InteractiveFly: GeneBrief

Tachykinin: Biological Overview | References

BIOLOGICAL OVERVIEW

Males of most species are more aggressive than females, but the neural mechanisms underlying this dimorphism are not clear. This study identified a neuron and a gene that control the higher level of aggression characteristic of Drosophila melanogaster males. Males, but not females, contain a small cluster of FruM⁺ neurons that express the neuropeptide tachykinin (Tk). Activation and silencing of these neurons increased and decreased, respectively, intermale aggression without affecting male-female courtship behavior. Mutations in both Tk and a candidate receptor, Takr86C, suppressed the effect of neuronal activation, whereas overexpression of Tk potentiated it. Tk neuron activation overcame reduced aggressiveness caused by eliminating a variety of sensory or contextual cues, suggesting that it promotes aggressive arousal or motivation. Tachykinin/Substance P has been implicated in aggression in mammals, including humans. Thus, the higher aggressiveness of Drosophila males reflects the sexually dimorphic expression of a neuropeptide that controls agonistic behaviors across phylogeny (Asahina, 2014).

Aggression is an innate, species-typical social behavior that is widespread in animal phylogeny. Expression of agonistic behavior is commonly observed between conspecific males in conflict over access to reproductively active females, food, territory, or other resources. In many animal species, aggression is often quantitatively higher in males than in females. In humans, violent aggression constitutes a major public health problem and its incidence is overwhelmingly higher among males than females. In addition, the behavioral expression of aggression is often qualitatively different between males and females, and may differ in the contexts in which it is exhibited (Asahina, 2014).

Despite recent progress, the neurobiological mechanisms underlying the evolutionarily conserved sexual dimorphism in aggressiveness remain poorly understood. Pheromones are known to play an important role in intermale aggression. However, in cases where the relevant receptors are known, dimorphic expression of these molecules does not appear to explain sex differences in aggressiveness. Studies in numerous vertebrate species have identified sexual dimorphisms in the size of brain nuclei or their constituent neuronal subpopulations that are controlled by gonadal steroid hormones in a manner that parallels the influence of these hormones on aggressive behavior. Recent studies have shown that genetic ablation of hypothalamic neurons expressing the progesterone receptor decreases both aggression and mounting in males, and mating behavior in females (Yang, 2013). These neurons display sexual dimorphisms in their projections, but whether this dimorphism is causally responsible for sex differences in levels of aggressiveness is not yet clear. As in other species, Drosophila males flies are more aggressive than females and also exhibit qualitative differences in agonistic behavior. These sex differences in aggression are known to be under the control of fruitless (fru), a master regulator of sexual differentiation of the brain. Although some efforts have been made to identify circuits through which fru exerts its influence on aggressive behavior, FruM⁺ neurons that are necessary, sufficient, and specific for male-type aggression have not yet been identified (Asahina, 2014).

This study has identified a small group of sexually dimorphic, FruM⁺ neurons that promote aggressiveness in Drosophila males but have no influence on male-female courtship behavior. These neurons enhance aggression, at least in part, through the release of a neuropeptide, Drosophila tachykinin (DTK) (Nassel, 2010; Winther, 2003). Tachykinin/Substance P has been implicated in certain forms of aggression in several mammalian species (Katsouni, 2009). Thus, the higher level of aggression that is characteristic of Drosophila males is promoted by sexually dimorphic neurons, which express a neuropeptide that regulates agonistic behavior across phylogeny (Asahina, 2014).

This study has identified a sexually dimorphic neuron and a gene that play a critical and specific role in the expression of intermale aggression in Drosophila. The gene encodes a neuropeptide homologous to mammalian Substance P, and its release from the identified neurons is important for aggression. Substance P has been implicated in aggression in several mammalian systems (Halasz, 2009; Katsouni, 2009; Siegel, 1997). Together, the data suggest that the higher level of aggressiveness in Drosophila males may be controlled by the expression in sexually dimorphic neurons of a neuropeptide that regulates forms of agonistic behavior across phylogeny (Asahina, 2014).

Previous studies have investigated the role of FruM⁺ neurons in aggression versus courtship. Selective masculinization of certain groups of neurons in females masculinized courtship behavior, but not aggression, suggesting that distinct subsets of FruM neurons may control these behaviors (Chan, 2007); however, a selective masculinization of aggression, but not courtship, was not observed. Feminization of most or all octopaminergic (OA) or cholinergic neurons, via expression of UAS-Tra, altered the balance between male-male courtship and aggression (Certel, 2007), or enhanced aggression (Mundiyanapurath, 2009), respectively. Feminization of a small subset of OA neurons increased male-male courtship, but not aggression (Certel, 2010). Specific OA and dopaminergic neurons that influence aggression have been identified (Alekseyenko, 2013; Zhou, 2008), but these neurons are not sexually dimorphic. The present results identify sexually dimorphic Tk-GAL4FruM neurons that are necessary, sufficient, and specific for the quantitatively higher level of aggressiveness that is characteristic of Drosophila males. The neurons responsible for the qualitative sex-specific differences in the behavioral expression of aggression remain to be identified (Asahina, 2014).

Studies in mice have localized aggression-promoting neurons to the ventrolateral subdivision of the ventromedial hypothalamus (VMHvl) (Lin, 2011). Genetic ablation of anatomically dimorphic neurons within VMHvl that express the progesterone receptor (PR) was shown to partially reduce aggressive behavior (Yang, 2013). However, this effect of this ablation was not specific to aggression, since male mating behavior and female mating behavior were attenuated as well. In contrast, the Tk-GAL4FruM neurons identified in this study control aggression, but not mating behavior. Unlike PR+ neurons, moreover, these cells are not detectable in females (Asahina, 2014).

The fact that the Tk-GAL4FruM neurons were not observed in females suggests that either the developmental generation of these neurons and/or their expression of the neuropeptide is male specific. Whatever the case, the absence of these neural elements from the female brain is likely to contribute to their lower level of aggressive behavior. The data suggest that sex-typical features of some innate behaviors in Drosophila may be achieved, at least in part, by the sexually dimorphic expression in specific neurons of neuropeptides that coordinate males-pecific behavioral subprograms. Dimorphic populations of FruM-expressing neurons also regulate sexually dimorphic behaviors through the release of classical fast neurotransmitters that act on sexually dimorphic chemical synapses (Asahina, 2014).

Several lines of evidence presented in this study argue that Tk-GAL4FruM neurons influence aggressive arousal or motivation, rather than simply acting as 'command neurons' for aggressive actions. First, activation of these neurons did not trigger a single aggressive action, as would be expected for a command neuron, but rather increased the frequency of multiple agonistic behaviors, including wing-threat, lunging, and tussling. Second, thermogenetic activation of these neurons supervened the requirement for several aggression-permissive conditions and cues, some of which (such as male-specific pheromones) could be construed as 'releasing signals'. The activation of Tk-GAL41 neurons was even able to promote lunging toward a moving dummy fly (albeit in a minority of trials). To the extent that increased arousal decreases the requirement for specific releasing signals to evoke innate behaviors, activation of Tk-GAL4FruM neurons may generate an arousal-like state that is specific for aggression. Alternatively, Tk-GAL4FruM neurons may enhance behavioral sensitivity to multiple releasing signals that characterize an attackable object, either at the level of parallel sensory processing pathways or at a locus downstream of the integration of these multisensory cues, analogous to the neuropeptide regulation of feeding behavior in C. elegans (Asahina, 2014).

Several lines of evidence presented in this study suggest that the release of DTK peptides indeed contributes to the aggression-promoting function of Tk-GAL4FruM neurons. Nevertheless, the release of a classical neurotransmitter, probably acetylcholine, likely contributes to the behavioral influence of Tk-GAL4FruM neurons as well. Furthermore, while the data implicate Takr86C as a receptor for Tk in the control of aggression, they do not exclude a role for Takr99D. Among three species of vertebrate Tachykinin neuropeptides, Substance P has been implicated, directly or indirectly, in various forms of aggression, including defensive rage and predatory attack in cats, and intermale aggression in rats. Although not all functions of Substance P are necessarily conserved (such as nociception in mammals and olfactory modulation in the fly, these data suggest that this neuropeptide is broadly involved in the control of agonistic behavior in both vertebrates and invertebrates. They therefore add to the growing list of neuropeptide systems that show a remarkable evolutionary conservation of functions in the regulation of innate 'survival behaviors' such as feeding and mating. Biogenic amines also control aggression across phylogeny. However, in the case of serotonin, the directionality of its influence is opposite in flies and humans (Asahina, 2014).

The findings of this study indicate that studies of agonistic behavior in Drosophila can identify aggression-regulating genes with direct relevance to vertebrates. Interestingly, in humans, the concentration of Substance P-like immunoreactivity in cerebrospinal fluid has been positively correlated with aggressive tendencies in patients with personality disorders. Substance P antagonists have been tested in humans as anxiolytic and antidepressant agents, although they failed to show efficacy . The present findings, taken together with mammalian animal studies, suggest that it may be worthwhile to investigate the potential of these antagonists for reducing violent aggression in humans (Asahina, 2014).

Starvation promotes concerted modulation of appetitive olfactory behavior via parallel neuromodulatory circuits

The internal state of an organism influences its perception of attractive or aversive stimuli and thus promotes adaptive behaviors that increase its likelihood of survival. The mechanisms underlying these perceptual shifts are critical to understanding of how neural circuits support animal cognition and behavior. Starved flies exhibit enhanced sensitivity to attractive odors and reduced sensitivity to aversive odors. This study shows that a functional remodeling of the olfactory map is mediated by two parallel neuromodulatory systems that act in opposing directions on olfactory attraction and aversion at the level of the first synapse. Short neuropeptide F sensitizes an antennal lobe glomerulus wired for attraction, while tachykinin (DTK) suppresses activity of a glomerulus wired for aversion. Thus this study shows parallel neuromodulatory systems functionally reconfigure early olfactory processing to optimize detection of nutrients at the risk of ignoring potentially toxic food resources (Ko, 2015).

This study demonstrates that shifts in the internal metabolic state of an animal lead to dramatic functional changes in its olfactory circuit and behaviors. Starved flies exhibit enhanced odor sensitivity in odorant receptor neurons (ORNs) that mediate behavioral attraction and decreased sensitivity in ORNs that mediate behavioral aversion. This functional remodeling of the olfactory map is mediated by parallel neuromodulatory systems that act in opposing directions on olfactory attraction and aversion. An earlier study showed that sNPFR signaling increases sensitivity in Or42b ORNs and thus enhances behavioral attraction (Root, 2011). The current study, however, shows that sNPFR signaling does not account for all changes induced by starvation in behavioral responses to a wider range of odor concentrations. Second, this study shows that starvation leads to a decreased sensitivity in the Or85a ORNs, an odorant channel that mediates behavioral aversion. Third, it was shown that DTKR signaling mediates the reduced sensitivity in the Or85a ORNs and partly accounts for enhanced behavioral attraction to high concentrations of vinegar. Fourth, eliminating DTKR and sNPFR signaling pathways together fully reverses the effect of starvation on behavioral attraction across all odor concentrations tested. Finally, evidence suggests that the same global insulin signal regulating sNPFR expression may also regulate DTKR expression (Ko, 2015).

In the wild, rotten fruits early in the fermentation process are more attractive to Drosophila than fresh or highly fermented fruits. In the laboratory, well fed flies display very little attraction to apple cider vinegar (Root, 2011). Low levels of vinegar are indicative of fresh fruit of limited nutritional value. Expanding odor sensitivity to lower concentrations of potential food odors may encourage flies to accept food sources of lower value. High odor concentrations typically accompany late stages of fermentation and are often aversive or uninteresting to flies. Starved flies are attracted to high concentrations of vinegar partly due to neuromodulatory mechanisms that enhance sensitivity in Or42b ORNs, an attractive odor channel, and partly through neuromodulatory mechanisms that reduce sensitivity in Or85a ORNs, an aversive odor channel. In the working model, behavioral attraction to higher odor concentrations of vinegar is the sum of the opposing effects of Or42b and Or85a. When flies face starvation, the balance of these inputs shifts to favor Or42b over Or85a inputs, as mediated by selective upregulation of sNPFR and DTKR in these ORNs, respectively. These processes could serve to encourage flies to risk ingestion of potentially toxic foods when under nutritional stress (Ko, 2015).

Given the broad array of glomeruli that can respond to odors such as vinegar, it may be surprising that the modulation of only two glomeruli is sufficient to significantly impact fly behavioral attraction. Whether these findings extend to a broad array of food associated odors and whether additional glomeruli are modulated by these neuromodulatory systems remain to be determined. In this context, it is noted that a recent correlational analysis predicts DM5 activity is highly correlated with behavioral attraction. However, this prediction has not been confirmed by direct testing of the DM5 glomerulus in behavioral experiments and is contradicted by more recent findings, as well as the data in this paper. Thus the current findings suggest that in starved flies the concentration range over which vinegar odor is attractive expands in both directions, with the acute need for caloric intake apparently outweighing considerations of food quality or risk (Ko, 2015).

This study highlights the importance of neuromodulators in shaping local neural circuit activity to accommodate the internal physiological state of an organism. The often unique expression patterns of specific GPCRs in sensory systems highlights the flexibility conferred by this evolutionarily ancient mechanism to translate neuroendocrine signals into local shifts in neuronal excitability and network properties that ultimately lead to adaptive behaviors. sNPF shares structural and functional similarities with its vertebrate homolog, NPY. Both neuropeptides show roles in controlling food intake and feeding behaviors in insects and vertebrates. Interestingly, NPY is also expressed in the vertebrate olfactory bulb and is thus positioned to shape olfactory processing during shifts in appetitive states as well. sNPF's broad expression pattern in the fly brain supports the possibility it is widely used to orchestrate changes across many different neuropils to shape appetitive behaviors. Indeed, sNPF and NPF, another NPY homolog in Drosophila, have been shown in the fly gustatory system to control sweet and bitter taste sensitivity, respectively, in parallel but opposing directions (Inagaki, 2014). The similar changes manifested by nutritional stress in both the olfactory and gustatory systems suggests complex networks of neuromodulators may shape sensory processing of aversive and attractive inputs differentially throughout the brain in a hunger state (Ko, 2015).

DTK and DTKR share homology with substance P and its receptor NK1, respectively. Interestingly, they seem to share roles in shaping the processing of stressful or negative sensory cues in both flies and mammals. For example, in rodents, emotional stressors cause long-lasting release of substance P to activate NK1 in the amygdala to generate anxiety-related behavior. In Drosophila, DTK signaling has also been shown to be critical for aggressive behaviors among male flies (Asahina, 2014). Previous work has shown Drosophila tachykinin mediates presynaptic inhibition in ORNs and detected expression in the LNs. This current study maps the locus of DTK's effects on behavioral responses to vinegar to the Or85a/DM5 ORNs using behavior and functional imaging. It was also confirmed that the source of the peptide is indeed the LNs as previous anatomical data had suggested. Thus, tachykinin's role in modulating stressful sensory inputs appears to extend to a glomerulus hardwired to behavioral aversion in the olfactory system (Ko, 2015).

The current results here resonate with discoveries in the gustatory system (Inagaki, 2014) and show that starvation changes the perception of both attractive and aversive sensory inputs beginning at the peripheral nervous system. Through the use of parallel neuromodulatory systems, the internal state of the organism functionally reconfigures early olfactory processing to optimize its detection of nutrients at the risk of ignoring potentially toxic food resources. It is certainly likely that neuromodulatory systems also impact and reconfigure central circuits in appetitive contexts. Thus, it will be of great interest to understand the contributions of peripheral and central circuits towards modifying appetitive behaviors (Ko, 2015).

Regulation of insulin-producing cells in the adult Drosophila brain via the tachykinin peptide receptor DTKR

Drosophila insulin-like peptides (DILPs) play important hormonal roles in the regulation of metabolic carbohydrates and lipids, but also in reproduction, growth, stress resistance and aging. In spite of intense studies of insulin signaling in Drosophila the regulation of DILP production and release in adult fruit flies is poorly understood. This study investigated the role of Drosophila tachykinin-related peptides (DTKs) and their receptors, DTKR (Tachykinin-like receptor at 99D) and NKD (Naked cuticle), in the regulation of brain insulin-producing cells (IPCs) and aspects of DILP signaling. First, DTK-immunoreactive axon terminations were shown close to the presumed dendrites of the IPCs, and DTKR immunolabeling was demonstrated in these cells. Second, targeted RNA interference was used to knock down expression of the DTK receptor, DTKR, in IPCs and the effects the on Dilp transcript levels were monitored in the brains of fed and starved flies. Dilp2 and Dilp3, but not Dilp5, transcripts were significantly affected by DTKR knockdown in IPCs, both in fed and starved flies. Both Dilp2 and Dilp3 transcripts increased in fed flies with DTKR diminished in IPCs whereas at starvation the Dilp3 transcript plummeted and Dilp2 increased. Trehalose and lipid levels were measured as well as survival in transgene flies at starvation. Knockdown of DTKR in IPCs leads to increased lifespan and a faster decrease of trehalose at starvation but has no significant effect on lipid levels. Finally, IPCs were targeted with RNAi or ectopic expression of the other DTK receptor, NKD, but no effect was found on survival at starvation. These results suggest that DTK signaling, via DTKR, regulates the brain IPCs (Birse, 2011).

This study investigated the effects of DTK signaling to IPCs in the Drosophila brain by monitoring Dilp transcript levels and survival at starvation, as well as trehalose and lipid levels in fed and starved flies. The brain IPCs are presumed to release DILP2, DILP3 and DILP5, orthologs of mammalian insulins. Since these insulin-like peptides have been shown to play a significant role in lifespan, in nutritional stress responses and in metabolic regulation, the DTK signaling onto these cells may be of significance for the regulation of vital physiological functions. However, the IPCs are also known to regulate feeding behavior, locomotor activity, sleep-wakefulness and ethanol sensitivity, and they may do so independent of the insulin signaling pathway. Thus, activation or inhibition of signaling in the IPCs may result in actions that are non-insulin mediated, either via other messengers released by the same cells or indirectly by the action of DILPs on specific neurons (see Root, 2011; Birse, 2011 and references therein).

The most direct evidence that DTK signaling affects the brain IPCs is that knock down of the receptor DTKR in IPCs leads to altered expression levels of Dilp2 and Dilp3 transcripts in fed flies and that Dilp3 RNA drops drastically in knockdown flies after 24 h starvation whereas Dilp2 levels increase. Interestingly, the Dilp5 transcript is not affected by DTKR-RNAi, but is the only one that seems affected by starvation both in controls and knockdown flies. It is known that restricted diet conditions in adult (control) flies alter the transcript level of Dilp5, but not Dilp2 and Dilp3 (Broughton, 2010), corroborating the current findings. These data are the first to quantify Dilp transcripts at complete starvation in adults, but an earlier report monitored Dilp transcripts by in situ hybridization of fed and starved third instar larvae. That study noted decreased Dilp3 and Dilp5 transcripts but unaffected levels of Dilp2. This difference could be either dependent on a difference in larval and adult functions of the IPCs or could be due to the difference in techniques used for monitoring transcript levels. Certainly the feeding behavior and metabolism differs greatly between larvae and adults in Drosophila. It should be noted here that insulin expression/signaling also involves autocrine or paracrine feedbacks so that DILP3 may act in stimulatory regulation of expression of DILP2 and DILP5 in the IPCs whereas DILP6 released from the fat body may negatively regulate the IPCs (Birse, 2011).

Unfortunately there are no reports that unequivocally demonstrate the release of DILPs into the circulation of Drosophila in a quantitative fashion. Thus, indirect measurements, such as Dilp transcript or DILP peptide-immunofluorescence levels in cell bodies of IPCs, have to be matched against the physiological effects seen after manipulations of IPCs. A few indicators of altered insulin signaling have been used here: levels of carbohydrate and lipids as well as effects on lifespan at starvation. As one of the functions of DILPs is to stimulate uptake of circulating blood sugar and thereby decreasing trehalose levels in the circulation, this study monitored whole-body trehalose levels in fed and starved flies after DTKR knockdown in IPCs. Knockdown of DTKR in IPCs had no effect on trehalose in fed flies, but induced an acute drop in trehalose after 5 h starvation, compared with controls, suggesting an increase in insulin signaling. Analysis of Dilp mutants or knockdown indicated that trehalose levels are regulated by DILP2 one of the peptides whose transcripts was indeed altered by DTKR knockdown at starvation. In the current experiments lipid levels were not affected by manipulations of DTKR on IPCs in fed or starved flies. Lipid metabolism may be regulated by multiple DILPs, including Dilp6 (Grönke, 2010), or by compensatory AKH signaling, and this may explain the lack of effect after manipulating only IPC activity (Birse, 2011).

Diminishment of DTKR expression on IPCs results in flies that display a shortened lifespan at starvation. This would also indicate increased insulin signaling, as deletion of IPCs or knocking down combinations of DILPs produce the opposite phenotype, and overexpression of Dilp2 in IPCs resulted in decreased resistance to starvation. A similar reduction of lifespan at starvation was seen after knock down of the inhibitory GABA_B receptor on IPCs (Enell, 2010). It is not clear which of the DILPs regulates the lifespan at dietary restriction or starvation, but DILP2 has been suggested as a candidate (Birse, 2011).

The second known DTK receptor, designated NKD, does not seem to play a role in the regulation of IPCs. NKD can be activated only by one of the DTKs, the N-terminally extended DTK-6, which has not been detected in the Drosophila brain, in contrast to the DTK-1-5 known to activate DTKR (Birse, 2011).

It can be mentioned that an earlier report shows that DTKR is expressed in renal (Malpighian) tubules where it regulates DILP5 signaling. It is proposed that this regulation is mediated by DTKs released hormonally from endocrine cells of the midgut. DTKs circulating locally act on DTKR expressed in principal cells of the renal tubules, resulting in a local activation of DILP5 signaling. The DTKR-regulated DILP5 signaling in renal tubules does not affect trehalose levels in fed or starved flies, but seems to be part of the defense against oxidative stress. Thus, this DTK-controlled DILP5 signaling in the tubules is probably independent of the paracrine DTK-mediated IPC regulation in the brain, but further studies of gut-derived DTK action are required to confirm this (Birse, 2011).

In summary these results indicate that in wild-type flies the activated DTKR inhibits insulin signaling in the brain IPCs, and knockdown of the receptor therefore leads to increased insulin signaling. This can be seen in the decreased lifespan and a considerable decrease in trehalose levels during short-term starvation compared with controls, similar to what is expected at increased DILP signaling. The most direct evidence that DTKR is involved in IPC regulation is the effect on Dilp2 and Dilp3 transcript levels seen after receptor knockdown in the IPCs in fed and starved flies. However, it is important for the future to develop a sensitive assay for quantifying hemolymph levels of individual DILPs to monitor how their release is affected by the DTKR signaling to IPCs (Birse, 2011).

Enteroendocrine cells support intestinal stem-cell-mediated homeostasis in Drosophila

Intestinal stem cells in the adult Drosophila midgut are regulated by growth factors produced from the surrounding niche cells including enterocytes and visceral muscle. The role of the other major cell type, the secretory enteroendocrine cells, in regulating intestinal stem cells remains unclear. This study shows that newly eclosed scute loss-of-function mutant flies are completely devoid of enteroendocrine cells. These enteroendocrine cell-less flies have normal ingestion and fecundity but shorter lifespan. Moreover, in these newly eclosed mutant flies, the diet-stimulated midgut growth that depends on the insulin-like peptide 3 expression in the surrounding muscle is defective. The depletion of Tachykinin-producing enteroendocrine cells or knockdown of Tachykinin leads to a similar although less severe phenotype. These results establish that enteroendocrine cells serve as an important link between diet and visceral muscle expression of an insulin-like growth factor to stimulate intestinal stem cell proliferation and tissue growth (Amcheslavsky, 2014).

Previous evidence shows that adult midgut mutant clones that have all the AS-C genes deleted are defective in EE formation while overexpression of scute (sc) or asense (ase) is sufficient to increase EE formation. Moreover, the Notch pathway with a downstream requirement of ase also regulates EE differentiation. To study the requirement of EEs in midgut homeostasis, attempts were made to delete all EEs by knocking down each of the AS-C transcripts using the ISC/EB driver esg-Gal4. The results show that sc RNAi was the only one that caused the loss of all EEs in the adult midgut. The esg-Gal4 driver is expressed in both larval and adult midguts, but the esg > sc RNAi larvae were normal while the newly eclosed adults had no EEs. Therefore, sc is likely required for all EE formation during metamorphosis when the adult midgut epithelium is reformed from precursors/stem cells (Amcheslavsky, 2014).

The sc6/sc10-1 hemizygous mutant adults were also completely devoid of midgut EEs, while other hemizygous combinations including sc1, sc3B, and sc5 were normal in terms of EE number. Df(1)sc10-1 is a small deficiency that has both ac and sc uncovered. sc1 and sc3B each contain a gypsy insertion in far-upstream regions of sc, while sc5 and sc6 are 1.3 and 17.4 kb deletions, respectively, in the sc 3' regulatory region. The sc6/sc10-1 combination may affect sc expression during midgut metamorphosis and thus the formation of all adult EEs (Amcheslavsky, 2014).

The atonal homolog 1 (Atoh1) is required for all secretory cell differentiation in mouse. However, esg-Gal4-driven atona; (ato) RNAi and the amorphic combination ato1/Df(3R)p13 showed normal EE formation. Nonetheless, older ato1/Df(3R)p13 flies exhibited a significantly lower increase of EE number, suggesting a role of ato in EE differentiation in adult flies (Amcheslavsky, 2014).

In sc RNAi guts, the mRNA expression of allatostatin (Ast), allatostatin C (AstC), Tachykinin (Tk), diuretic hormone (DH31), and neuropeptide F (NPF) was almost abolished, consistent with the absence of all EEs. On the other hand, the mRNA expression of the same peptide genes in heads showed no significant change. Even though the EEs and regulatory peptides were absent from the midgut, the flies were viable and showed no apparent morphological defects. There was no significant difference in the number of eggs laid and the number of pupae formed from control and sc RNAi flies, suggesting that the flies probably have sufficient nutrient uptake to support the major physiological task of reproduction. However, when the longevity of these animals was examined, the EE-less flies after sc RNAi showed significantly shorter lifespan. In addition, when the number of EEs was increased in adult flies by esgGal4;tubGal80ts (esg^ts)-driven sc overexpression, an even shorter lifespan was observed. These results suggest that a balanced number of EEs is essential for the long-term health of the animal. Moreover, there may be important physiological changes in these EE-less flies that are yet to be uncovered, such as reduced intestinal growth described in detail below (Amcheslavsky, 2014).

One of the phenotypic changes found for the sc RNAi/EE-less flies was that under normal feeding conditions, their midguts had a significantly narrower diameter than that of control midguts. When reared in poor nutrition of 1% sucrose, both wild-type (WT) and EE-less flies had thinner midguts. When reared in normal food, WT flies had substantially bigger midgut diameter, while EE-less flies had grown significantly less. The cross-section area of enterocytes in the EE-less midguts was smaller, suggesting that there is also a growth defect at the individual cell level (Amcheslavsky, 2014).

A series of experiments showed that ingestion of food dye by the sc RNAi/EE-less flies was not lower than control flies. The measurement of food intake by optical density (OD) of gut dye contents also showed similar ingestion. The measurement of excretion by counting colored deposits and visual examination of dye clearing from guts showed that there was no significant change in food passage. The normal fecundity also suggested that the mutant flies likely had absorbed sufficient nutrient for reproduction. Nonetheless, another phenotype that was detected was a substantial reduction of intestinal digestive enzyme activities including trypsin, chymotrypsin, aminopeptidase, and acetate esterase. These enzyme activities exhibit strong reduction after starvation of WT flies. The EE-less flies therefore have a physiological response as if they experience starvation although they are provided with a normal diet (Amcheslavsky, 2014).

A previous report has established that newly eclosed flies respond to nutrient availability by increasing ISC division that leads to a jump start of intestinal growth. When newly eclosed flies were fed on the poor diet of 1% sucrose, both WT and sc RNAi/EE-less guts had a very low number of p-H3-positive cells, which represent mitotic ISCs because ISCs are the only dividing cells in the adult midgut. When fed on normal diet, the WT guts had significantly higher p-H3 counts, but the sc RNAi/EE-less guts were consistently lower at all the time points. The sc6/sc10-1 hemizygous mutant combination exhibited a similarly lower mitotic activity on the normal diet (Amcheslavsky, 2014).

When possible signaling defects were investigated in the EE-less flies,in addition to other gut peptide mRNAs, the level of Dilp3 mRNA was also found to be highly decreased in these guts while the head Dilp3 was normal. This is somewhat surprising, because Dilp3 is expressed not in the epithelium or EEs but in the surrounding muscle. Dilp3 promoter-Gal4-driven upstream activating sequence (UAS)-GFP expression (Dilp3 > GFP) was used to visualize the expression in muscle. Both control and sc RNAi under this driver showed normal muscle GFP expression, demonstrating that sc does not function within the smooth muscle to regulate Dilp3 expression. The esg-Gal4 and Dilp3-Gal4, and the control UAS-GFP samples showed the expected expression in both midgut precursors and surrounding muscles. When these combined Gal4 drivers were used to drive sc RNAi, the smooth muscle GFP signal was clearly reduced. These guts also exhibited no Prospero staining and overall fewer cells with small sizes as expected from esg > sc RNAi (Amcheslavsky, 2014).

A previous report showed an increase of Dilp3 expression from the surrounding muscle in newly eclosed flies under a well-fed diet. This muscle Dilp3 expression precedes brain expression and is essential for the initial nutrient stimulated intestinal growth. The EE-less flies show similar growth and Dilp3 expression defects, suggesting that EE is a link between nutrient sensing and Dilp3 expression during this early growth phase (Amcheslavsky, 2014).

WT and AS-C deletion (scB57) mutant clones in adult midguts did not exhibit a difference in their cell numbers. Moreover, esg^ts > sc RNAi in adult flies for 3 days but did not undergo a decrease of mitotic count or EE number. Together, these results suggest that sc is not required directly in ISC for proliferation, and they imply that the ISC division defects observed in the sc mutant/EE-less flies is likely due to the loss of EEs. To investigate this idea further, the esg^ts > system to was used to up- and downshift the expression of sc at various time points, and the correlation of sc expression, EE number, and ISC mitotic activity were measured. The overexpression of sc after shifting to 29°C for a few days correlated with increased EE number, expression of gut peptides, and increased ISC activity. Then, flies were downshifted back to room temperature to allow the Gal80ts repressor to function again. The sc mRNA expression was quickly reduced within 2 days and remained low for 4 days. Although there was no working antibody to check the Sc protein stability, the expression of a probable downstream gene phyllopod showed the same up- and downregulation, revealing that Sc function returned to normal after the temperature downshift. Meanwhile, the number of Pros+ cells and p-H3 count remained higher after the downshift. Therefore, the number of EEs, but not sc mRNA or function, correlates with ISC mitotic activity (Amcheslavsky, 2014).

Another experiment that was independent of sc expression or expression in ISCs was performed. The antiapoptotic protein p35 was driven by the pros-Gal4 driver, which is expressed in a subset of EEs in the middle and posterior midgut. This resulted in a significant albeit smaller increase in EE number and a concomitant increase in mitotic activity, which was counted only in the middle and posterior midgut due to some EC expression of this driver in the anterior region. Therefore, the different approaches show consistent correlation between EE number and ISC division (Amcheslavsky, 2014).

Dilp3 expression was significantly although modestly increased in flies that had increased EE number after sc overexpression, similar to that observed in fed versus fasted flies. Whether Dilp3 was functionally important in this EE-driven mitotic activity was tested. Flies were generated that contained a ubiquitous driver with temperature controlled expression, i.e., tub-Gal80ts/UAS-sc; tub-Gal4/UAS-Dilp3RNAi. These fly guts showed a significantly lower number of p-H3+ cells than that in the tub-Gal80ts/UAS-sc; tub-Gal4/+ control flies. These results demonstrate that the EE-regulated ISC division is partly dependent on Dilp3. The expression of an activated insulin receptor by esg-Gal4 could highly increase midgut proliferation, and this effect was dominant over the loss of EEs after scRNAi, which is consistent with an important function of insulin signaling in the midgut (Amcheslavsky, 2014).

Normally hatched flies did not lower their EE number after esg^ts > sc RNAi, perhaps due to redundant function with other basic-helix-loop-helix proteins in adults. The expression of proapoptotic proteins by the prosts-Gal4 also could not reduce the EE number. Thus other drivers were screened and a Tk promoter Gal4 (Tk-Gal4) was identifed that had expression recapitulating the Tk staining pattern representing a subset of EEs. More importantly, when used to express the proapoptotic protein Reaper (Rpr), this driver caused a significant reduction in the EE number, Tk and Dilp3 mRNA, and mitotic count. The Tk-Gal4-driven expression of another proapoptotic protein, Hid, caused a less efficient killing of EEs and subsequently no reduction of p-H3 count. The knockdown of Tk itself by Tk-Gal4 also caused significant reduction of p-H3 count. A previous report revealed the expression by antibody staining of a Tk receptor (TkR86C) in visceral muscles, and the knockdown of TkR86C in smooth muscle by Dilp3-Gal4 or Mef2-Gal4 showed a modest but significant decrease in ISC proliferation. There was a concomitant reduction of Dilp3 mRNA in guts of all these experiments, while the head Dilp3 mRNA had no significant change in all these experiments. As a comparison, TkR99D or NPFR RNAi did not show the same consistent defect (Amcheslavsky, 2014).

In conclusion, this study has shown that among the AS-C genes, sc is the one essential for the formation of all adult midgut EEs and is probably required during metamorphosis when the midgut is reformed. In newly eclosed flies, EEs serve as a link between diet-stimulated Dilp3 expression in the visceral muscle and ISC proliferation. Depletion of Tk-expressing EEs caused similar Dilp3 expression and ISC proliferation defects, although the defects appeared to be less severe than that in the sc RNAi/EE-less guts. The results together suggest that Tk-expressing EEs are part of the EE population required for this regulatory circuit. The approach reported in this study has established the Drosophila midgut as a model to dissect the function of EEs in intestinal homeostasis and whole-animal physiology (Amcheslavsky, 2014).

Control of lipid metabolism by Tachykinin in Drosophila

The intestine is a key organ for lipid uptake and distribution, and abnormal intestinal lipid metabolism is associated with obesity and hyperlipidemia. Although multiple regulatory gut hormones secreted from enteroendocrine cells (EEs) regulate systemic lipid homeostasis, such as appetite control and energy balance in adipose tissue, their respective roles regarding lipid metabolism in the intestine are not well understood. This study demonstrates that Tachykinins (TKs), one of the most abundant secreted peptides expressed in midgut EEs, regulate intestinal lipid production and subsequently control systemic lipid homeostasis in Drosophila and that TKs repress lipogenesis in enterocytes (ECs) associated with TKR99D receptor and protein kinase A (PKA) signaling. Interestingly, nutrient deprivation enhances the production of TKs in the midgut. Finally, unlike the physiological roles of TKs produced from the brain, gut-derived TKs do not affect behavior, thus demonstrating that gut TK hormones specifically regulate intestinal lipid metabolism without affecting neuronal functions (Song, 2014).

Previous studies in mammals have indicated that a few gut secretory hormones, like GLP1 and GLP2, are involved in intestinal lipid metabolism. However, due to gene and functional redundancy, mammalian genetic models for gut hormones and/or their receptors with severe metabolic defects are not available. This study has establish that Drosophila TKs produced from EEs coordinate midgut lipid metabolic processes. The studies clarify the roles of TK hormones in intestinal lipogenesis and establish Drosophila as a genetic model to study the regulation of lipid metabolism by gut hormones (Song, 2014).

Six mature TKs, TK1-TK6, are processed and secreted from TK EEs in both the brain and midgut (Reiher, 2011). Using a specific Gal4 driver line, gene expression in TK EEs was specifically manipulated, leading to the demonstration that loss of gut TKs results in an increase in midgut lipid production. Further, this study showed that TKs regulate intestinal lipid metabolism associated with TKR99D, but not TKR86C, which is consistent with the expression of these receptors. Consistent with previous reports that TK/TKR99D signaling regulates cAMP level and PKA activation, loss of gut TKs is associated with a reduction in PKA activity in ECs, and overexpression of a PKA catalytic subunit was able to reverse the increased intestinal lipid production associated with loss of TKR99D. In addition, the transcription factor SREBP that triggers lipogenesis was controlled by TK/TKR99D/PKA signaling. Taken together, these results suggest that TKs produced from EEs regulate midgut lipid metabolism via TKR99D/PKA signaling and regulation of, at least, SREBP-induced lipogenesis in ECs (Song, 2014).

Interestingly, this study reveals that TKs derived from either the brain or gut exhibit distinct functions: TKs derived from gut control intestinal lipid metabolism, whereas TKs derived from brain control behavior. This is reminiscent of the distinct functions of mammalian secreted regulatory peptides, where different spatial expressions or deliveries of peptides like Ghrelin can result in distinct physiological functions. In addition, some prohormones encode multiple mature peptides that can have multiple functions. For example, processing of proglucagon in the pancreas α cells preferentially gives rise to glucagon, which antagonizes the effect of insulin. In intestine L cells, however, proglucagon is mostly processed into GLP1 to promote insulin release. These studies of TKs exemplify how secreted regulatory peptides derived from different tissues can be associated with fundamentally diverse physiological functions. Clearly, additional studies examining the function of secreted peptides in a cell-type- and tissue-specific manner are needed to fully appreciate and unravel their complex roles both in flies and mammals (Song, 2014).

There is a growing body of studies emphasizing that intestinal lipid metabolism is key to the control of systemic lipid homeostasis. For example, chemicals such as orlistat, designed to inhibit dietary lipid digestion/absorption in the intestine, efficiently reduce obesity. In addition, mammalian inositol-requiring enzyme 1β deficiency-induced abnormal chylomicron assembly in the small intestine results in hyperlipidemia. Similarly, in Drosophila, dysfunction of intestinal lipid digestion/absorption caused by Magro/LipA deficiency eventually decreases whole-body lipid storage and starvation resistance in Drosophila. Further, intestinal lipid transport, controlled by lipoproteins, is essential for systemic lipid distribution and energy supply in other tissues. Consistent with these observations, this study demonstrates that increased midgut lipid synthesis associated with gut TK deficiency is sufficient to elevate systemic lipid storage. Although TK ligands and TK receptors show high homologies between mammals and fruit flies, whether mammalian TK signaling plays a similar role in intestinal lipid metabolism is largely unknown. Future studies will reveal whether mammalian TK signaling affects intestinal lipid metabolism as in Drosophila. If this is the case, it may provide a therapeutic opportunity for the treatment of intestinal lipid metabolic disorder and obesity (Song, 2014).

Production and secretion of gut hormones are precisely regulated under various physiological conditions. Similar to previous observations that starvation induces gut TK secretion in other insects, this study found that nutrient deprivation promotes TK production in EEs. Interestingly, feeding of amino-acid-enriched yeast, but not coconut oil or sucrose, potently suppressed gut TK levels, indicating that amino acids may act directly on TK production in EEs. It has been reported that dietary nutrients regulate gut hormone production through certain receptors located on the cell membrane of EEs in mammals. Future studies will be necessary to elucidate the detailed mechanism by which nutrients regulate TK production from EEs (Song, 2014).

Tachykinin acts upstream of autocrine Hedgehog signaling during nociceptive sensitization in Drosophila

Pain signaling in vertebrates is modulated by neuropeptides like Substance P (SP). To determine whether such modulation is conserved and potentially uncover novel interactions between nociceptive signaling pathways SP/Tachykinin signaling was examined in a Drosophila model of tissue damage-induced nociceptive hypersensitivity. Tissue-specific knockdowns and genetic mutant analyses revealed that both Tachykinin and Tachykinin-like receptor (DTKR99D) are required for damage-induced thermal nociceptive sensitization. Electrophysiological recording showed that DTKR99D is required in nociceptive sensory neurons for temperature-dependent increases in firing frequency upon tissue damage. DTKR overexpression caused both behavioral and electrophysiological thermal nociceptive hypersensitivity. Hedgehog, another key regulator of nociceptive sensitization, was produced by nociceptive sensory neurons following tissue damage. Surprisingly, genetic epistasis analysis revealed that DTKR function was upstream of Hedgehog-dependent sensitization in nociceptive sensory neurons. These results highlight a conserved role for Tachykinin signaling in regulating nociception and the power of Drosophila for genetic dissection of nociception (Im, 2015).

This study establishes that Tachykinin signaling regulates UV-induced thermal allodynia in Drosophila larvae. It is envisioned that UV radiation either directly or indirectly activates Tachykinin expression and/or release from peptidergic neuronal projections - likely those within the CNS that express DTK and are located near class IV axonal tracts. Following release, it is speculated that Tachykinins diffuse to and ultimately bind DTKR on the plasma membrane of class IV neurons. This activates downstream signaling, which is mediated at least in part by a presumed heterotrimer of α G alpha (Gαq, CG17760), a G β (Gβ5), and a G γ (Gγ1) subunit. One likely downstream consequence of Tachykinin receptor activation is Dispatched-dependent autocrine release of Hh from these neurons. It is envisioned that Hh then binds to Patched within the same class IV neurons, leading to derepression of Smo and activation of downstream signaling through this pathway. One new aspect of the thermal allodynia response dissected in this study is that the transcription factors Cubitius interruptus and Engrailed act downstream of Smo, suggesting that, as in other Hh-responsive cells, activation of target genes is an essential component of thermal allodynia. Finally, activation of Smo impinges upon Painless through as yet undefined mechanisms to regulate thermal allodynia. Some of the implications of this model for Tachykinin signaling, Hh signaling, and their conserved regulation of nociceptive sensitization are discussed below (Im, 2015).

The results demonstrate that Tachykinin is required for UV-induced thermal allodynia. UV radiation may directly or indirectly trigger Tachykinin expression and/or release from the DTK-expressing neurons. Given the transparent epidermis and cuticle, direct induction mechanisms are certainly plausible. Indeed in mammals, UV radiation causes secretion of SP and CGRP from both unmyelinated c fibers and myelinated Aγ fibers nociceptive sensory afferents. Furthermore, in the Drosophila intestine Tachykinin release is induced by nutritional and oxidative stress, although the effect of UV has not been examined. The exact mechanism of UV-triggered neuropeptide release remains unclear; however, it is speculated that UV causes depolarization and activation of exocytosis of Tachykinin-containing vesicles (Im, 2015).

In heterologous cells synthetic Tachykinins (DTK1-5) can activate DTKR. Immunostaining analysis of dTk and genetic analysis of tissue-specific function of dtkr supports the model that Tachykinins from brain peptidergic neurons bind to DTKR expressed on class IV neurons. Pan-neuronal, but not class IV neuron- specific knockdown of dTk reduced allodynia, whereas modulation of DTKR function in class IV neurons could either decrease (RNAi) or enhance (overexpression) thermal allodynia. How do brain-derived Tachykinins reach DTKR expressed on the class IV neurons? The cell bodies and dendritic arbors of class IV neurons are located along the larval body wall, beneath the barrier epidermal cells. However, the axonal projection of each nociceptive neuron extends into the ventral nerve cord (VNC) of the CNS in close proximity to Tachykinin-expressing axons. Because neuropeptide transmission does not depend on specialized synaptic structures, it is speculated given their proximity that Tachykinin signaling could occur via perisynaptic or volume transmission. An alternative possibility is that Tachykinins are systemically released into the circulating hemolymph as neurohormones following UV irradiation, either from the neuronal projections near class IV axonal tracts or from others further afield within the brain (Im, 2015).

Indeed the gain-of-function behavioral response induced by overexpression of DTKR, a receptor that has not been reported to have ligand-independent activity, suggests that class IV neurons may be constitutively exposed to a low level of subthreshold DTK peptide in the absence of injury. The direct and indirect mechanisms of DTK release are not mutually exclusive and it will be interesting to determine the relative contribution of either mechanism to sensitization (Im, 2015).

Like most GPCRs, DTKR engages heterotrimeric G proteins to initiate downstream signaling. Gq/11 and calcium signaling are both required for acute nociception and nociceptive sensitization. The survey of G protein subunits identified a putative Gαq, CG17760. It was demonstrated that DTKR activation leads to an increase in Ca2+, strongly pointing to Gαq as a downstream signaling component. To date, CG17760 is one of three G alpha subunits encoded in the fly genome that has no annotated function in any biological process. For the G β and G γ classes, Gβ5 and Gγ1 were identified. Gβ5 was one of two G β subunits with no annotated physiological function. Gγ1 regulates asymmetric cell division and gastrulation, cell division, wound repair, and cell spreading dynamics. The combination of tissue-specific RNAi screening and specific biologic assays, as employed in this study, has allowed assignment of a function to this previously 'orphan' gene in thermal nociceptive sensitization. The findings raise a number of interesting questions about Tachykinin and GPCR signaling in general in Drosophila: Are these particular G protein subunits downstream of other neuropeptide receptors? Are they downstream of DTKR in biological contexts other than pain? Could RNAi screening be used this efficiently in other tissues/behaviors to identify the G protein trimers relevant to those processes (Im, 2015)?

To date three signaling pathways were found that regulate UV-induced thermal allodynia in Drosophila: TNF, Hedgehog, and Tachykinin. All are required for a full thermal allodynia response to UV but genetic epistasis tests reveal that TNF and Tachykinin act in parallel or independently, as do TNF and Hh. This could suggest that in the genetic epistasis contexts, which rely on class IV neuron-specific pathway activation in the absence of tissue damage, hyperactivation of one pathway (say TNF or Tachykinin) compensates for the lack of the function normally provided by the other parallel pathway following tissue damage. While TNF is independent of Hh and DTKR, analysis of DTKR versus Hh uncovered an unexpected interdependence (Im, 2015).

This study showed that Hh signaling is downstream of DTKR in the context of thermal allodynia. Two pieces of genetic evidence support this conclusion. First, flies transheterozygous for dTk and smo displayed attenuated UV-induced thermal allodynia. Thus, the pathways interact genetically. Second, and more important for ordering the pathways, loss of canonical downstream Hh signaling components blocked the ectopic sensitization induced by DTKR overexpression. It was previously shown that loss of these same components also blocks allodynia induced by either UV or Hh hyperactivation (Babcock, 2011), suggesting that these downstream Hh components are also downstream of DTKR. The fact that Smo is activated upon overexpression of DTKR within the same cell argues that class IV neurons may need to synthesize their own Hh following a nociceptive stimulus such as UV radiation. The data supporting an autocrine model of Hh production are three fold: (1) only class IV neuron-mediated overexpression of Hh caused thermal allodynia suggesting this tissue is fully capable of producing active Hh ligand; (2) expression of UAS-dispRNAi within class IV neurons blocked UV- and DTKR-induced thermal allodynia, implicating a role for Disp-driven Hh secretion in these cells, and (3) the combination of UAS-dispRNAi and UV irradiation caused accumulation of Hh punctae within class IV neurons. Disp is not canonically viewed as a downstream target of Smo and indeed, blocking disp did not attenuate UAS-PtcDN-induced or UAS-TNF-induced allodynia, indicating that Disp is specifically required for Hh production between DTKR and Smo. Thus, Tachykinin signaling leads to Hh expression, Disp-mediated Hh release, or both (Im, 2015).

Autocrine release of Hh has only been demonstrated in a few non-neuronal contexts to date. This signaling architecture differs from what has been found in Drosophila development in two main ways. One is that DTKR is not known to play a patterning role upstream of Smo. The second is that Hh-producing cells are generally not thought to be capable of responding to Hh during the formation of developmental compartment boundaries (Im, 2015).

What happens downstream of Smoothened activation to sensitize class IV neurons? Ultimately, a sensitized neuron needs to exhibit firing properties that are different from those seen in the naïve or resting state. Previously, sensitization was examined only at the behavioral level. This study also monitored changes through extracellular electrophysiological recordings. These turned out to correspond remarkably well to behavioral sensitization. In control UV-treated larvae, nearly every temperature in the low 'allodynic' range showed an increase in firing frequency in class IV neurons upon temperature ramping. Dtkr knockdown in class IV neurons abolished the UV-induced increase in firing frequency seen with increasing temperature and overexpression of DTKR increased the firing rate comparable to UV treatment. This latter finding provides a tidy explanation for DTKR-induced 'genetic allodynia.' The correspondence between behavior and electrophysiology argues strongly that Tachykinin directly modifies the firing properties of nociceptive sensory neurons in a manner consistent with behavioral thermal allodynia (Im, 2015).

Genetically, knockdown of painless blocks DTKR- or PtcDN-induced ectopic sensitization suggesting that, ultimately, thermal allodynia is mediated in part via this channel. Indeed, the SP receptor Neurokinin-1 enhances TRPV1 function in primary rat sensory neurons. Tachykinin/Hh activation could lead to increased Painless expression, altered Painless localization, or to post-translational modification of Painless increasing the probability of channel opening at lower temperatures. Because thermal allodynia evoked by UV and Hh-activation requires Ci and En, the possibility is favored that sensitization may involve a simple increase in the expression level of Painless, although the above mechanisms are not mutually exclusive. Altered localization has been observed with a different TRP channel downstream of Hh stimulation; Smo activation leads to PKD2LI recruitment to the primary cilium in fibroblasts, thus regulating local calcium dynamics of this compartment. The exact molecular mechanisms by which nociceptive sensitization occurs is the largest black box in the field and will take a concerted effort by many groups to precisely pin down (Im, 2015).

Tachykinin and Substance P as regulators of nociception: What is conserved and what is not? The results establish that Tachykinin/SP modulation of nociception is conserved across phyla. However, there are substantial differences in the architecture of this signaling axis between flies and mammals. In mammals, activation of TRP channels in the periphery leads to release of SP from the nerve termini of primary afferent C fibers in the dorsal horn. SP and spinal NK-1R have been reported to be required for moderate to intense baseline nociception and inflammatory hyperalgesia although some discrepancies exist between the pharmacological and genetic knockout data. The most profound difference of Drosophila Tachykinin signaling anatomically is that DTK is not expressed and does not function in primary nociceptive sensory neurons. Rather, DTK is expressed in brain neurons and the larval gut, and DTKR functions in class IV neurons to mediate thermal pain sensitization. Indeed, this raises an interesting possibility for mammalian SP studies, because nociceptive sensory neurons themselves express NK-1R and SP could conceivably activate the receptor in an autocrine fashion. A testable hypothesis that emerges from these studies is that NK-1R in vertebrates might play a sensory neuron-autonomous role in regulating nociception. This possibility, while suggested by electrophysiology and expression studies, has not been adequately tested by genetic analyses in mouse to date (Im, 2015).

In summary, this study discovered a conserved role for systemic Tachykinin signaling in the modulation of nociceptive sensitization in Drosophila. The sophisticated genetic tools available in Drosophila have allowed uncovering both a novel genetic interaction between Tachykinin and Hh signaling and an autocrine function of Hh in nociceptive sensitization. This work thus provides a deeper understanding of how neuropeptide signaling fine-tunes an essential behavioral response, aversive withdrawal, in response to tissue damage (Im, 2015).

A neural circuit linking two sugar sensors regulates satiety-dependent fructose drive in Drosophila

In flies, neuronal sensors detect prandial changes in circulating fructose levels and either sustain or terminate feeding, depending on internal state. This study describes a three-part neural circuit that imparts satiety-dependent modulation of fructose sensing. Dorsal fan-shaped body neurons display oscillatory calcium activity when hemolymph glucose is high, and these oscillations require glutamatergic input from SLP-AB or 'Janus' neurons projecting from the protocerebrum to the asymmetric body. Suppression of activity in this circuit, either by starvation or by genetic silencing, promotes specific drive for fructose ingestion. This is achieved through neuropeptidergic signaling by tachykinin, which is released from the fan-shaped body when glycemia is high. Tachykinin, in turn, signals to Gr43a-positive fructose sensors to modulate their response to fructose. Together, these results demonstrate how a three-layer neural circuit links the detection of two sugars to produce precise satiety-dependent control of feeding behavior (Musso, 2021).

Regulation of energy intake is a complex process involving food search, an animal's internal state, and the sensory qualities of food. In flies, fructose, either consumed directly or rapidly metabolized from precursors, promotes feeding through activation of a brain fructose sensor called Gr43a. This study describes how a neuronal network composed of neurons in the FB and asymmetric body contributes to energy homeostasis by detecting satiety-dependent changes in hemolymph glucose and modulating fructose drive (Musso, 2021).

The central complex, which is composed of the FB, the protocerebral bridge (PB), the ellipsoid body, and the noduli, is regarded as a center for sensorimotor integration that functions in goal-directed behavior. The FB is organized in nine horizontal layers and nine vertical columns. FB large field neurons of layers 1 to 3, and inputs to these layers from the PB, encode flight direction and general sensory orientation. FB layers 6 and 7 are well known to regulate sleep and arousal, locomotor control, courtship, visual memory, and decision-making related to taste. Layer 6 also plays a role in avoiding conditioned odors, while layers 1, 2, 4, and 5 respond to electric stimuli and are required for innate odor avoidance. However, the function of the most dorsal FB layers (8 and 9), mostly innervated local tangential neurons and AB-FBl8 (or vΔA_a), remained poorly understood. The results demonstrate a role for these layers in feeding regulation (Musso, 2021).

dFB oscillations were found to be require glutamatergic input from Janus neuron projections to the asymmetric body. Described for the first time in 2004, very little is known about AB function; 92.4% of flies display asymmetry in the AB, with the body present only in the right hemisphere, while 7.6% also have a body on the left side. It is noted that oscillations in the dFB display a tendency to be faster on the right side, with clearly asynchronous activity between the two sides that may reflect their asymmetric input from Janus neurons. The small proportion of flies displaying symmetry in the AB have defects in LTM, a process that is known to require energy. It is speculated that these symmetric flies may have a dysfunctional Janus neurons-to-dFB connection, resulting in impaired Tk release. This could affect LTM either directly or through changes in feeding. A role for TK in memory has been demonstrated in honeybees and mammals, and TkR86C appears to be expressed in serotonergic paired neurons known to interact with MB-MP1 neurons required for LTM formation. Tk also acts through TkR99D to modulate activity in neurons producing insulin-like peptides, which affect LTM formation (Musso, 2021).

Modulation of dFB oscillations by Janus neurons requires glutamatergic signaling through a group of glutamate receptors including KaiR1D, NmdaR1, NmdaR2, and GluClα, but not AMPA receptors. Both KaiR1D receptors, which are homomeric, and N-methyl-D-aspartate (NMDA) receptors, which are heteromeric complexes between subunits 1 and 2, pass Ca2+ current. NMDA receptors (NMDAR) are well known for their role in mediating synaptic plasticity and can also trigger oscillatory activity. NMDAR function as molecular coincidence detectors, requiring simultaneous ligand binding and membrane depolarization for activation. It is possible that dFB neuron oscillations are triggered by the coincident detection of glutamate from Janus neurons and glucose from the hemolymph; however, because the FB are receiving many inputs from other brain region, it is suspect that dFB oscillations require additional inputs as well. The chloride channel GluClα is also required for dFB oscillations. GluClα has been previously implicated in on/off responses of the visual system of flies and memory retention in honeybees, demonstrating a role in regulating cell excitability. Perhaps, GluClα functions in repolarization of the dFB neurons between calcium bursts. Further study will be required to fully understand how the suite of glutamate receptors function together to drive oscillations, along with the source of input to Janus neurons in the protocerebrum (Musso, 2021).

Because glucose is the primary circulating energy source, one might intuitively expect that enhancing feeding in response to postingestive glucose detection would be the most efficient means of optimizing energy uptake. However, using elevation of hemolymph glucose as a signal to continue feeding is problematic because glucose levels are tightly regulated and elevated glucose serves as a signal of satiety. On the other hand, internal fructose can vary widely in response to ingestion and can therefore be a more reliable indicator of recent sugar intake. Thus, the separation of glucose as a satiety indicator and fructose as marker of sugar consumption removes the potential ambiguity of each as a signal. Moreover, fructose typically coexists with other nutritive sugars in common food sources. Therefore, it may not be the case that flies specifically benefit from fructose intake but rather that fructose serves as an effective proxy for general carbohydrate ingestion. By using fructose and the narrowly tuned Gr43a fructose receptor to survey sugar consumption, flies can effectively benefit from both a fructose-mediated positive feedback loop and glucose-mediated negative feedback to co-operatively ensure appropriate energy intake (Musso, 2021).

The finding that dFB glucose sensing modulates fructose feeding via Gr43a brain neurons fits with the established model of Gr43a brain neurons as central fructose sensors. For this mechanism to effectively sustain feeding on a rich sugar source, ingested sugars must rapidly increase fructose signaling to Gr43a brain neurons, which then must acutely promote feeding. While the precise kinetics of internal fructose elevation after sugar consumption have not been quantified, fructose levels in the head rapidly increase 10-fold after fructose feeding and then return to baseline. The role of direct fructose sensing by Gr43a brain neurons is highlighted by the observation that Gr43a knockdown in those neurons results in markedly lower relative intake of fructose compared to glucose. Unexpectedly, knockdown of Gr64a, another sugar receptor expressed in the same neurons, produced the opposite effect. This could be because Gr64a contributes to modulation of Gr43a brain neurons by other sugar cues, and the absence of this activity makes Gr43a-mediated fructose responses more pronounced. Alternatively, Gr43a may be expressed more strongly after Gr64a knockdown, leading to an increased fructose response (Musso, 2021).

Little is known about the mechanisms downstream of Gr43a brain neurons that promote feeding. All Gr43a brain neurons express the peptide Crz, but knockdown of Crz expression produced no significant effect on fructose preference over glucose. This suggests an important functional role for another neurotransmitter, although it is also possible that the RNAi knockdown was not effective. Irrespective of mechanism, two experiments support the idea that activation of Gr43a neurons acutely enhances feeding. First, silencing of dFB neurons by genetic manipulation or prolonged starvation produces Gr43a-dependent fructose preference within the first 10 min of a flyPAD assay. Second, closed-loop optogenetic activation of Gr43a brain neurons was sufficient to produce a strong positive preference within 10 min in the STROBE (Musso, 2021).

The separable functions of glucose and fructose sensing in flies bear notable resemblance to the differential effects of these two sugars in the mammalian hypothalamus. In particular, AMPK expression in the arcuate nucleus of the hypothalamus is known to link energy levels to food drive. When glycemia is low, AMPK is activated and thereby promotes feeding through orexigenic AgRP/NPY neuron activity. Glucose administration suppresses activity in these peptidergic neurons, while fructose can have the opposite effect and promote further feeding. The first description of fly Gr43a neurons noted their orexinegenic activity and suggested a potential functional homology with the hypothalamus. In the present study, a multilayered neural system centered on a brain energy sensor (dFB), was uncovered whose activation by glucose leads to anorexigenic behavior through inhibition of the brain fructose sensor Gr43a. Thus, the results are consistent with at least partial functional homology between the mammalian hypothalamus and brain Gr43a neurons of the fly (Musso, 2021).

Insulin production and signaling in renal tubules of Drosophila is under control of tachykinin-related peptide and regulates stress resistance

The insulin-signaling pathway is evolutionarily conserved in animals and regulates growth, reproduction, metabolic homeostasis, stress resistance and life span. In Drosophila seven insulin-like peptides (DILP1-7) are known, some of which are produced in the brain, others in fat body or intestine. This study shows that DILP5 is expressed in principal cells of the renal tubules of Drosophila (see Three Overviews of the Drosophila Malpighian Tubule for information on Malpighian tubule structure) and affects survival at stress. Renal (Malpighian) tubules regulate water and ion homeostasis, but also play roles in immune responses and oxidative stress. This study investigated the control of DILP5 signaling in the renal tubules by Drosophila tachykinin peptide (DTK) and its receptor Tachykinin-like receptor at 99D during desiccative, nutritional and oxidative stress. The DILP5 levels in principal cells of the tubules are affected by stress and manipulations of DTKR expression in the same cells. Targeted knockdown of DTKR, DILP5 and the insulin receptor dInR in principal cells or mutation of Dilp5 resulted in increased survival at either stress, whereas over-expression of these components produced the opposite phenotype. Thus, stress seems to induce hormonal release of DTK that acts on the renal tubules to regulate DILP5 signaling. Manipulations of S6 kinase and superoxide dismutase (SOD2) in principal cells also affect survival at stress, suggesting that DILP5 acts locally on tubules, possibly in oxidative stress regulation. These findings are the first to demonstrate DILP signaling originating in the renal tubules and that this signaling is under control of stress-induced release of peptide hormone (Söderberg, 2011).

The insulin-signaling pathway is evolutionarily conserved in multicellular animals and insulin-like peptides (ILPs) regulate growth, reproduction and metabolism and play important roles in stress resistance and regulation of life span. In Drosophila genetic ablation of cells in the brain producing ILPs, or mutations in the ILP receptor (dInR) and other insulin signaling components, lead to an increase in stress tolerance and extension of life span at the expense of fertility and body size. Also carbohydrate and lipid homeostasis is affected by these manipulations. Seven Drosophila ILPs (DILP1-7) have been identified and some of these are expressed in the brain, others in fat body or intestine. Although much has been learned about insulin signaling downstream of the insulin receptor, it is not clear how the production and release of DILPs is regulated in adult Drosophila in response to nutritional or stress signals. Nutritional sensing appears to take place in adipose tissue, the fat body, and recently it was shown that there is a humoral link between the fat body and insulin-producing cells (IPCs) in the brain. Thus, availability of nutrients sensed by the fat body is an important factor in regulation of DILP release. In addition recent evidence suggest that the IPCs can sense glucose levels autonomously (Söderberg, 2011).

It is likely that hormonal or neural signals also regulate production and release of DILPs by IPCs of the adult insect, as has been shown to be the case in pancreatic beta-cells in mammals. However, such hormones have not yet been identified in the fly, although recently neurons expressing, short neuropeptide F, GABA or serotonin were suggested as regulators of DILP production in IPCs of the brain. The role of DILPs in stress responses is intriguing and this study sought to investigate hormonal signaling pathways that mediate regulation of release of DILPs during stress in Drosophila (Söderberg, 2011).

For nutritional and osmotic stress one possible hormonal route is signaling from endocrine cells of the intestine. The intestine could provide further sensors to monitor metabolic status and it has been shown that midgut endocrine cells in insects release peptide hormone at starvation. A few candidate peptide hormones have been identified in endocrine cells of the Drosophila intestine. This study focused on peptides encoded by the gene Tachykinin (Tk, Dtk or CG14734), the five Drosophila tachykinin-related peptides DTKs, and the role of their receptors in regulation of DILPs in the fly. The reason for this focus is that a novel set of cells was detected that produce DILP5 and also express one of the two known receptors for DTKs (Söderberg, 2011).

This study shows that the main epithelial cells of the renal tubules (Malpighian tubules), the principal cells, express both DILP5 and the DTK receptor DTKR, suggesting that these insulin-producing cells are targets of circulating DTKs. Indeed, it was found that DTK signaling regulates levels of DILP5 in principal cells under nutritional stress. Since the renal tubules are not innervated, DTK can only reach them as a circulating hormone, likely to be released from the intestine (Söderberg, 2011).

In Drosophila the renal tubules display high metabolic activity and play roles, not only in water and ion transport, but also in oxidative stress, detoxification and immune responses. Encouraged by this and by the likely importance of insulin signaling in the physiology of the kidneys of mammals, the roles were investigated of DILP5 signaling locally in the renal tubules. Interference with the expression levels of DTKR, DILP5, dInR and some further components of the insulin-signaling pathway in principal cells during metabolic and oxidative stress all lead to altered lifespan. Furthermore, knockdown of superoxide dismutase (SOD2) in principal cells leads to decreased lifespan at desiccation and oxidative stress, suggesting a possible link between insulin signaling and oxidative stress responses. It is proposed that insulin signaling in the tubules may be part of an autocrine regulation of renal function that in turn is controlled by hormonal DTK signaling from the intestine at metabolic and oxidative stress (Söderberg, 2011).

This study has identified the renal tubules as a novel site of insulin production and signaling in Drosophila. The principal cells of these tubules produce DILP5 and express the ubiquitous DILP receptor, dInR. From these findings it is suggested that DILP5 may signal locally within the epithelium of the renal tubules. This local DILP signaling appears to be under hormonal regulation during desiccative, nutritional and oxidative stress by means of the peptide DTK acting on the receptor, DTKR, localized on the principal cells. These findings, that diminished DTKR, DILP5 and dInR extend life span, suggest an involvement of this signaling pathway in tubules in desiccation, nutritional and oxidative stress responses in adult Drosophila. Finally, manipulations of dS6K, 4E-BP and SOD (SOD2) in principal cells altered life span of flies at stress supporting that insulin signaling acts within the tubules, probably in regulation of oxidative stress responses. Interestingly, the signaling within the renal tubules affects the survival of the whole organism as shown also for mitochondrial function in tubules at oxidative stress (Söderberg, 2011).

The roles of DILPs in stress resistance and regulation of life span are well established in Drosophila, but hormonal mechanisms for regulation of production and release of DILPs in IPCs of adult flies have not been reported. Thus this demonstration of DTKs acting on IPCs in the renal tubules is a first identification of a hormonal factor regulating DILP release in adult insects. Interestingly, there is evidence for actions of tachykinins on IPCs also in mammals: the tachykinin substance P has been shown to increase insulin secretion from the pancreas of rat and pig and this effect is reversed in the diabetic rat (Söderberg, 2011 and references therein).

Since the renal tubules are not innervated, peptide receptors in this tissue can only be activated by hormonal messengers. One source of hormonal DTKs in Drosophila is a population of endocrine cells in the intestine (midgut) located close to the attachment of the renal tubules. In locust and cockroach similar cells have been identified and it was shown that at starvation tachykinin-related peptide was released into the circulation (Söderberg, 2011).

Renal tubules in insects have been primarily investigated with respect to their function in water and ion transport and several peptide hormones have been implicated in the control of diuresis. The current findings suggest that peptide hormones that target the renal tubules may play roles other than in direct regulation of diuresis. The Drosophila renal tubules express an impressive array of genes and combined with experimental analysis it is suggestive that this tissue partakes in detoxification processes, oxidative stress, dietary osmotic stress and immune responses (Söderberg, 2011 and references therein).

How does DTK signaling to the renal tubules produce a response that affects sensitivity to desiccation and starvation? The DTK signal may be a general metabolic stress signal that reaches the renal tubules. In these experiments this stress signaling is amplified with the over-expression of DTKR in principal cells and diminished by its knockdown leading to changes in lifespan. The role of DTK may be to regulate factors in principal cells involved in local metabolism, oxidative stress resistance or immune responses at the cost of decreased life span when in over-drive. One such a factor may be DILP5. Both in Drosophila and C. elegans immune response genes are expressed in the intestine (including renal tubules in the fly) and recent work has shown that these genes are under control of insulin signaling. In Drosophila the DILP signaling pathway is involved in infection-induced wasting (loss of energy stores) where reduced signaling leads to reduction in pathology (Söderberg, 2011).

Also oxidative stress resistance is linked to insulin signaling in Drosophila. Superoxide dismutases (SOD) are key enzymes protecting proteins from reactive oxygen species and are thought to be regulated by insulin signaling: SOD activity is elevated in chico (dInR substrate) mutants of Drosophila and Daf-2 mutants of C. elegans. Also in yeast insulin-signaling mutations affect lifespan via SOD. The knockdown of Sod2 (encoding MnSOD), but not Sod1, in renal tubules decreased lifespan at desiccation and oxidative stress in Drosophila. Thus, it is possible that DILP signaling in tubules target mitochondrial SOD2 and affects resistance to oxidative stress. Interestingly, diminishing oxidative stress resistance via Sod2 locally in the principal cells of Drosophila renal tubules is sufficient to shorten the lifespan of the fly during stress. This is similar to findings in a study of genetical impairment of a mitochondrial inner membrane ATP/ADP exchanger in the same cells (Söderberg, 2011),

In conclusion, this study presents evidence for DTK controlled insulin signaling in the renal tubules of Drosophila being important for survival at metabolic and oxidative stress. The findings of this study may suggest an autocrine regulatory loop within the tubules with a role in renal function. Local signaling within Drosophila renal tubules has previously been demonstrated with endogenously produced tyramine and nitric oxide, that regulate chloride permeability and innate immune responses, respectively. It is possible that the insulin signaling in the renal tubules is part of the epithelial immune system or oxidative stress defense via SOD, but it cannot be excluded that the dInRs on principal cells regulate DILP5 production or release and that additional DILP5 targets are located outside the renal tubules (Söderberg, 2011).

Metabolic stress responses in Drosophila are modulated by brain neurosecretory cells that produce multiple neuropeptides

In Drosophila, neurosecretory cells that release peptide hormones play a prominent role in the regulation of development, growth, metabolism, and reproduction. Several types of peptidergic neurosecretory cells have been identified in the brain of Drosophila with release sites in the corpora cardiaca and anterior aorta. In adult flies this study shows that the products of three neuropeptide precursors are colocalized in five pairs of large protocerebral neurosecretory cells in two clusters (designated ipc-1 and ipc-2a): Drosophila tachykinin (DTK), short neuropeptide F (sNPF) and ion transport peptide (ITP). These peptides were detected by immunocytochemistry in combination with GFP expression driven by the enhancer trap Gal4 lines c929 and Kurs-6, both of which are expressed in ipc-1 and 2a cells. This mix of colocalized peptides with seemingly unrelated functions is intriguing and prompted an initiation of analysis of the function of the ten neurosecretory cells. The role of peptide signaling from large ipc-1 and 2a cells in stress responses was investigated by monitoring the effect of starvation and desiccation in flies with levels of DTK or sNPF diminished by RNA interference. Using the Gal4-UAS system the peptide knockdown this study targeted specifically to ipc-1 and 2a cells with the c929 and Kurs-6 drivers. Flies with reduced DTK or sNPF levels in these cells displayed decreased survival time at desiccation and starvation, as well as increased water loss at desiccation. These data suggest that homeostasis during metabolic stress requires intact peptide signaling by ipc-1 and 2a neurosecretory cells (Kahsai, 2010).

Presynaptic peptidergic modulation of olfactory receptor neurons in Drosophila

The role of classical neurotransmitters in the transfer and processing of olfactory information is well established in many organisms. Neuropeptide action, however, is largely unexplored in any peripheral olfactory system. A subpopulation of local interneurons (LNs) in the Drosophila antennal lobe is peptidergic, expressing Drosophila tachykinins (DTKs). This study shows that olfactory receptor neurons (ORNs) express the DTK receptor (DTKR). Using two-photon microscopy, it was found that DTK applied to the antennal lobe suppresses presynaptic calcium and synaptic transmission in the ORNs. Furthermore, reduction of DTKR expression in ORNs by targeted RNA interference eliminates presynaptic suppression and alters olfactory behaviors. Opposite behavioral phenotypes were detected after reduction and over-expression of DTKR in ORNs. These findings suggest a presynaptic inhibitory feedback to ORNs from peptidergic LNs in the antennal lobe (Ignell, 2009).

Drosophila ORNs express DTKR, which appears to serve in a feedback circuit from local peptidergic interneurons, LNs, of the antennal lobe. These LNs express the peptide products, DTK1-5, of the dtk gene (Winther, 2003). Two-photon imaging and behavioral data, using RNAi and over-expression of the DTK receptor, provide evidence that ORNs are modulated presynaptically by DTKs. This peptidergic presynaptic inhibition of ORNs is detected behaviorally only at high odorant concentrations, and may thus serve to modulate the dynamic range in sensitivity to relevant odors (Ignell, 2009).

Recent studies of peripheral olfactory signal processing in Drosophila have shown that there are afferent and local excitatory cholinergic circuits, combined with presynaptic and postsynaptic GABAergic inhibition of ORNs and interneurons. This study demonstrates a second presynaptic inhibitory pathway mediated by DTK-expressing LNs, providing an additional modulation mechanism in peripheral olfactory processing. Given the importance of presynaptic inhibition future experiments will be necessary to demonstrate a synaptic connection between LNs and ORNs. However, in studies of the cockroach Periplaneta americana both GABAergic and other LNs were found presynaptic to ORNs (Ignell, 2009).

There is ample physiological evidence to suggest that vertebrate and invertebrate ORN axon terminals can be presynaptically modulated by GABA and inhibitory LNs, but this study is unique in that peptidergic presynaptic inhibition of ORNs by local interneurons has been demonstrated. In animals other than Drosophila there is only immunocytochemical data to suggest such circuitry. For example, in the rat, periglomerular cells, interneurons that modulate the first synaptic relay in olfactory processing, have been shown to express 2 neuropeptides: somatostatin and cholecystokinin. Moreover, somatostatin receptor immunoreactivity in axons of the rat olfactory nerve has been demonstrated. These morphological studies may suggest that presynaptic peptidergic modulation of ORNs is not exclusive to Drosophila. In addition, there are examples of peptidergic modulation in the olfactory system by efferent neurons. Centrifugal peptidergic modulation has been demonstrated in the olfactory epithelia of a salamander, where neuropeptide Y was shown to enhance responses evoked by a food-related odor in hungry animals. The peptide FMRFamide has been shown to modulate the activity of ORNs in the olfactory epithelium of the mouse and the salamander, but the circuitry is not clear (Ignell, 2009).

Recently, a study of how specific glomeruli mediate olfactory-guided behavior showed that the activation of the DM1 glomerulus is necessary and sufficient to mediate fly attraction to vinegar odor. This study tested, physiologically and behaviorally, the response to 2 other food related odors. Both odors elicited activation of DM1 and in flies with down regulated DTKR levels an increased activation of this specific glomerulus was detected. In line with this, it was observed that DTKR knock down flies were more attracted to the 2 odors, suggesting that the behavioral responses to these odors may be linked to DTK mediated inhibition of DM1 (Ignell, 2009).

Neuropeptides often colocalize with classical neurotransmitters in neurons and may act as cotransmitters at synapses. This study found that DTK is expressed in a population partly overlapping with GAD1-expressing LNs, likely to be GABAergic. Therefore, experiments were conducted to determine whether GABA and DTK act synergistically on ORNs. Preliminary data, however, suggest that the 2 compounds may act independently. This is in contrast to findings in the crayfish visual system where GABA hyperpolarizes photoreceptors and a tachykinin-like peptide potentiates this response (Glantz, 2000). In the crayfish, it is not clear which GABA receptor type that mediates the response, although a Cl- conductance appears to be activated, whereas in Drosophila ORNs the presynaptic inhibition is mediated by metabotropic GABAB receptors (Ignell, 2009).

In summary, these findings suggest a presynaptic inhibitory feedback to ORNs from a population of peptidergic LNs in the antennal lobe. These LNs express DTKs, and the ORNs of the antennae express the DTK receptor. Evidence is provided for peptidergic modulation in the antennal lobe before the olfactory signals are relayed via projection neurons to higher brain centers, possibly acting as a mechanism to control olfactory sensitivity (Ignell, 2009).

Widely distributed Drosophila G-protein-coupled receptor (CG7887) is activated by endogenous tachykinin-related peptides

Neuropeptides related to vertebrate tachykinins have been identified in Drosophila. Two Drosophila G-protein-coupled receptors (GPCRs), designated NKD (CG6515) and DTKR (CG7887), cloned earlier, display sequence similarities to mammalian tachykinin receptors. However, they were not characterized with the endogenous Drosophila tachykinins (DTKs). The present study characterizes one of these receptors, DTKR. It was determined that HEK-293 cells transfected with DTKR displayed dose-dependent increases in both intracellular calcium and cyclic AMP levels in response to the different DTK peptides. DTK peptides also induced internalization of DTKR-green fluorescent protein (GFP) fusion constructs in HEK-293 cells. Specific antireceptor antisera were generated and DTKR was shown to be widely distributed in the adult brain and more scarcely in the larval CNS. The distribution of the receptor in brain neuropils corresponds well with the distribution of its ligands, the DTKs. These findings suggest that DTKR is a DTK receptor in Drosophila and that this ligand-receptor system plays multiple functional roles (Birse, 2006).

Several experimental approaches used in this study have provided data to support the hypothesis that the Drosophila GPCR, DTKR (CG7887), is involved in signaling by endogenous tachykinin ligands, the DTKs. The most compelling evidence was furnished by the dual assays of Ca2²⁺ and cAMP levels. The assays of intracellular Ca2²⁺ levels indicated that the DTKR receptor, when stably expressed in HEK-293 cells, displays a distinct activation by all six DTK isoforms. In the assay of cAMP levels, DTKR displayed a distinct activation by DTK-1 to 5 (DTK-6 was not tested). In both cases the responses are clearly dose-dependent (Birse, 2006).

Activation of DTKR was specific to the six DTK isoforms, because no other peptides that were tested produced any responses. It appears that DTKR has a slight preferential affinity for DTK-1, while the other DTKs display almost an order of magnitude lower activity levels in the Ca2²⁺ assay. The responses by DTKR to DTKs measured in the cAMP assay required approximately 10-fold more peptide than in the Ca2²⁺ assay. Likewise, the stable fly (Stomoxys calcitrans) tachykinin receptor, STKR, appears to couple both to changes in Ca2²⁺ levels and in cAMP but with ~10-fold less sensitivity. These results beg the question of whether DTKR normally signals through cAMP. There are many precedents by which single receptors have been demonstrated to couple to multiple second messengers (Birse, 2006).

When these results are reconsidered with future experiments in native Drosophila cells, it will be useful to test the hypothesis that both pathways are regulated by DTKR activation. In the original identification of DTKR, insect tachykinins were not known and the mammalian tachykinin substance P was used at very high doses to activate the receptor when expressed in Xenopus oocytes (Li, 1991). The currents monitored indicated an activation of the PLC pathway, a signaling pathway commonly recruited by tachykinin receptors in mammals. It is noteworthy that the mammalian tachykinins (with FXGLMamide C-terminus) need to be applied in micromolar concentrations to activate invertebrate tachykinin GPCRs, whereas invertebrate TKs (with FXGXRamide) are active even in sub-nanomolar concentrations on the same receptors (Birse, 2006).

Further support for the role of DTKR as a DTK receptor derives from ligand-induced GPCR-GFP internalization experiments. Using HEK-293 cells transfected with a DTKR-GFP fusion protein internalization of the receptor could be detected after the application of 1 microM of DTK-1. Internalization of GPCRs from the plasma membrane after prolonged activation by specific ligands is a common phenomenon serving to terminate peptide activation of the receptor complex. In this process of desensitization β-arrestin is recruited to the plasma membrane. An earlier investigation, using a different approach, demonstrated that a GFP-coupled β-arrestin could be translocated to the plasma membrane of HEK-293 cells transfected with DTKR and activated by 1 microM of DTK-1. This technique measures the desensitization of an activated GPCR regardless of the second messenger pathway(s) that is activated. DTK-1 specifically activated the DTKR receptor in this assay, while no response could be seen with any of 12 other unrelated peptides tested (Birse, 2006).

The expression of DTKR was establised in different portions of adult flies and in the larva by specific antisera to part of the receptor protein. Immunocytochemistry indicated that the relative abundance of DTKR and number of immunoreactive cell bodies in the brain are considerably greater than in thoracic abdominal ganglia both in adults and larvae. The cellular localization of the DTKR immunoreactivity in the intestine was hard to resolve, although Western blotting clearly indicated presence of immunoreactive material in this tissue. Microarray analysis has shown that CG7887 transcript is present in low but significant amounts in Malpighian tubules of flies. However, the current study could not detect reproducible DTKR immunoreactive cells in larval tubules, but only diffuse labeling in adult tubules (Birse, 2006).

A distinct pattern of distribution of DTKR immunoreactivity was found in larvae and adults. In the adult brain the immunolabeling in synaptic neuropil closely resembles that of arborizing neuronal processes containing DTKs. Thus there is a good match between putative release sites of DTKs and the distribution of their receptor DTKR. The distribution of DTKs has been studied in Drosophila both by immunocytochemistry and in situ hybridization in the brain and intestine (see Siviter, 2000; Nassel, 2002; Winther, 2003). In the adult brain TK-immunoreactivity is seen in varicose processes in synaptic neuropils of the antennal lobe, superior protocerebrum (including pars intercerebralis), in the central body, subesophageal ganglion, and in the medulla of the optic lobe. These brain sites also express tachykinin receptor protein of DTKR type. The DTKR receptor distribution would thus suggest that DTKs and one of their receptors are involved in modulation of chemosensory and visual processing, control of hormone release (superior protocerebrum), and modulation of higher locomotor activities (central body) (Birse, 2006).

In conclusion, this study has shown that one of the proposed Drosophila tachykinin receptors, DTKR, is indeed likely to be involved in tachykinin (DTK) signaling and is expressed widely in the CNS. The DTKR receptor appears to have a slight preferential affinity for the DTK-1 isoform compared to DTK-2 to 6 and couples to phospholipase C and to adenylate cyclase in HEK-293 cells. It would be interesting to determine the distribution of the other presumed tachykinin receptor, NKD, to reveal whether the NKD and DTKR receptors display differential distributions in the adult brain and intestine, thus suggesting a functional and spatial separation of DTK signaling (Birse, 2006).

Neuronal expression of tachykinin-related peptides and gene transcript during postembryonic development of Drosophila

The gene Dtk, encoding the prohormone of tachykinin-related peptides (TRPs), has been identified from Drosophila. This gene encodes five putative tachykinin-related peptides (DTK-1 to 5) that share the C-terminal sequence FXGXRamide (where X represents variable residues) as well as an extended peptide (DTK-6) with the C-terminus FVAVRamide). By mass spectrometry (MALDI-TOF-MS), ion signals with masses identical to those of DTK-1 to 5 were identified in specific brain regions. The distribution of the Dtk transcript and peptides was analyzed by in situ hybridization and immunocytochemistry during postembryonic development of the central nervous system (CNS) of Drosophila. Antiserum against a cockroach TRP that cross-reacts with the DTKs was used for immunocytochemistry. Expression of transcript and peptides was detected from first to third instar larvae, through metamorphosis to adult flies. Throughout postembryonic development, it was possible to follow the strong expression of TRPs in a pair of large descending neurons with cell bodies in the brain. The number of TRP-expressing neuronal cell bodies in the brain and ventral nerve cord increases during larval development. In the early pupa (stage P8), the number of TRP-expressing cell bodies is lower than in the third instar larvae. The number drastically increases during later pupal development, and in the adult fly about 200 TRP-expressing neurons can be seen in the CNS. The continuous expression of TRPs in neurons throughout postembryonic development suggests specific functional roles in both larval and imaginal flies and possibly also in some neurons during pupal development (Winther, 2003).

Expression and functional characterization of a Drosophila neuropeptide precursor with homology to mammalian preprotachykinin A

Peptides structurally related to mammalian tachykinins have recently been isolated from the brain and intestine of several insect species, where they are believed to function as both neuromodulators and hormones. Further evidence for the signaling role of insect tachykinin-related peptides was provided by the cloning and characterization of cDNAs for two tachykinin receptors from Drosophila melanogaster. However, no endogenous ligand has been isolated for the Drosophila tachykinin receptors to date. Analysis of the Drosophila genome resulted in identification of a putative tachykinin-related peptide prohormone (prepro-DTK) gene. A 1.5-kilobase pair cDNA amplified from a Drosophila head cDNA library contained an 870-base pair open reading frame, which encodes five novel Drosophila tachykinin-related peptides (called DTK peptides) with conserved C-terminal FXGXR-amide motifs common to other insect tachykinin-related peptides. The tachykinin-related peptide prohormone gene (Dtk) is both expressed and post-translationally processed in larval and adult midgut endocrine cells and in the central nervous system, with midgut expression starting at stage 17 of embryogenesis. The predicted Drosophila tachykinin peptides have potent stimulatory effects on the contractions of insect gut. These data provide additional evidence for the conservation of both the structure and function of the tachykinin peptides in the brain and gut during the course of evolution (Siviter, 2000).

Search PubMed for articles about Drosophila Tachykinin

Alekseyenko, O. V., Chan, Y. B., Li, R. and Kravitz, E. A. (2013). Single dopaminergic neurons that modulate aggression in Drosophila. Proc Natl Acad Sci U S A 110: 6151-6156. PubMed ID: 23530210

Amcheslavsky, A., Song, W., Li, Q., Nie, Y., Bragatto, I., Ferrandon, D., Perrimon, N. and Ip, Y. T. (2014). Enteroendocrine cells support intestinal stem-cell-mediated homeostasis in Drosophila. Cell Rep 9(1):32-9. PubMed ID: 25263551

Asahina, K., Watanabe, K., Duistermars, B. J., Hoopfer, E., Gonzalez, C. R., Eyjolfsdottir, E. A., Perona, P. and Anderson, D. J. (2014). Tachykinin-expressing neurons control male-specific aggressive arousal in Drosophila. Cell 156: 221-235. PubMed ID: 24439378

Babcock, D. T., Shi, S., Jo, J., Shaw, M., Gutstein, H. B. and Galko, M. J. (2011). Hedgehog signaling regulates nociceptive sensitization. Curr Biol 21: 1525-1533. PubMed ID: 21906949

Birse, R. T., et al. (2006). Widely distributed Drosophila G-protein-coupled receptor (CG7887) is activated by endogenous tachykinin-related peptides. J. Neurobiol. 66(1): 33-46. PubMed ID: 16193493

Birse, R. T., Soderberg, J. A., Luo, J., Winther, A. M. and Nassel, D. R. (2011). Regulation of insulin-producing cells in the adult Drosophila brain via the tachykinin peptide receptor DTKR. J Exp Biol 214: 4201-4208. PubMed ID: 22116763

Certel, S. J., Savella, M. G., Schlegel, D. C. and Kravitz, E. A. (2007). Modulation of Drosophila male behavioral choice. Proc Natl Acad Sci U S A 104: 4706-4711. PubMed ID: 17360588

Certel, S. J., Leung, A., Lin, C. Y., Perez, P., Chiang, A. S. and Kravitz, E. A. (2010). Octopamine neuromodulatory effects on a social behavior decision-making network in Drosophila males. PLoS One 5: e13248. PubMed ID: 20967276

Chan, Y. B. and Kravitz, E. A. (2007). Specific subgroups of Fru^M neurons control sexually dimorphic patterns of aggression in Drosophila melanogaster. Proc Natl Acad Sci U S A 104: 19577-19582. PubMed ID: 18042702

Halasz, J., Toth, M., Mikics, E., Hrabovszky, E., Barsy, B., Barsvari, B. and Haller, J. (2008). The effect of neurokinin1 receptor blockade on territorial aggression and in a model of violent aggression. Biol Psychiatry 63: 271-278. PubMed ID: 17678879

Ignell, R., Root, C. M., Birse, R. T., Wang, J. W., Nässel, D. R., Winther, A. M. (2009). Presynaptic peptidergic modulation of olfactory receptor neurons in Drosophila. Proc. Natl. Acad. Sci. 106(31): 13070-13075. PubMed ID: 19625621

Im, S. H., Takle, K., Jo, J., Babcock, D. T., Ma, Z., Xiang, Y. and Galko, M. J. (2015). Tachykinin acts upstream of autocrine Hedgehog signaling during nociceptive sensitization in Drosophila. Elife 4. PubMed ID: 26575288

Inagaki, H. K., Panse, K. M. and Anderson, D. J. (2014). Independent, reciprocal neuromodulatory control of sweet and bitter taste sensitivity during starvation in Drosophila. Neuron 84: 806-820. PubMed ID: 25451195

Kahsai, L., Kapan, N., Dircksen, H., Winther, A. M. and Nassel, D. R. (2010). Metabolic stress responses in Drosophila are modulated by brain neurosecretory cells that produce multiple neuropeptides. PLoS One 5: e11480. PubMed ID: 20628603

Katsouni, E., Sakkas, P., Zarros, A., Skandali, N. and Liapi, C. (2009). The involvement of substance P in the induction of aggressive behavior. Peptides 30: 1586-1591. PubMed ID: 19442694

Ko, K. I., Root, C. M., Lindsay, S. A., Zaninovich, O. A., Shepherd, A. K., Wasserman, S. A., Kim, S. M. and Wang, J. W. (2015). Starvation promotes concerted modulation of appetitive olfactory behavior via parallel neuromodulatory circuits. Elife 4. PubMed ID: 26208339

Lin, D., Boyle, M. P., Dollar, P., Lee, H., Lein, E. S., Perona, P. and Anderson, D. J. (2011). Functional identification of an aggression locus in the mouse hypothalamus. Nature 470: 221-226. PubMed ID: 21307935

Mundiyanapurath, S., Chan, Y. B., Leung, A. K. and Kravitz, E. A. (2009). Feminizing cholinergic neurons in a male Drosophila nervous system enhances aggression. Fly (Austin) 3: 179-184. PubMed ID: 19556850

Musso, P. Y., Junca, P. and Gordon, M. D. (2021). A neural circuit linking two sugar sensors regulates satiety-dependent fructose drive in Drosophila. Sci Adv 7(49): eabj0186. PubMed ID: 34851668

Nassel, D. R. and Winther, A. M. (2010). Drosophila neuropeptides in regulation of physiology and behavior. Prog Neurobiol 92: 42-104. PubMed ID: 20447440

Reiher, W., Shirras, C., Kahnt, J., Baumeister, S., Isaac, R. E. and Wegener, C. (2011). Peptidomics and peptide hormone processing in the Drosophila midgut. J Proteome Res 10: 1881-1892. PubMed ID: 21214272

Siegel, A., Schubert, K. L. and Shaikh, M. B. (1997). Neurotransmitters regulating defensive rage behavior in the cat. Neurosci Biobehav Rev 21: 733-742. PubMed ID: 9415898

Root, C. M., Ko, K. I., Jafari, A. and Wang, J. W. (2011). Presynaptic facilitation by neuropeptide signaling mediates odor-driven food search. Cell 145: 133-144. PubMed ID: 21458672

Siviter, R. J., Coast, G. M., Winther, A. M., Nachman, R. J., Taylor, C. A., Shirras, A. D., Coates, D., Isaac, R. E. and Nassel, D. R. (2000). Expression and functional characterization of a Drosophila neuropeptide precursor with homology to mammalian preprotachykinin A. J Biol Chem 275: 23273-23280. PubMed ID: 10801863

Soderberg, J. A., Birse, R. T. and Nassel, D. R. (2011). Insulin production and signaling in renal tubules of Drosophila is under control of tachykinin-related peptide and regulates stress resistance. PLoS One 6: e19866. PubMed ID: 21572965

Song, W., Veenstra, J. A. and Perrimon, N. (2014). Control of lipid metabolism by Tachykinin in Drosophila. Cell Rep 9(1): 40-7. PubMed ID: 25263556

Winther, A. M., Siviter, R. J., Isaac, R. E., Predel, R. and Nassel, D. R. (2003). Neuronal expression of tachykinin-related peptides and gene transcript during postembryonic development of Drosophila. J Comp Neurol 464: 180-196. PubMed ID: 12898611

Yang, C. F., Chiang, M. C., Gray, D. C., Prabhakaran, M., Alvarado, M., Juntti, S. A., Unger, E. K., Wells, J. A. and Shah, N. M. (2013). Sexually dimorphic neurons in the ventromedial hypothalamus govern mating in both sexes and aggression in males. Cell 153: 896-909. PubMed ID: 23663785

Zhou, C., Rao, Y. and Rao, Y. (2008). A subset of octopaminergic neurons are important for Drosophila aggression. Nat Neurosci 11: 1059-1067. PubMed ID: 19160504

date revised: 11 June 2022

Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.