritional stress. The data on Crz immunolabeling intensity corroborate these earlier findings and suggest that Crz is released to restore metabolic homeostasis by mobilizing energy stores from the fat body to fuel food search behavior. After restoring nutritional and osmotic stress the Crz signaling decreases and thus the proposed inhibition of CAPA release from Va neurons is lifted (Zandawala, 2021).

The question as to how the Crz neurons sense nutrient deficiency or whether they can detect changes in osmolarity. However, it is known that Crz-producing DLPs express an aquaporin, Drip, and the carbohydrate-sensing gustatory receptors, Gr43a and Gr64a. A subset of the Crz neurons also express a glucose transporter (Glut1) that is involved in glucose-sensing. Thus, imbalances in internal nutritional and maybe even osmotic status could either be sensed cell autonomously by the Crz neurons or indirectly by signals relayed to them via other pathways (Zandawala, 2021).

A few other findings might support roles of Crz in ameliorating nutrient stress. This study found that Crz neurons innervate the antennal lobe (AL), and that the CrzR is strongly expressed in local interneurons of the AL. Possibly, Crz modulates odor sensitivity in hungry flies to increase food search, similar to peptides like short neuropeptide F (sNPF), tachykinin and SIFamide. Another peptide hormone, adipokinetic hormone (AKH), has been shown to be critical in initiating locomotor activity and food search in food deprived flies and AKH also affects sensitivity of gustatory neurons to glucose. The effect of AKH on increasing locomotor activity is evident only after 36h of starvation and may correlate with the proposed action of Crz during starvation. Possibly Crz acts in concert with AKH to allocate fuel during metabolic stress. Since it was found that the CrzR is not expressed in AKH-producing cells, it is likely that these two peptides act in parallel rather than in the same circuit/pathway. However, the DLPs also produce sNPF and this peptide is known to act on the AKH producing cells and thereby modulate glucose homeostasis and possibly, sensitivity of gustatory neurons. Thus, the DLPs may act systemically with Crz and by paracrine signaling with sNPF in the corpora cardiaca to act on AKH cells. Thereby the Crz and AKH systems could be linked by sNPF. In addition, while this manuscript was under revision, a paper (Koyama, 2021) was posted that added an interesting angle to the role of AKH signaling. That study revealed that the AKH producing cells (APCs) express the CAPA receptor and that CAPA acts on APCs to decrease AKH release, which diminishes lipolysis in adipocytes. Thus, in the fed fly, CAPA not only induces diuresis, it also diminishes energy mobilization. The same study also showed that the Va cells in flies are active after sucrose feeding or drinking, and that CAPA triggers nutrient uptake and peristalsis in the intestine. This interaction between CAPA-AKH signaling can also explain the direction of the phenotypes seen following CrzR knockdown in Va neurons. For instance, knockdown of CrzR in Va neurons results in increased CAPA release and one would predict decreased desiccation survival if CAPA is exclusively promoting excretion. However, increased CAPA signaling could also result in reduced AKH signaling that in turn promotes starvation survival. Thus, the direction of the phenotypes observed following CrzR knockdown in Va neurons can be explained by CAPA actions on excretion and AKH-release from APCs. Lastly, the possibility was not ruled out that other neuropeptides possibly coexpressed with CAPA could also contribute to the observed phenotypes (Zandawala, 2021).

In conclusion, it is suggested that Crz regulates acute metabolic stress-associated physiology and behavior via the fat body to ensure nutrient allocation to power food search and feeding during prolonged starvation. During food search and feeding, excretion is blocked by Crz acting on the Va cells to inhibit CAPA release. Following food intake and ensuing need for diuresis, Crz signaling ceases and CAPA can be released to ensure restoration of water and ion homeostasis. Taken together, these findings and those of previous studies indicate that Crz acts on multiple neuronal and peripheral targets to coordinate and sustain water, ion and metabolic homeostasis. It might even be possible that an ancient role of the common ancestor of Crz and AKH signaling systems was to modulate stress-associated physiology and that these paralogous signaling systems have sub-functionalized and neo-functionalized over evolution (Zandawala, 2021).

ER-Ca2+ sensor STIM regulates neuropeptides required for development under nutrient restriction in Drosophila

Neuroendocrine cells communicate via neuropeptides to regulate behaviour and physiology. This study examines how STIM (Stromal Interacting Molecule), an ER-Ca2+ sensor required for Store-operated Ca2+ entry, regulates neuropeptides required for Drosophila development under nutrient restriction (NR). Two STIM-regulated peptides, Corazonin and short Neuropeptide F, were found to be required for NR larvae to complete development. Further, a set of secretory DLP (Dorso lateral peptidergic) neurons which co-express both peptides was identified. Partial loss of dSTIM caused peptide accumulation in the DLPs, and reduced systemic Corazonin signalling. Upon NR, larval development correlated with increased peptide levels in the DLPs, which failed to occur when dSTIM was reduced. Comparison of systemic and cellular phenotypes associated with reduced dSTIM, with other cellular perturbations, along with genetic rescue experiments, suggested that dSTIM primarily compromises neuroendocrine function by interfering with neuropeptide release. Under chronic stimulation, dSTIM also appears to regulate neuropeptide synthesis (Megha, 2019).

Metazoan cells commonly use ionic Ca2+ as a second messenger in signal transduction pathways. To do so, levels of cytosolic Ca2+ are dynamically managed. In the resting state, cytosolic Ca2+ concentration is kept low and maintained thus by the active sequestration of Ca2+ into various organelles, the largest of which is the ER. Upon activation, ligand-activated Ca2+ channels on the ER, such as the ryanodine receptor or inositol 1,4,5-trisphosphate receptor (IP3R), release ER-store Ca2+ into the cytosol. Loss of ER-Ca2+ causes STromal Interacting Molecule (STIM), an ER-resident transmembrane protein, to dimerize and undergo structural rearrangements. This facilitates the binding of STIM to Orai, a Ca2+ channel on the plasma membrane, whose pore then opens to allow Ca2+ from the extracellular milieu to flow into the cytosol. This type of capacitative Ca2+ entry is called Store-operated Ca2+ entry (SOCE). Of note, key components of SOCE include the IP3R, STIM and Orai, that are ubiquitously expressed in the animal kingdom, underscoring the importance of SOCE to cellular functioning. Depending on cell type and context, SOCE can regulate an array of cellular processes (Megha, 2019).

Neuronal function in particular is fundamentally reliant on the elevation of cytosolic Ca2+. By tuning the frequency and amplitude of cytosolic Ca2+ signals that are generated, distinct stimuli can make the same neuron produce outcomes of different strengths. The source of the Ca2+ influx itself contributes to such modulation as it can either be from internal ER-stores or from the external milieu, through various activity-dependent voltage gated Ca2+ channels (VGCCs) and receptor-activated Ca2+ channels or a combination of the two. Although the contributions of internal ER-Ca2+ stores to neuronal Ca2+ dynamics are well recognized, the study of how STIM and subsequently, SOCE-mediated by it, influences neuronal functioning, is as yet a nascent field (Megha, 2019).

Mammals have two isoforms of STIM, STIM1 and STIM2, both which are widely expressed in the brain. As mammalian neurons also express multiple isoforms of Orai and IP3R, it follows that STIM-mediated SOCE might occur in them. Support for this comes from studies in mice, where STIM1-mediated SOCE has been reported for cerebellar granule neurons and isolated Purkinje neurons, while STIM2-mediated SOCE has been shown in cortical and hippocampal neurons. STIM can also have SOCE-independent roles in excitable cells, that are in contrast to its role via SOCE. In rat cortical neurons and vascular smooth muscle cells, Ca2+ release from ER-stores prompts the translocation of STIM1 to ER-plasma membrane junctions, and binding to the L-type VGCC, CaV1.2. Here STIM1 inhibits CaV1.2 directly and causes it to be internalized, reducing the long-term excitability of these cells. In cardiomyocyte-derived HL1 cells, STIM1 binds to a T-type VGCC, CaV1.3, to manage Ca2+ oscillations during contractions. These studies indicate that STIM regulates cytosolic Ca2+ dynamics in excitable cells, including neurons and that an array of other proteins determines if STIM regulation results in activation or inhibition of neurons. Despite knowledge of the expression of STIM1 and STIM2 in the hypothalamus, the major neuroendocrine centre in vertebrates, studies on STIM in neuroendocrine cells are scarce. This study therefore used Drosophila melanogaster to address this gap (Megha, 2019).

Neuroendocrine cells possess elaborate machinery for the production, processing and secretion of neuropeptides (NPs), which perhaps form the largest group of evolutionarily conserved signalling agents. Inside the brain, NPs typically modulate neuronal activity and consequently, circuits; when released systemically, they act as hormones. Drosophila is typical in having a vast repertoire of NPs that together play a role in almost every aspect of its behaviour and physiology. Consequently, NP synthesis and release are highly regulated processes. As elevation in cytosolic Ca2+ is required for NP release, a contribution for STIM-mediated SOCE to NE function was hypothesized (Megha, 2019).

Drosophila possess a single gene for STIM, IP3R and Orai, and all three interact to regulate SOCE in Drosophila neurons. In dopaminergic neurons, dSTIM is important for flight circuit maturation, with dSTIM-mediated SOCE regulating expression of a number of genes, including Ral, which controls neuronal vesicle exocytosis. In glutamatergic neurons, dSTIM is required for development under nutritional stress and its' loss results in down-regulation of several ion channel genes which ultimately control neuronal excitability. Further, dSTIM over-expression in insulin-producing NE neurons could restore Ca2+ homeostasis in a non-autonomous manner in other neurons of an IP3R mutant, indicating an important role for dSTIM in NE cell output, as well as compensatory interplay between IP3R and dSTIM. At a cellular level, partial loss of dSTIM impairs SOCE in Drosophila neurons as well as mammalian neural precursor cells. Additionally, reducing dSTIM in Drosophila dopaminergic neurons attenuates KCl-evoked depolarisation and as well as vesicle release. Because loss of dSTIM specifically in dimm+ NE cells results in a pupariation defect on nutrient restricted (NR) media, this study used the NR paradigm as a physiologically relevant context in which to investigate STIM's role in NE cells from the cellular as well as systemic perspective (Megha, 2019).

This study employed an in vivo approach coupled to a functional outcome, in order to broaden understanding of how STIM regulates neuropeptides. A role for dSTIM-mediated SOCE in Drosophila neuroendocrine cells for survival on NR was previously established. The previous study offered the opportunity to identify SOCE-regulated peptides, produced in these neuroendocrine cells, that could be investigated in a physiologically relevant context (Megha, 2019).

In Drosophila, both Crz and sNPF have previously been attributed roles in many different behaviours. Crz has roles in adult metabolism and stress responses, sperm transfer and copulation, and regulation of ethanol sedation. While, sNPF has been implicated in various processes including insulin regulation circadian behaviour, sleeping and feeding. Thus, the identification of Crz and sNPF in coping with nutritional stress is perhaps not surprising, but a role for them in coordinating the larval to pupal transition under NR is novel (Megha, 2019).

A role for Crz in conveying nutritional status information is supported by this study. In larvae, Crz+ DLPs are known to play a role in sugar sensing and in adults, they express the fructose receptor Gr43a. Additionally, they express receptors for neuropeptides DH31, DH44 and AstA, which are made in the gut as well as larval CNS. Together, these observations and are strongly indicative of a role for Crz+ DLPs in directly or indirectly sensing nutrients, with a functional role in larval survival and development in nutrient restricted conditions (Megha, 2019).

Several neuropeptides and their associated signalling systems are evolutionarily conserved. The similarities between Crz and GnRH (gonadotrophin-releasing hormone), and sNPF and PrRP (Prolactin-releasing peptide), at the structural, developmental and receptor level therefore, is intriguing. Structural similarity of course does not imply functional conservation, but notably, like sNPF, PrRP has roles in stress response and appetite regulation. This leads to the conjecture that GnRH and PrRP might play a role in mammalian development during nutrient restriction (Megha, 2019).

dSTIM regulates Crz and sNPF at the levels of peptide release and likely, peptide synthesis upon NR. It is speculated that neuroendocrine cells can use these functions of STIM, to fine tune the amount and timing of peptide release, especially under chronic stimulation (such as 24hrs NR), which requires peptide release over a longer timeframe. Temporal regulation of peptide release by dSTIM may also be important in neuroendocrine cells that co-express peptides with multifunctional roles, as is the case for Crz and sNPF. It is conceivable that such different functional outcomes may require distinct bouts of NP release, varying from fast quantile release to slow secretion. As elevation in cytosolic Ca2+ drives NP vesicle release, neurons utilise various combinations of Ca2+ influx mechanisms to tune NP release. For example, in Drosophila neuromuscular junction, octopamine elicits NP release by a combination of cAMP signalling and ER-store Ca2+, and the release is independent of activity-dependent Ca2+ influx. In the mammalian dorsal root ganglion, VGCC activation causes a fast and complete release of NP vesicles, while activation of TRPV1 causes a pulsed and prolonged release. dSTIM-mediated SOCE adds to the repertoire of mechanisms that can regulate cytosolic Ca2+ levels and therefore, vesicle release. This has already been shown for Drosophila dopaminergic neurons and this study extends the scope of release to peptides. Notably, dSTIM regulates exocytosis via Ral in neuroendocrine cells, like in dopaminergic neurons (Megha, 2019).

In Drosophila larval Crz+ DLPs, dSTIM appears to have a role in both fed, as well as NR conditions. On normal food, not only do Crz+ DLPs exhibit small but significant levels of neuronal activity but also, loss of dSTIM in these neurons reduced Crz signalling. Thus, dSTIM regulates Ca2+ dynamics and therefore, neuroendocrine activity, under basal as well as stimulated conditions. This is consistent with observations that basal SOCE contributes to spinogenesis, ER-Ca2+ dynamics as well as transcription. This regulation appears to have functional significance only in NR conditions as pupariation of larvae, with reduced levels of dSTIM in Crz+ neurons, is not affected on normal food. In a broader context, STIM is a critical regulator of cellular Ca2+ homeostasis as well as SOCE, and a role for it in the hypothalamus has been poorly explored. Because STIM is highly conserved across the metazoan phyla, this study predicts a role for STIM and STIM-mediated SOCE in peptidergic neurons of the hypothalamus. There is growing evidence that SOCE is dysregulated in neurodegenerative diseases. In neurons derived from mouse models of familial Alzheimer's disease and early onset Parkinson's, reduced SOCE has been reported. How genetic mutations responsible for these diseases manifest in neuroendocrine cells is unclear. If they were to also reduce SOCE in peptidergic neurons, it's possible that physiological and behavioural symptoms associated with these diseases, may in part stem from compromised SOCE-mediated NP synthesis and release (Megha, 2019).

The evolution and nomenclature of GnRH-type and corazonin-type neuropeptide signaling systems

Gonadotropin-releasing hormone (GnRH) was first discovered in mammals on account of its effect in triggering pituitary release of gonadotropins and the importance of this discovery was recognized forty years ago in the award of the 1977 Nobel Prize for Physiology or Medicine. Investigation of the evolution of GnRH revealed that GnRH-type signaling systems occur throughout the chordates, including agnathans (e.g. lampreys) and urochordates (e.g., sea squirts). Furthermore, the discovery that adipokinetic hormone (AKH) is the ligand for a GnRH-type receptor in the arthropod Drosophila melanogaster provided evidence of the antiquity of GnRH-type signaling. However, the occurrence of other AKH-like peptides in arthropods, which include corazonin and AKH/corazonin-related peptide (ACP), has complicated efforts to reconstruct the evolutionary history of this family of related neuropeptides. Genome/transcriptome sequencing has revealed that both GnRH-type receptors and corazonin-type receptors occur in lophotrochozoan protostomes (annelids, mollusks) and in deuterostomian invertebrates (cephalochordates, hemichordates, echinoderms). Furthermore, peptides that act as ligands for GnRH-type and corazonin-type receptors have been identified in mollusks. However, what has been lacking is experimental evidence that distinct GnRH-type and corazonin-type peptide-receptor signaling pathways occur in deuterostomes. Two neuropeptides that act as ligands for either a GnRH-type receptor or a corazonin-type receptor have been identified in an echinoderm species - the common European starfish Asterias rubens. Discovery of distinct GnRH-type and corazonin-type signaling pathways in this deuterostomian invertebrate has demonstrated for the first time that the evolutionarily origin of these paralogous systems can be traced to the common ancestor of protostomes and deuterostomes. Furthermore, lineage-specific losses of corazonin signaling (in vertebrates, urochordates and nematodes) and duplication of the GnRH signaling system in arthropods (giving rise to the AKH and ACP signaling systems) and quadruplication of the GnRH signaling system in vertebrates (followed by lineage-specific losses or duplications) accounts for the phylogenetic distribution of GnRH/corazonin-type peptide-receptor pathways in extant animals. A standardized nomenclature for GnRH/corazonin-type neuropeptides is proposed wherein peptides are either named 'GnRH' or 'corazonin', with the exception of the paralogous GnRH-type peptides that have arisen by gene duplication in the arthropod lineage and which are referred to as 'AKH' (or red pigment concentrating hormone, 'RCPH', in crustaceans) and 'ACP' (Zandawala, 2017).

Loss of Atg16 delays the alcohol-induced sedation response via regulation of Corazonin neuropeptide production in Drosophila

Autophagy defects lead to the buildup of damaged proteins and organelles, reduced survival during starvation and infections, hypersensitivity to stress and toxic substances, and progressive neurodegeneration. This study shows that, surprisingly, Drosophila mutants lacking the core autophagy gene Atg16 are not only defective in autophagy but also exhibit increased resistance to the sedative effects of ethanol, unlike Atg7 or Atg3 null mutant flies. This mutant phenotype is rescued by the re-expression of Atg16 in Corazonin (Crz)-producing neurosecretory cells that are known to promote the sedation response during ethanol exposure, and RNAi knockdown of Atg16 specifically in these cells also delays the onset of ethanol-induced coma. Atg16 and Crz colocalize within these neurosecretory cells, and both Crz protein and mRNA levels are decreased in Atg16 mutant flies. Thus, Atg16 promotes Crz production to ensure a proper organismal sedation response to ethanol (Varga, 2016).

High doses of ethanol induce a typical motor impairment and sedation in both mammalian and Drosophila models, similar to the effects of severe ethanol intoxication in humans. The risk for alcoholism is higher for people who show increased resistance to the sedating effects of ethanol, partly because they can consume more alcohol. Thus, although sedation is not a model for alcohol addiction, gaining more insight into the regulation of ethanol responses may be relevant for human studies as well (Varga, 2016).

Recent work in Drosophila revealed that the transcription factor apontic mediates ethanol-induced sedation via regulating the production of the neuropeptide hormone Crz by a small population of neurosecretory cells in the fly brain. However, the molecular mechanisms that control this response to ethanol still remain poorly understood (Varga, 2016).

This work identified the core autophagy gene Atg16 as a key component promoting ethanol-induced sedation in Drosophila. Atg16 is a subunit of an E3-like protein complex involved in Atg8a lipidation. Surprisingly, this study found that it acts independent of both the E1-like Atg7 and the E2-like Atg3 enzymes that function in the same pathway during autophagy in the larval fat body. Cell type-specific knockdown and genetic rescue experiments reveal that the function of Atg16 also maps to Crz-producing neurosecretory cells, which were previously implicated in the ethanol sedation response in fruit flies (Varga, 2016).

Importantly, a polymorphism of Atg16 is a genetic risk factor for inflammatory bowel disease. Atg16 has been suggested to affect the secretory function of Paneth cells, which may influence the intestinal microbiota and sensitize affected human patients and mice to chronic inflammation of the gut. Interestingly, this study found that Atg16 regulates Crz expression in Drosophila: both its protein and mRNA levels are reduced in the absence of Atg16 function. The colocalization of Atg16 and Crz within these cells may seem to support a secretory role for Atg16 in this setting, too, but one would expect to see increased rather than decreased level of Crz if there was a secretory defect. It seems more likely that the loss of Atg16 somehow affects crz transcription or mRNA stability. Future studies will be necessary to understand the molecular mechanisms of how Atg16 controls Crz production (Varga, 2016).

Taken together, this study generated and characterized novel Drosophila mutants for Atg16, and showed that in addition to its role autophagy, it also functions in the ethanol-induced sedation response by promoting the production of the neuropeptide Crz in a small group of neurosecretory cells (Varga, 2016).

A small group of neurosecretory cells expressing the transcriptional regulator apontic and the neuropeptide corazonin mediate ethanol sedation in Drosophila

In the fruit fly Drosophila melanogaster, as in mammals, acute exposure to a high dose of ethanol leads to stereotypical behavioral changes beginning with increased activity, followed by incoordination, loss of postural control, and eventually, sedation. The mechanism(s) by which ethanol impacts the CNS leading to ethanol-induced sedation and the genes required for normal sedation sensitivity remain largely unknown. This study identified the gene apontic (apt), an Myb/SANT-containing transcription factor that is required in the nervous system for normal sensitivity to ethanol sedation. Using genetic and behavioral analyses, it was shown that apt mediates sensitivity to ethanol sedation by acting in a small set of neurons that express Corazonin (Crz), a neuropeptide likely involved in the physiological response to stress. The activity of Crz neurons regulates the behavioral response to ethanol, as silencing and activating these neurons affects sedation sensitivity in opposite ways. Furthermore, this effect is mediated by Crz, as flies with reduced crz expression show reduced sensitivity to ethanol sedation. Finally, both apt and crz were found to be rapidly upregulated by acute ethanol exposure. Thus, two genes and a small set of peptidergic neurons were identified that regulate sensitivity to ethanol-induced sedation. It is proposed that Apt regulates the activity of Crz neurons and/or release of the neuropeptide during ethanol exposure (McClure, 2013).

This work identifies apt and crz that mediate the fly's sensitivity to ethanol-induced sedation. Flies with reduced expression of either gene display dramatically decreased ethanol sedation sensitivity; thus, both apt and crz normally promote ethanol sedation. Normal sensitivity to ethanol sedation requires apt expression in neurons during two distinct life stages, metamorphosis and adulthood. Apt function in a subset of crz-expressing neurons (approximately 6 of the 12-16 crz-expressing cells) is necessary and sufficient for normal sensitivity to ethanol sedation. Acute manipulations of the activity of crz neurons led to altered ethanol sedation sensitivity, demonstrating that these neurons play an active role in regulating the behavioral response to ethanol-induced sedation. The neuropeptide Crz is also involved in ethanol sedation, as flies with reduced crz expression, specifically during adulthood, show dramatically decreased ethanol sedation sensitivity. Finally, in response to acute ethanol exposure the expression of both apt and crz are rapidly upregulated during ethanol exposure. It is hypothesized that the Apt-Crz system, functioning in a very small group of neurosecretory cells, may be an early target of ethanol in the fly brain whose function is crucial for normal sensitivity to ethanol (McClure, 2013).

How does Apt regulate ethanol-induced sedation? Although Apt's role could be to regulate the expression of crz, no such function was observed. It is thus postulated that Apt functions to regulate the activity of crz neurons and/or neuropeptide release. For example, Apt, acting as a transcription factor, could regulate the transcription of proteins required for synthesis, packaging, and/or release of Crz and possibly other neuropeptides. Alternatively, Apt could regulate the expression of proteins required for synapse formation. In support of these two possibilities, apt mutant embryos were observed to have defective synaptic transmission at the neuromuscular junction, as well as fewer numbers of active zones within motoneurons, indicating a presynaptic defect (McClure, 2013).

Another possible function for Apt in regulating ethanol sedation behavior may be found in its neuronal requirement during metamorphosis, a time of intense remodeling to construct the adult CNS. During embryogenesis, Apt functions in multiple morphogenetic processes, including tracheal, head, CNS, and heart morphogenesis, as well as border cell migration. It is therefore possible that during metamorphosis Apt establishes proper development and neuronal connectivity of the adult CNS, and in particular the Crz neurons. However, this possibility seems somewhat unlikely given that the adult CNS in apt^13-66 flies appeared normal, as was the number and morphology of Crz-expressing neurons. Additionally, it was found that adult-specific expression of apt in neurons was necessary for normal sedation sensitivity. However, there may be subtle defects in the adult CNS of apt mutant flies, which were not detected, that could contribute to their altered sedation sensitivity (McClure, 2013).

Apt shows highest sequence conservation with the human FSBP, a negative regulator of transcription of the gamma chain of fibrinogen (see Starz-Gaiano, 2008). Sequence conservation between apt and FSBP is observed within the DNA-binding domain. Interestingly, moderate alcohol consumption in humans has been known to exert a cardioprotective effect, in part by lowering levels of circulating Fibrinogen. The mechanism for how alcohol consumption regulates Fibrinogen is currently unknown, but in light of the current findings it is speculated that it may occur at the level of transcription. Ethanol exposure in flies was found to acutely upregulate apt expression. A similar situation may occur in humans, whereby alcohol consumption could upregulate the transcription of FSBP, ultimately leading to negative regulation of the gamma chain of fibrinogen and lowered levels of circulating Fibrinogen, which in turn would provide cardioprotection (McClure, 2013).

The observations implicate crz-expressing neurons in the regulation of ethanol sedation behavior, a function not previously attributed to these neurons. This study demonstrated that adult-specific silencing of crz neurons significantly reduced ethanol sedation sensitivity, while increasing their activity resulted in the opposite phenotype, an increase in ethanol sedation sensitivity. Based on the observation that inhibiting crz expression also reduced ethanol sedation sensitivity, it is believed that the phenotypes associated with crz neuronal manipulations reflect changes in the release of the neuropeptide Crz and activation of its signaling pathway. However, a few Crz neurons also express the short Neuropeptide F (sNPF). sNPF is considered to be a multifunctional neuropeptide due to its broad expression in diverse neuronal types, and its possible role in crz-expressing neurons and in ethanol sedation sensitivity has not been excluded. However, the observation that Apt function is required in a subset of crz neurons to promote ethanol sedation behavior, firmly establishes the importance of these neurons in mediating the behavioral response to ethanol. The data also suggest that the function of both genes, crz and apt, overlaps in a small set of neurons likely located in the pars lateralis (PL) to mediate the behavioral response to ethanol-induced sedation (McClure, 2013).

It has been hypothesized that Crz is released in response to various types of stress in insects (Veenstra, 2009; Boerjan, 2010), and that this could explain its pleiotropic effects. This hypothesis was bolstered by a recent study showing that flies deficient in Crz are resistant to metabolic, osmotic, and oxidative stress, as measured by survival (Zhao, 2010). In addition, Crz plays a role in stress physiology through its association with well characterized stress hormones. For instance, crz-expressing neurons in the PL also express receptors for two diuretic hormones, DH⁴⁴ and DH³¹. By virtue of receptor similarity, DH⁴⁴ and DH³¹ are related to corticotrophin-releasing factor (CRF) and calcitonin-gene related peptide (CGRP), respectively, both of which mediate the mammalian physiological and behavioral responses to stress. Interestingly, both CRF and CGRP act to inhibit secretion of GnRH in the mammalian hypothalamus. This is significant because Crz is thought to be the homolog of mammalian GnRH, and suggests that analogous regulation occurs in Drosophila (Cazzamali, 2002). It is thus possible that in flies a stress signal or the animal's stress status may be relayed to Crz neurons and alters their function. Thus, based on its functional and molecular associations with stress physiology, it is tempting to speculate that the role of Crz signaling in ethanol sedation sensitivity is related to a stress response. A previous study has shown that stress, in the form of heat shock, induces tolerance to a subsequent ethanol exposure, and that ethanol tolerance relies on the gene hangover, a large nuclear zinc-finger protein, that mediates various other stress responses (Scholz, 2005). In addition, several genes related to stress responses have been shown to be upregulated by ethanol exposure in transcriptional profiling studies, including nearly half of all Drosophila heat shock protein genes, as well as genes involved in the regulation of oxidative stress and aging. Importantly, a maladaptive response to stress has been shown in humans to be a major and common element contributing to drug addiction. Finally, an increase in ethanol self-administration has been observed in animal models with physical, social, and emotional stress. In light of these findings, it will be interesting to further explore the role of Crz and its function in stress physiology and the regulation of ethanol-related behaviors (McClure, 2013).

Neuropeptides are diverse signaling molecules that mediate a broad spectrum of physiological and behavioral processes. Several studies have linked neuropeptides to behavioral responses to ethanol. For instance, one of the first ethanol sensitivity mutants described in Drosophila, amnesiac, encodes a neuropeptide homologous to the vertebrate pituitary adenylate cyclase-activating peptide. In addition, mice lacking either neuropeptide Y (NPY), a neuromodulator abundantly expressed in many regions of the CNS, or its Y1 receptor subtype, display increased ethanol consumption and resistance to ethanol sedation, whereas animals overexpressing NPY show the opposite behavioral phenotypes. Neuropeptide F (NPF), the sole member of the NPY family in Drosophila, and its receptor NPFR1, has similarly been shown to mediate the fly's sensitivity to ethanol-induced sedation. Finally, flies with neuronal perturbations in the insulin signaling pathway displayed increased ethanol sedation sensitivity. These studies, implicating the neuropeptide Crz in sensitivity to ethanol sedation, suggest that neuropeptides are important regulators of the behavioral response to ethanol, and it would therefore be interesting to survey all known Drosophila neuropeptides and their downstream signaling components for possible role(s) in ethanol-related behaviors (McClure, 2013).

Diverse roles for the fructose sensor Gr43a

The detection of nutrients, both in food and within the body, is crucial for the regulation of feeding behavior, growth and metabolism. While the molecular basis for sensing food chemicals by the taste system has been firmly linked to specific taste receptors, relatively little is known about the molecular nature of the sensors that monitor nutrients internally. Recent reports of taste receptors expressed in other organ systems, foremost in the gastrointestinal tract of mammals and insects, has led to the proposition that some taste receptors may also be used as sensors of internal nutrients. Indeed, direct evidence has been provided that the Drosophila gustatory receptor 43a (Gr43a) plays a critical role in sensing internal fructose levels in the fly brain. In addition to the brain and the taste system, Gr43a is also expressed in neurons of the proventricular ganglion and the uterus. This paper discusses the multiple potential roles of Gr43a in the fly. Evidence is provided that its activation in the brain is likely mediated by the neuropeptide Corazonin. Finally, it is posited that Gr43a may represent only a precedent for other taste receptors that sense internal nutrients, not only in flies but, quite possibly, in other animals, including mammals (Miyamoto, 2013b).

Omnivores consume a wide selection of nutrients such as carbohydrates, amino acids, fatty acids and numerous salts to meet their needs for energy expenditure, growth and development. Absence of a single group of nutrients can results in stunted growth, morbidity, metabolic dysfunction and premature death. The sense of taste plays a central role for evaluating the palatability of potential food sources, and recent progress in uncovering the molecular and cellular principle that underlie taste perception have led to a broad understanding of how mammals and insects identify and discriminate among different food chemicals and avoid the many non-nutritious, toxic chemicals which often taste bitter.1 Intriguingly, it has been demonstrated recently in both vertebrates and insects that at least some of these nutrients can be sensed not only by the taste systems, but also by internal sensors present in the gastrointestinal system and the brain. For example, mammals appear to sense glucose (and other sugars) in the gut using the T1R2/ T1R3 taste receptors, and the glucose transporter GLUT2 mediates glucose uptake in the pancreas and probably also in selected hypothalamic and other neurons in the brain. These glucose-sensing processes are essential for the regulation of nutrient metabolisms and behaviors via the secretion of insulin, glucagon and numerous neuropeptides. In insects, the G-protein coupled receptor BOSS was proposed to function as a glucose sensor in the fat body to regulate insulin signaling (Kohyama-Koganeya, 2008). Gastrointestinal systems have also been implicated in sensing bitter substances, since gut endothelial cells of mammals and insects express T2R and Gr bitter taste receptors, respectively. Sodium is probably sensed by the mechanosensory channel TRPV1 and the atypical sodium channel NaX in the brain, while PKD2L1, a sour taste sensor, is expressed in the neurons surrounding the central canal of the spinal cord. Finally, both mammals and insects can sense internal levels of amino acids, which is used to modulate their feeding behavior; specifically, uncharged tRNAs are suggested to be mediators of amino acid sensing in brain neurons of mammals (Miyamoto, 2013b).

The Drosophila gustatory receptor 43a (Gr43a), one of the most conserved insect taste receptors, is expressed not only in taste neurons, but also in neurons associated with internal organs such as the brain, the proventricular ganglion and the uterus (Miyamoto, 2012). Expression in these organs was established with a GAL4 knock-in allele (Gr43aGAL4), in which the Gr43a coding sequence was replaced with that of the GAL4 gene. Using Ca2+ imaging, Gr43a was found to function as a narrowly tuned receptor for fructose. These observations raised the intriguing possibility that fructose is not only sensed as a dietary component by the taste system, but also serves as a carbohydrate component in the hemolymph to reflect the internal nutrient status. This paper discusses potential functions of Gr43a expressing neurons in each of the organ system where its expression has been established. This study also shows that the Gr43a expressing uterus neurons respond to fructose in a manner similar to the brain neurons. Finally, evidence is provided that Gr43a expressing neurons use distinct modes of neurotransmission in different organs to propagate stimulation by fructose. Specifically, the Gr43a expressing brain neurons co-express Corazonin, a highly conserved insect neuropeptide, suggesting that downstream neurons, which mediate Gr43a activity, express the Corazonin receptor (Miyamoto, 2013b).

Fly taste neurons, referred to as gustatory receptor neurons (GRNs), are organized in taste sensilla and taste pegs, located in various appendages. The two labial palps harbor close to 80 taste sensilla and taste pegs, and there are ~30 to 40 taste sensilla on each leg and about 20 on each anterior wing margin; while the function of labial and tarsal taste sensilla in mediating feeding responses is well established, the contribution to taste of sensilla on the wing margin are largely unknown. Most taste sensilla contain four GRNs, each thought to detect distinct groups of food and other chemicals: the sweet neuron senses various sugars; the bitter/high salt neuron responds to various non-nutritious and often harmful organic chemicals, as well as high concentration of salt > 400 mM; a third neuron responds to low salt solutions, and the last neuron responds to water. In addition, the fly harbors internal taste neurons, located in three pharyngeal structures, the labral, dorsal and ventral cibarial sense organs (LSO, 18 neurons; DCSO, 6 and VCSO, 8) (Miyamoto, 2013b).

The most prominent expression of Gr43a in the taste system is observed in the legs: Gr43aGAL4 is expressed in a single GRN of two taste sensilla located on the 5th tarsal segment of each leg. These GRNs also express several members of the sugar gustatory receptor (sugar Gr) subfamily consisting of Gr5a, Gr61a and Gr64a-f and are broadly tuned to and activated by most sugars, as determined by Ca2+ imaging. Lack of Gr43a reduces the response specifically to fructose, while absence of the sugar Gr genes abolishes the response to all sugars except fructose and sucrose (disaccharide of fructose and glucose). Finally, lack of all sugar Gr genes and Gr43a completely abolishes fructose and sucrose response, and transgene expression of Gr43a alone is sufficient to restore both responses. Thus, Gr43a functions as a secondary tarsal fructose receptor. It is noted that the Gr43a expressing neuron housed in the 5V1 sensillum is significantly more sensitive to sugars, especially to fructose and sucrose, than other sweet sensing GRNs that do not express Gr43a (Miyamoto, 2013a). A possible explanation for the large contribution of Gr43a to fructose sensing in this sensillum is its high level of expression: analysis from tarsal tissue shows that Gr43a transcripts are represented approximately 10 times more than transcripts of any of the classical sugar receptor genes, even though the former is expressed in fewer cells than the latter. Behavioral relevance for the high sensitivity of Gr43a expressing neurons has yet to be established, as the standard behavioral proboscis extension reflex (PER) response is not significantly affected for any sugars in Gr43a mutant flies when compared with wild type flies (Miyamoto, 2013b).

Gr43a is expressed in ~8 GRNs in the labial palp. Surprisingly, Gr64f, a marker expressed in virtually all sugar neurons, is not co-expressed with Gr43a in these neurons, and Gr66a, a receptor for caffeine and a marker for bitter sensing GRNs, is not co-expressed with Gr43a either. Thus, by default, these Gr43aGAL4 expressing neurons appear to correspond to water or low salt sensing neurons. Single neuron Ca2+ imaging has not yet been possible on sensilla located in the palps and, hence, the response properties of these Gr43a expressing neurons are not known. Compared with other taste organs (i.e., tarsal neurons or pharyngeal neurons; see below), the expression level of Gr43aGAL4 in the labial palp is much lower, and it is therefore also possible that Gr43a has no obvious function in these neurons (Miyamoto, 2013b).

Gr43a is expressed in two neurons located in the LSO and the VCSO. A putative sugar receptor gene, Gr64f is also co-expressed in two of the Gr43aGAL4 positive LSO neurons, but not in the VCSO neurons (Miyamoto, 2012). The only established role for pharyngeal taste neurons has been reported for the VCSO, where bitter sensing (Gr66a expressing) neurons contribute to egg laying preference on lobeline containing food substrates. This is interesting, because bitter chemicals sensed by labial or tarsal neurons suppress feeding responses in proboscis extension reflex (PER) assays. Regardless, no co-expression between Gr43a and Gr66a was observed in any pharyngeal taste neurons. Based on partial co-expression with Gr64f, the role of Gr43a is likely to be related to sensing sugars while food is ingested, but new behavioral paradigms will have to be developed to assess their specific role in feeding (Miyamoto, 2013b).

In addition to gustatory neurons, Gr43a is also expressed in defined sets of neurons of the proventricular ganglion, the brain and the uterus (Miyamoto, 2012). Ca2+ imaging experiments using ex-vivo preparations of brains and uterus confirmed that Gr43a also functions as a fructose receptor in these organs (Miyamoto, 2013b).

The taste organs examine food chemicals before they enter the digestive system, but it is now well documented that both nutrients and potential toxins are also re-evaluated when they are in the gastrointestinal tract. For example, in mammals, sugar taste receptors expressed in the gastrointestinal tract stimulate glucagon-like peptide 1 secretion in response to sugar ingestions, and bitter taste receptors are also expressed in gut epithelial cells of both mammals and insects. In the mouse, activation of T2Rs in the gut leads to secretion of cholecystokinin from enteroendocrine cells, which limits absorption of dietary toxins. In addition, cholecystokinin signaling also increases expression of the ABCB1 efflux transporter, thereby actively limiting absorption of bitter-tasting toxins. In Drosophila, ingested food passes through the pharynx and the foregut and is initially deposited in the crop. The stored food is then moved into the midgut through the proventriculus, a muscular organ that separates foregut and midgut. The proventricular ganglion is located on the dorsal side of the foregut. This ganglion contains about 30-40 neurons, six of which express Gr43a; they send dendritic terminals into the lumen of the foregut, but not into the crop duct. One group of neurons sends axonal projections to the SOG along the esophagus through the brain, forming a nerve bundle with axons of GRNs located in the LSO and the VCSO. The other group of neurons extends axons posteriorly, where they innervate cells in the midgut (Miyamoto, 2012). The anatomy and location of processes of these neurons suggests that fructose content of food may be monitored immediately before entering the crop and the midgut. While the neurons projecting to the SOG may serve similar roles as taste neurons (PER, food intake), the neurons projecting to the midgut may regulate food transport and/or secretion of neuropeptides/hormones in response to sugar consumption. Expression of Gr43a in the gastrointestinal system appears to be conserved across different insect species; the Gr43a orthologs in the silkworm Bombyx mori (BmGr-9) and in the cotton bollworm Helicoverpa armigera (HaGR9) are also expressed in their digestive systems (Miyamoto, 2013b).

Gr43a is expressed in 2-4 neurons located in the posterior superior lateral protocerebrum of each brain hemisphere (Miyamoto, 2012). Two neurons are easily identifiable using live GFP imaging, but two additional neurons are characterized by lower level of expression and are only detected using antibody staining of dissected brains. The Gr43a expressing neurons were shown to respond to fructose, using Ca2+ imaging of ex vivo brain preparations at levels as low as 5 mM. Indeed, hemolymph sugar measurements have revealed that fructose levels increase to at least ~5 mM after flies feed on various nutritious carbohydrates, suggesting that these neurons are activated after ingestion of a carbohydrate rich meal. The steep increase of hemolymph fructose can be observed regardless of the type of sugar present in the meal, as long as it is metabolized, whereas non-nutritious carbohydrates (sucralose, xylose, arabinose) fail to increase hemolymph fructose. This observation suggests that a fraction of dietary, nutritious sugar is converted into fructose after ingestion, probably via the polyol pathway, and that this conversion is used to signal to the brain that carbohydrates are consumed. This signaling event is thought to be integrated with the feeding status (hungry vs. satiated), thereby establishing positive or negative valence (Miyamoto, 2013b).

Animals can sense the nutritional value of food and regulate their food intake through non-taste mechanisms. Feeding experiments of wild type flies and flies in which Gr43a expression was restricted to specific cells revealed that the brain neurons function as a nutrient sensor: in hungry flies, the Gr43a brain neurons are necessary and sufficient to promote feeding, while in satiated flies, they function to suppress feeding (Miyamoto, 2012). Based on these observations, it is proposed that ingestion of nutritious carbohydrates rapidly increases circulating fructose, resulting in activation of Gr43a-expressing brain neurons. This activation is perceived positively in hungry flies and reinforces feeding, but it is perceived negatively in satiated flies, leading to termination/ suppression of feeding. Evoking opposite perceptions by a single group of neurons is unusual, but not unprecedented. In mice, a small set of neurons in the piriform cortex, a higher order olfactory integration center, mediates opposite valence (attraction vs. avoidance), depending on the nature of stimuli during conditioning (Choi, 2011). The mechanisms by which piriform neurons in the mouse or Gr43a brain neurons in the fly accomplish such binary valence are unknown. In the fly, it is possible that the Gr43a brain neurons communicate with two, functionally distinct, group of target neurons, a notion that is supported by distinct projection tracts of subsets of these neurons (Miyamoto, 2013b).

One role of the insect uterus is to provide a receptacle for sperm and seminal fluid during copulation. In the fly, the seminal fluid not only contains sperm to fertilize the egg, but it serves also as a source of signals that induce numerous changes in the female's behavior. In addition, the uterus also serves as a storage space for the egg prior deposition, and as a contractible muscle for expunging the mature egg (Miyamoto, 2013b).

The uterus harbors three neuronal clusters, and Gr43a is expressed in approximately 4 out of 10 neurons in one of them. These neurons send dendritic and axonal projections to the uterus lumen and the abdominal ganglion, respectively. To assess and confirm ligand specificity of Gr43a expressing uterus neurons, an ex vivo preparation was established, and Ca2+ imaging studies were performed. These experiments demonstrate that fructose specifically activates these neurons, though the sensitivity and magnitude is lower than that of leg or brain neurons (Miyamoto, 2013b).

Activation of the sex peptide receptor (SPR) in neurons of the uterus is essential for females to undergo various postmating changes, such as increase in egg production, reduction in mating activity and a switch from a carbohydrate-rich diet to one containing more protein. SPR was shown to be activated by sex peptide (SP), one of several small proteins present in seminal fluid, which is transferred to the female reproductive tract during mating. While SPR is broadly expressed throughout the nervous system, expression in the uterus neurons alone is sufficient to induce changes in post-mating behaviors. It is noted that fructose is abundantly present in the seminal fluids of mammals and some insects, albeit it is currently unknown whether it is found in Drosophila seminal fluid (Miyamoto, 2013b).

Interestingly, this study found that Gr43a and SPR are co-expressed in the uterus neurons, suggesting that these neurons may sense several cues present in male seminal fluid. Interaction of SP with SPR leads to silencing of the neuron and hence, simultaneous binding of SPR and Gr43a to their ligand might have counteracting (inhibitory and excitatory) effects on these cells, leading to modulation of one system by the other. However, it is also possible that hemolymph fructose is sensed by these neurons, which would imply that feeding on carbohydrate can modulate postmating responses through counteracting SPR mediated silencing. Future studies will be necessary to elucidate the physiological role of Gr43a in uterus neurons for post-mating behavior (Miyamoto, 2013b).

Fructose and its receptor play roles in Drosophila nutrient sensing in multiple organ systems. A well-defined function is currently only evident in the brain, where it provides distinct valence to the experience of food intake. To dissect the mechanism of satiation-dependent valence setting, it will be crucial to define the neural circuit that is governed by Gr43a. A first and important step toward this goal is to identify the neurotransmitter that is released in response to Gr43a neural activation. A striking similarity of brain expression patterns was noticed between Gr43aGAL4 and Corazonin (Crz). Therefore potential co-expression of these two genes was examined, and it was observed that all Gr43aGAL4-positive neurons also express this peptide. Crz, a short neuropeptide/ hormone, and its receptor (crzR) are orthologs of the mammalian gonadotropin releasing hormone and its receptor, respectively. In Drosophila, a role for Crz and crzR has been reported in resistance to alcohol sedation and the regulation of sperm and seminal fluid transfer during mating. Interestingly, the latter study provided evidence that Crz acts as a neurotransmitter, rather than a hormone. Thus, the next goal is to identify the brain neurons that express the crzR gene, which will provide critical information about the downstream targets in this neural circuit. It is noted that Crz is not expressed in other internal Gr43aGAL4 expressing neurons (uterus, proventriculus) or in taste neurons and, therefore, another neurotransmitter must be used to mediate Gr43a activity in these organ systems (Miyamoto, 2013b).

The role of fructose as a nutrient signal is not well understood. The identification of an internal fructose sensor and the observation that fructose is a regulated component of Drosophila hemolymph, features likely to be conserved in many other insect species, will stimulate at least three avenues of future studies. First, other Gr proteins known to function as taste receptors are expressed in internal organs, including the brain, and are therefore likely employed to sense internal signaling molecules (other sugars, especially glucose and trehalose, as well as amino acids). In this regard, it is noteworthy that numerous members of the Gr28 gene family are expressed in many neuronal and non-neuronal cell populations43 throughout development and in the adult, and several putative bitter and sugar receptors were found to be expressed in the gastrointestinal tract of larvae. Thus, identification of their ligands and their specific function in feeding and other behaviors will be of great interest. Second, the Gr43a brain neurons represent a highly tractable and relatively simple case of a brain structure that mediates opposite valence, and they therefore provide an ideal case to dissect the mechanism of choice behavior encoded in the insect brain. And third, the emergence of hemolymph fructose in insects warrants efforts to explore the potential role of this sugar as a nutrient ligand in other organisms, including mammals. In humans, the increase of fructose consumption is strongly associated with a steady increase in obesity, insulin resistance and other metabolic syndromes. In addition, several studies in humans and mice suggest that fructose and glucose, the most common dietary sugars, are absorbed in distinct regions of the gastrointestinal tract and metabolized differently, and that fructose and glucose affect brain activity and feeding behavior in a disparate manner. Thus, it will be interesting to see whether this sugar (and a specific receptor for it) also plays a role in nutrient sensing in mammals (Miyamoto, 2013b).

Regulation of ethanol-related behavior and ethanol metabolism by the Corazonin neurons and Corazonin receptor in Drosophila melanogaster.

Impaired ethanol metabolism can lead to various alcohol-related health problems. Key enzymes in ethanol metabolism are alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH); however, neuroendocrine pathways that regulate the activities of these enzymes are largely unexplored. This study has identified a neuroendocrine system involving Corazonin (Crz) neuropeptide and its receptor (CrzR) as important physiological regulators of ethanol metabolism in Drosophila. Crz-cell deficient (Crz-CD) flies displayed significantly delayed recovery from ethanol-induced sedation that is referred to as hangover-like phenotype. Newly generated mutant lacking Crz Receptor (CrzR⁰¹) and CrzR-knockdown flies showed even more severe hangover-like phenotype, which is causally associated with fast accumulation of acetaldehyde in theCrzR⁰¹ mutant following ethanol exposure. Higher levels of acetaldehyde are likely due to 30% reduced ALDH activity in the mutants. Moreover, increased ADH activity was found in the CrzR⁰¹ mutant, but not in the Crz-CD flies. Quantitative RT-PCR revealed transcriptional upregulation of Adh gene in the CrzR⁰¹. Transgenic inhibition of cyclic AMP-dependent protein kinase (PKA) also results in significantly increased ADH activity and Adh mRNA levels, indicating PKA-dependent transcriptional regulation of Adh by CrzR. Furthermore, inhibition of PKA or cAMP response element binding protein (CREB) in CrzR cells leads to comparable hangover-like phenotype to the CrzR⁰¹ mutant. These findings suggest that CrzR-associated signaling pathway is critical for ethanol detoxification via Crz-dependent regulation of ALDH activity and Crz-independent transcriptional regulation of ADH. This study provides new insights into the neuroendocrine-associated ethanol-related behavior and metabolism (Sha, 2014).

Depending on the concentrations, effects of ethanol consumption in humans include euphoria, impaired motor function and speech, followed by vomiting, coma and even death in certain cases. Upon cessation of drinking, hangover is characterized by unpleasant physical pains such as headache, sensory problems such as vertigo, gastrointestinal symptoms such as nausea and vomiting, and disruption of sleep and biological rhythms. These symptoms are mainly from acetaldehyde accumulation, as supported by high incidence of ethanol intoxication in Eastern Asian populations due to the polymorphic deficiency of functional ALDH2 allele and ALDH2-knockout mice. In line with this, drugs inhibiting ALDH, such as disulfiram, have been used to treat chronic alcoholism by causing adverse symptoms from ethanol intak. Despite these reports, relatively little is known about the regulation of ethanol metabolism by physiological factors (Sha, 2014).

Like mammals, D. melanogaster metabolizes ethanol to acetaldehyde and acetate catalyzed by ADH and ALDH, respectively, and Adh and Aldh mutant flies showed dramatically reduced tolerance to ethanol. Intriguingly, our present data suggest that a neuroendocrine system involving Crz plays an important role in regulating ADH and ALDH activities. CrzR activation leads to the PKA-dependent transcriptional repression of Adh and to post-transcriptional activation of ALDH via unknown pathways, which together suppress acetaldehyde accumulation (Sha, 2014).

In contrast to CrzR⁰¹, Crz-CD did not affect ADH activity levels, but reduced ALDH activity. Such a difference could explain that the Crz-CD flies displayed milder hangover-like phenotype than CrzR⁰¹ did. One might argue that Crz-CD still has residual Crz function. Although this is a possibility, undetectable Crz neurosecretory cells in the Crz-CD brains indicate near lack of Crz function. This is much more severe than RNAi-induced Crz knockdown with respect to the level of Crz expression. Another caveat is that Crz-CD phenotype is due to an elimination of other co-existing transmitters. In fact, a subset of Crz neurons was shown to co-express small neuropeptide F (sNPF). However, since sNPF-producing neurons are so widely distributed in the brain, ablation of a few neurons co-expressing Crz and sNPF is unlikely to affect sNPF functions. Although more definitive evidence for the Crz await Crz-null mutant, the data with CrzR-null mutant support a role for Crz in the hangover-like phenotype (Sha, 2014).

Assuming that Crz-CD eliminates Crz function nearly entirely, it is proposed that CrzR mediates two separate signaling pathways; Crz-dependent up-regulation of ALDH activity and Crz-independent down-regulation of Adh transcription. The latter pathway might involve a distinct ligand that activates CrzR. One possible candidate is adipokinetic hormone (AKH), as the AKH receptor is structurally related to the CrzR with 56% amino acid sequence similarity. However, CrzR showed very little affinity for AKH, indicating that AKH is unlikely to be a natural ligand for CrzR. Alternatively, CrzR might have an intrinsic activity that is not required for ligand binding. It is not uncommon that some GPCRs have intrinsic (or spontaneous) activities in the absence of ligand binding (Sha, 2014).

Although the identity of the second ligand for CrzR is speculative, a growing body of evidence suggests that a GPCR couples to different G-proteins in response to different agonists; such molecular flexibility is often referred to as 'functional selectivity' of GPCR. According to this hypothesis, binding properties of different ligands induce and stabilize a unique conformational status of GPCR, which in turn shifts coupling preference to different G-proteins. Recently, CrzR isolated from the silkworm, Bombyx mori, was shown to couple dually to the Gq and Gs proteins in cell-based assays. However, the current results indicate that Gq-associated PKC activation is unlikely to be involved in the regulation of Adh transcription in Drosophila. Thus it is proposed that Gs-led PKA activation is the major in vivo signaling pathway of the CrzR at least for the Adh regulation. Nevertheless, it seems that CrzR is an excellent model system to unravel the physiological dynamics of the GPCR (Sha, 2014).

Essential role of grim-led programmed cell death for the establishment of corazonin-producing peptidergic nervous system during embryogenesis and metamorphosis in Drosophila melanogaster

In Drosophila melanogaster, combinatorial activities of four death genes, head involution defective (hid), reaper (rpr), grim, and sickle (skl), have been known to play crucial roles in the developmentally regulated programmed cell death (PCD) of various tissues. However, different expression patterns of the death genes also suggest distinct functions played by each. During early metamorphosis, a great number of larval neurons unfit for adult life style are removed by PCD. Among them are eight pairs of corazonin-expressing larval peptidergic neurons in the ventral nerve cord (vCrz). To reveal death genes responsible for the PCD of vCrz neurons, extant and recently available mutations were examined, as well as RNA interference that disrupt functions of single or multiple death genes. grim was found to be a chief proapoptotic gene and skl and rpr as minor ones. The function of grim is also required for PCD of the mitotic sibling cells of the vCrz neuronal precursors (EW3-sib) during embryonic neurogenesis. An intergenic region between grim and rpr, which, it has been suggested, may enhance expression of three death genes in embryonic neuroblasts, appears to play a role for the vCrz PCD, but not for the EW3-sib cell death. The death of vCrz neurons and EW3-sib is triggered by ecdysone and the Notch signaling pathway, respectively, suggesting distinct regulatory mechanisms of grim expression in a cell- and developmental stage-specific manner (Lee, 1013).

Identified peptidergic neurons in the Drosophila brain regulate insulin-producing cells, stress responses and metabolism by coexpressed short neuropeptide F and corazonin

Insulin/IGF-like signaling regulates the development, growth, fecundity, metabolic homeostasis, stress resistance and lifespan in worms, flies and mammals. Eight insulin-like peptides (DILP1-8) are found in Drosophila. Three of these (DILP2, 3 and 5) are produced by a set of median neurosecretory cells (insulin-producing cells, IPCs) in the brain. Activity in the IPCs of adult flies is regulated by glucose and several neurotransmitters and neuropeptides. One of these, short neuropeptide F (sNPF), regulates food intake, growth and Dilp transcript levels in IPCs via the sNPF receptor (sNPFR1) expressed on IPCs. This study identified a set of brain neurons that utilizes sNPF to activate the IPCs. These sNPF-expressing neurons (dorsal lateral peptidergic neurons, DLPs) also produce the neuropeptide corazonin (CRZ) and have axon terminations impinging on IPCs. Knockdown of either sNPF or CRZ in DLPs extends survival in flies exposed to starvation and alters carbohydrate and lipid metabolism. Expression of sNPF in DLPs in the sNPF mutant background is sufficient to rescue wild-type metabolism and response to starvation. Since CRZ receptor RNAi in IPCs affects starvation resistance and metabolism, similar to peptide knockdown in DLPs, it is likely that also CRZ targets the IPCs. Knockdown of sNPF, but not CRZ in DLPs decreases transcription of Dilp2 and 5 in the brain, suggesting different mechanisms of action on IPCs of the two co-released peptides. These findings indicate that sNPF and CRZ co-released from a small set of neurons regulate IPCs, stress resistance and metabolism in adult Drosophila (Kapan, 2012).

A neuropeptide circuit that coordinates sperm transfer and copulation duration in Drosophila

Innate behaviors are often executed in concert with accompanying physiological programs. How this coordination is achieved is poorly understood. Mating behavior and the transfer of sperm and seminal fluid (SSFT) provide a model for understanding how concerted behavioral and physiological programs are coordinated. This study identified a male-specific neural pathway that coordinates the timing of SSFT with the duration of copulation behavior in Drosophila. Silencing four abdominal ganglion (AG) interneurons (INs) that contain the neuropeptide corazonin (Crz) both blocked SSFT and substantially lengthened copulation duration. Activating these Crz INs caused rapid ejaculation in isolated males, a phenotype mimicked by injection of Crz peptide. Crz promotes SSFT by activating serotonergic (5-HT) projection neurons (PNs) that innervate the accessory glands. Activation of these PNs in copulo caused premature SSFT and also shortened copulation duration. However, mating terminated normally when these PNs were silenced, indicating that SSFT is not required for appropriate copulation duration. Thus, the lengthened copulation duration phenotype caused by silencing Crz INs is independent of the block to SSFT. It is concluded that four Crz INs independently control SSFT and copulation duration, thereby coupling the timing of these two processes (Tayler, 2012).

Corazonin neurons function in sexually dimorphic circuitry that shape behavioral responses to stress in Drosophila

All organisms are confronted with dynamic environmental changes that challenge homeostasis, which is the operational definition of stress. Stress produces adaptive behavioral and physiological responses, which, in the Metazoa, are mediated through the actions of various hormones. Based on its associated phenotypes and its expression profiles, a candidate stress hormone in Drosophila is the corazonin neuropeptide. This study evaluated the potential roles of corazonin in mediating stress-related changes in target behaviors and physiologies through genetic alteration of corazonin neuronal excitability. Ablation of corazonin neurons confers resistance to metabolic, osmotic, and oxidative stress, as measured by survival. Silencing and activation of corazonin neurons lead to differential lifespan under stress, and these effects showed a strong dependence on sex. Additionally, altered corazonin neuron physiology leads to fundamental differences in locomotor activity, and these effects were also sex-dependent. The dynamics of altered locomotor behavior accompanying stress was likewise altered in flies with altered corazonin neuronal function. Corazonin transcript expression is altered under starvation and osmotic stress, and triglyceride and dopamine levels are equally impacted in corazonin neuronal alterations and these phenotypes similarly show significant sexual dimorphisms. Notably, these sexual dimorphisms map to corazonin neurons. These results underscore the importance of central peptidergic processing within the context of stress and place corazonin signaling as a critical feature of neuroendocrine events that shape stress responses and may underlie the inherent sexual dimorphic differences in stress responses (Zhao, 2010).

Spatial regulation of Corazonin neuropeptide expression requires multiple cis-acting elements in Drosophila melanogaster

Although most invertebrate neuropeptide-encoding genes display distinct expression patterns in the central nervous system (CNS), the molecular mechanisms underlying spatial regulation of the neuropeptide genes are largely unknown. Expression of the neuropeptide Corazonin (Crz) is limited to only 24 neurons in the larval CNS of Drosophila melanogaster, and these neurons have been categorized into three groups, namely, DL, DM, and vCrz. To identify cis-regulatory elements that control transcription of Crz in each neuronal group, reporter gene expression patterns driven by various 5' flanking sequences of Crz were analyzed to assess their promoter activities in the CNS. The 504-bp 5' upstream sequence is the shortest promoter directing reporter activities in all Crz neurons. Further dissection of this sequence revealed two important regions responsible for group specificity: -504::-419 for DM expression and -380::-241 for DL and vCrz expression. The latter region is further subdivided into three sites (proximal, center, and distal), in which any combinations of the two are sufficient for DL expression, whereas both proximal and distal sites are required for vCrz expression. Interestingly, the TATA box does not play a role in Crz transcription in most neurons. S 434-bp 5' upstream sequence of the D. virilis Crz gene, when introduced into the D. melanogaster genome, drives reporter expression in the DL and vCrz neurons, suggesting that regulatory mechanisms for Crz expression in at least two such neuronal groups are conserved between the two species (Choi, 2008).

Developmental regulation and functions of the expression of the neuropeptide corazonin in Drosophila melanogaster.

Although the corazonin gene (Crz) has been molecularly characterized, little is known concerning the function of this neuropeptide in Drosophila melanogaster. To gain insight into Crz function in Drosophila, this study investigated the developmental regulation of Crz expression and the morphology of corazonergic neurons. From late embryo to larva, Crz expression is consistently detected in three neuronal groups: dorso-lateral Crz neurons (DL), dorso-medial Crz neurons (DM), and Crz neurons in the ventral nerve cord (vCrz). Both the vCrz and DM groups die via programmed cell death during metamorphosis, whereas the DL neurons persist to adulthood. In adults, Crz is expressed in a cluster of six to eight neurons per lobe in the pars lateralis (DLP), in numerous neuronal cells in the optic lobes, and in a novel group of four abdominal ganglionic neurons present only in males (ms-aCrz). The DLP group consists of two subsets of cells having different developmental origins: embryo and pupa. In the optic lobes, both Crz transcripts and Crz promoter activity were detected, but no Crz-immunoreactive products, suggesting a post-transcriptional regulation of Crz mRNA. Projections of the ms-aCrz neurons terminate within the ventral nerve cord, implying a role as interneurons. Terminals of the DLP neurons are found in the retrocerebral complex that produces juvenile hormone and adipokinetic hormone. Significant reduction of trehalose levels in adults lacking DLP neurons suggests that DLP neurons are involved in the regulation of trehalose metabolism. Thus, the tissue-, stage-, and sex-specific expression of Crz and the association of Crz with the function of the retrocerebral complex suggest diverse roles for this neuropeptide in Drosophila (Lee, 2008).

Corazonin receptor signaling in ecdysis initiation

Corazonin is a highly conserved neuropeptide hormone of wide-spread occurrence in insects yet is associated with no universally recognized function. After discovery of the corazonin receptor in Drosophila, this study identified its ortholog in the moth, Manduca sexta, as a prelude to physiological studies. The corazonin receptor cDNA in M. sexta encodes a protein of 436 amino acids with seven putative transmembrane domains and shares common ancestry with its Drosophila counterpart. The receptor exhibits high sensitivity and selectivity for corazonin when expressed in Xenopus oocytes (EC(50) approximately 200 pM) or Chinese hamster ovary cells (EC(50) approximately 75 pM). Northern blot analysis locates the receptor in peripheral endocrine Inka cells, the source of preecdysis- and ecdysis-triggering hormones. Injection of corazonin into pharate larvae elicits release of these peptides from Inka cells, which induce precocious preecdysis and ecdysis behaviors. In vitro exposure of isolated Inka cells to corazonin (25-100 pM) induces preecdysis- and ecdysis-triggering hormone secretion. Using corazonin receptor as a biosensor, this study showed that corazonin concentrations in the hemolymph 20 min before natural preecdysis onset range from 20 to 80 pM and then decline over the next 30-40 min. These findings support the role of corazonin signaling in initiation of the ecdysis behavioral sequence. A model for peptide-mediated interactions between Inka cells and the CNS underlying this process in insect development (Kim, 2004).

Identification of G protein-coupled receptors for Drosophila PRXamide peptides, CCAP, corazonin, and AKH supports a theory of ligand-receptor coevolution

G-protein coupled receptors (GPCRs) are ancient, ubiquitous sensors vital to environmental and physiological signaling throughout organismal life. With the publication of the Drosophila genome, numerous 'orphan' GPCRs have become available for functional analysis. This study analyzes two groups of GPCRs predicted as receptors for peptides with a C-terminal amino acid sequence motif consisting of PRXamide (PRXa). Assuming ligand-receptor coevolution, two alternative hypotheses were constructed and tested. The insect PRXa peptides are evolutionarily related to the vertebrate peptide neuromedin U (NMU), or are related to arginine vasopressin (AVP), both of which have PRXa motifs. Seven Drosophila GPCRs related to receptors for NMU and AVP were cloned and expressed in Xenopus oocytes for functional analysis. Four Drosophila GPCRs in the NMU group (CG11475, CG8795, CG9918, CG8784) are activated by insect PRXa pyrokinins (FXPRXamide), Cap2b-like peptides (FPRXamide), or ecdysis triggering hormones (PRXamide). Three Drosophila GPCRs in the vasopressin receptor group respond to crustacean cardioactive peptide (CCAP), corazonin, or adipokinetic hormone (AKH), none of which are PRXa peptides. These findings support a theory of coevolution for NMU and Drosophila PRXa peptides and their respective receptors (Park, 2002).

Examination of the three Drosophila GPCRs homologous to the AVP receptor yielded serendipitous findings. CG6111, orthologous to the vasopressin receptor, is activated by CCAP and AKH. CG10698 and CG11325 are activated by corazonin and AKH, respectively. The EC50 values for receptors in the AVP group are consistently lower than those observed in the NMU PRXa group (Park, 2002).

It is surprising that CG6111, an orthologous gene of AVP receptor, is activated by CCAP and AKH, but not by AVP. The presence of an insect vasopressin-like peptide was reported in locust, but searches of the Drosophila genome sequence to locate a candidate AVP-like peptide sequence have been unsuccessful. CCAP and AVP both are C-terminally amidated, disulfide bridged peptides, but share no significant sequence similarity. The current data set favors assignment of CG6111 as an authentic CCAP receptor because of ligand cross-reactivity within this group of GPCRs. It seems reasonable to have residual functional cross-activity within recent evolutionarily diverged GPCRs. Further work is needed to verify whether CG6111 is an authentic CCAP receptor or is a receptor for unidentified Drosophila AVP-like peptide cross-reacting to the CCAP (Park, 2002).

CG10698 is activated by corazonin with an EC50 of 1 nM. Similarly, CG11325, previously cloned by its homology to GNRHR, is activated by AKH with an EC50 of 0.3 nM. These evolutionarily related GPCRs, activated by structurally similar signaling peptides, reveals a clear case of receptor-ligand coevolution (Park, 2002).

CCAP, corazonin, and AKH have overlapping biological functions, and thus it is not unexpected that their receptors would fall into an evolutionarily related group. CCAP was initially identified by its cardioacceleratory action on the heart of the shore crab and in the tobacco hawkmoth, Manduca. The primary structure of this peptide appears to be strictly conserved across the arthropods. Additional functions of CCAP include myotropic actions, induction of AKH release in corpora cardiaca of locust, and induction of ecdysis behaviors. Corazonin is known for its cardioactive function in cockroach and pigment modulation in locust. AKH and related peptides, grouped with red pigment concentrating hormone of crustacea are cardioacceleratory and have metabolic functions such as lipid and carbohydrate mobilization (Park, 2002).

The present findings favor a hypothesis that the PRXa motif is an evolutionarily conserved signature in both vertebrate NMU and insect PRXa peptides. Examination of potential cross-activity of NMU and insect PRXa peptides for their receptors may provide further support for the theory of ligand-receptor coevolution in the PRXa peptide-receptor group. Another clear case of ligand-receptor coevolution has been shown for recently diverging corazonin and AKH, and their receptors (Park, 2002).

The ligand-activated GPCR responses described in this report provide an important first step in defining authentic physiological roles for these signaling peptides. These findings help to promote a subset of GPCRs from 'orphan' to 'putative' receptors, and provide a direction for further characterization of receptors for PRXa peptides, CCAP, corazonin, and AKH. Expanded studies in Drosophila and in other insects will help to validate these initial findings (Park, 2002).

The multiple ligand sensitivity exhibited by certain GPCRs such as CG8795 and CG6111 raises the obvious question of physiological significance. One possible interpretation is that, in contrast to a one ligand-one receptor model, certain receptors may be involved in the transduction of multiple peptide signals, thus providing a pleiotropic model of functional regulation. This possibility deserves careful examination in more physiologically relevant bioassays, as well as in vivo (Park, 2002).

Search PubMed for articles about Drosophila Corazonin

Bergland, A. O., Chae, H. S., Kim, Y. J. and Tatar, M. (2012). Fine-scale mapping of natural variation in fly fecundity identifies neuronal domain of expression and function of an aquaporin. PLoS Genet 8(4): e1002631. PubMed ID: 22509142

Boerjan, B., Verleyen, P., Huybrechts, J., Schoofs, L. and De Loof, A. (2010). In search for a common denominator for the diverse functions of arthropod corazonin: a role in the physiology of stress? Gen Comp Endocrinol 166: 222-233. PubMed ID: 19748506

Cazzamali, G., Saxild, N. and Grimmelikhuijzen, C. (2002). Molecular cloning and functional expression of a Drosophila corazonin receptor. Biochem Biophys Res Commun 298: 31-36. PubMed ID: 12379215

Chintapalli, V. R., Wang, J. and Dow, J. A. (2007). Using FlyAtlas to identify better Drosophila melanogaster models of human disease. Nat Genet 39(6): 715-720. PubMed ID: 17534367

Choi, S. H., Lee, G., Monahan, P. and Park, J. H. (2008). Spatial regulation of Corazonin neuropeptide expression requires multiple cis-acting elements in Drosophila melanogaster. J Comp Neurol 507(2): 1184-1195. PubMed ID: 18181151

Davies, S. A., Cabrero, P., Povsic, M., Johnston, N. R., Terhzaz, S. and Dow, J. A. (2013). Signaling by Drosophila capa neuropeptides. Gen Comp Endocrinol 188: 60-66. PubMed ID: 23557645

Johnson, E. C., Shafer, O. T., Trigg, J. S., Park, J., Schooley, D. A., Dow, J. A. and Taghert, P. H. (2005). A novel diuretic hormone receptor in Drosophila: evidence for conservation of CGRP signaling. J Exp Biol 208(Pt 7): 1239-1246. PubMed ID: 15781884

Kapan, N., Lushchak, O. V., Luo, J. and Nassel, D. R. (2012). Identified peptidergic neurons in the Drosophila brain regulate insulin-producing cells, stress responses and metabolism by coexpressed short neuropeptide F and corazonin. Cell Mol Life Sci 69(23): 4051-4066. PubMed ID: 22828865

Kim, Y. J., Spalovska-Valachova, I., Cho, K. H., Zitnanova, I., Park, Y., Adams, M. E. and Zitnan, D. (2004). Corazonin receptor signaling in ecdysis initiation. Proc Natl Acad Sci U S A 101(17): 6704-6709. PubMed ID: 15096620

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date revised: 12 June 2021

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