InteractiveFly: GeneBrief

Drosulfakinin: Biological Overview | References

BIOLOGICAL OVERVIEW

Aggressive behavior is regulated by various neuromodulators such as neuropeptides and biogenic amines. This study found that the neuropeptide Drosulfakinin (Dsk) modulates aggression in Drosophila melanogaster. Knock-out of Dsk or Dsk receptor CCKLR-17D1 reduced aggression. Activation and inactivation of Dsk-expressing neurons increased and decreased male aggressive behavior, respectively. Moreover, data from transsynaptic tracing, electrophysiology and behavioral epistasis reveal that Dsk-expressing neurons function downstream of a subset of P1 neurons (P1(a)-splitGAL4) to control fighting behavior. In addition, winners show increased calcium activity in Dsk-expressing neurons. Conditional overexpression of Dsk promotes social dominance, suggesting a positive correlation between Dsk signaling and winning effects. The mammalian ortholog CCK has been implicated in mammal aggression, thus this work suggests a conserved neuromodulatory system for the modulation of aggressive behavior (Wu, 2020).

Aggression is a common innate behavior in most vertebrate and invertebrate species and a major driving force for natural and sexual selections. It is a critical behavior for defense against conspecifics to obtain food resources and mating partners (Wu, 2020).

Aggressive behavior of fruit flies was first reported by Alfred Sturtevant. Since then, a number of ethological and behavioral studies in flies pave the way for using Drosophila as a genetic system to study aggression. Drosophila provides an excellent system to manipulate genes and genetically defined populations of neurons, leading to the identification of multiple genes and neural circuits that control aggression. The neural circuits of aggression involve the peripheral sensory systems that detect male-specific pheromones and auditory cues necessary for aggression, a subset of P1 neurons (Hoopfer, 2015), pCd (Jung, 2020) in the central brain controlling aggressive arousal, and AIP neurons controlling threat displays (Duistermars, 2018). Aggression is modulated by various monoamines and neuropeptides. Octopamine, serotonin and dopamine are important neuromodulators for fly aggression and the specific aminergic neurons that control aggression have been identified. Neuropeptides such as tachykinin and neuropeptide F are required for normal male aggression. Cholecystokinin (CCK) is a neuropeptide that is linked to a number of psychiatric disorders and involved in various emotional behaviors in humans and other mammals. Infusion of CCK induces panic attack in humans. Enhanced CCK level is detected in a rat model of social defeat. CCK is implicated to act in the periaqueductal gray to potentiate defensive rage behavior in cats. In addition, CCK is a satiety signal in a number of species. Silencing CCK-like peptide Drosulfakinin could decrease satiety signaling and increase intake of food in flies. Co-injection of nesfatin-1 and CCK8 decreased food intake in Siberian sturgeon (Acipenser baerii) (Wu, 2020).

This study investigated the roles of cholecysokinin-like peptide Drosulfakinin (Dsk) in Drosophila aggression. Knock-outs and GAL4 knock-ins were generated for Dsk and candidate Dsk receptors. Loss-of-function in either Dsk or Dsk receptor CCKLR-17D1 reduces aggression. Thermogenetic activation of DskGAL4 neurons promotes aggression, while silencing these neurons suppresses aggression. Transsynaptic tracing, electrophysiology and behavioral epistasis experiments were performed to illustrate that Dsk-expressing neurons are functionally connected with a subset of P1 neurons (P1a-splitGAL4, 8 ~ 10 pairs of P1 Neurons) and act downstream of a subset of P1 neurons to control fighting behavior. Furthermore, this study found that winners show increased calcium activity in Dsk-expressing neurons and that conditional overexpression of Dsk promotes winning effects, implicating an important role of the Dsk system in the establishment of social hierarchy during fly fighting. Previously the mamalian ortholog CCK has been implicated in aggression, thus this work suggests a potentially conserved neural pathway for the modulation of aggressive behavior (Wu, 2020).

This study has systematically dissected the neuromodulatory roles of the Dsk system in fly aggression. At the molecular level, Dsk neuropeptide and its receptor CCKLR-17D1 are important for fly aggression. At the circuit level, Dsk-expressing neurons function downstream of a subset of P1 neurons (P1a-splitGAL4, 8 ~ 10 pairs of P1 Neurons) to control aggression. Furthermore, winners show increased calcium activity of Dsk-expressing neurons. Conditional overexpression of Dsk promotes winner effects, suggesting that Dsk is closely linked to the establishment of dominance. Taken together, these results elucidate the molecular and circuit mechanism underlying male aggression and suggest that cholecystokinin-like neuropeptide is likely to be evolutionarily conserved for the neuromodulation of aggression (Wu, 2020).

A neural circuitry controlling aggression should be composed of multiple modules that extend from sensory inputs to motor outputs. A variety of peptidergic and aminergic neurons are implicated in fly aggression, but it is not clear how these modulatory neurons integrate input signals from other neural circuits to signal specific physiological states. The current data from circuit tracing, functional connectivity and behavioral epistasis suggest that Dsk-expressing neurons function downstream of a subset of P1 neurons and likely summate inputs from a subset of P1 neurons to signal an internal state of aggression. Activation of a subset of P1 neurons triggers both aggression and courtship. Interestingly, while the aggression-promoting effect of activating a subset of P1 neurons is dramatically suppressed by the loss of the Dsk gene, the courtship-promoting effect remains intact in the ΔDsk mutant background. On the other hand, recent study suggested that Dsk neurons might function to antagonize P1 neurons on regulating male courtship (Wu, 2019). This dissociation suggests that while a subset of P1 neurons signal an arousal state facilitating both aggression and courtship, the Dsk system acts downstream of a subset of P1 neurons specifically required for aggression. It worth mentioning that the P1a-splitGAL4 used in those studies not only labeled a small subset of Fru+ neurons but also several Fru- neurons, and previous study on pC1 neurons suggested that Fru+ pC1 neurons promote courtship and Fru- pC1 neurons promote aggression (Koganezawa, 2016), so further studies are needed to characterize whether different subset of P1a-splitGAL4 labeled neurons are function differently on aggression and how Dsk system are involved. In addition, it remains unknown whether the Dsk system is responsible for integrating the sensory inputs and arousal state related to aggression, and how it connects to other components of the aggression circuitry, such as Tk neurons and AIP neurons (Wu, 2020).

As a caveat, it has been reported that Dsk is involved in feeding behavior (Nassel, 2014; Williams, 2014). The current experiment also reproduced the result that ΔDsk mutants show increased food consumption in the Capillary Feeder (CAFE) essay. Previous studies reported a positive correlation between the body size of flies and the aggression level, suggesting that the modulational effects of DSK neurons on aggression and feeding can be separated. Further research is required to disentangle the relationship between DSK neurons modulating aggression and those regulating feeding (Wu, 2020).

This study classified the eight DSK neurons into three subtypes (Type I, II and III) based on the morphology of the neurites or two subtypes (DSK-M and DSK-L) based on the location of the cell bodies. Interestingly, these subtypes also show functional difference in modulating aggression and differential connectivity with the a subset of P1 neurons. Note that Type I and II neurons correspond to DSK-M and Type III neurons correspond to DSK-L. The finding that DSK-M neurons showed stronger responses to a subset of P1 neurons activation is consistent with the behavioral results of the flip-out experiment, in which Type I and II neurons, but not Type III, are critical to aggression. In future research, it would be interesting to use intersect method to more specifically label and manipulate the DSK neuron subtypes (Wu, 2020).

Previous study implicated that the cholecystokinin system is closely linked with various human psychiatric disorders, such as bipolar disorder and panic attacks. Interestingly, verbal aggression is promoted by the administration of cholecystokinin tetrapeptide in human subjects. In cats, cholecystokinin agonists potentiate the defensive rage behavior while the cholecystokinin antagonists suppress it. These results reveal that cholecystokinin-like peptide Dsk and Dsk receptor CCKLR-17D1 are important for Drosophila aggression. In addition, increased calcium activity in Dsk-expressing neurons coincides with winner states. Thus, the cholecystokinin system is linked to aggressive behavior in a variety of species and is likely to be an evolutionarily conserved pathway for modulating aggressiveness (Wu, 2020).

It has long been noticed that hierarchical relationships could be established during fly fights, with winners remaining highly aggressive and winning the subsequent encounters, and losers retreating and losing second fights. The winner state is perceived as a reward signal while losing experience is aversive (Kim, 2018). The establishment of social hierarchy is only observed in males, and this male-specific feature of fly aggression is specified by fruitless. However, neural correlates of dominance have not been reported. In this study, Using a transcriptional reporter of intracellular calcium (TRIC), it was found that winners display increased calcium activity in the median Dsk-expressing neurons. Moreover, conditional overexpression of Dsk specifically in the adult stage increases the flies' aggressiveness and makes them more likely to win against opponents without Dsk overexpression. Thus, both the enhanced Dsk signaling in the brain and the winning-promoting effect of conditional overexpression supported that the Dsk system may be involved in the establishment of social hierarchy during fly aggression (Wu, 2020).

A descending inhibitory mechanism of nociception mediated by an evolutionarily conserved neuropeptide system in Drosophila

Nociception is a neural process that animals have developed to avoid potentially tissue-damaging stimuli. While nociception is triggered in the peripheral nervous system, its modulation by the central nervous system is a critical process in mammals, whose dysfunction has been extensively implicated in chronic pain pathogenesis. The peripheral mechanisms of nociception are largely conserved across the animal kingdom. However, it is unclear whether the brain-mediated modulation is also conserved in non-mammalian species. This study shows that Drosophila has a descending inhibitory mechanism of nociception from the brain, mediated by the neuropeptide Drosulfakinin (DSK), a homolog of cholecystokinin (CCK) that plays an important role in the descending control of nociception in mammals. Mutants lacking dsk or its receptors are hypersensitive to noxious heat. Through a combination of genetic, behavioral, histological, and Ca(2+) imaging analyses, neurons involved in DSK-mediated nociceptive regulation were subsequently revealed at a single-cell resolution, and a DSKergic descending neuronal pathway was identified that inhibits nociception. This study provides the first evidence for a descending modulatory mechanism of nociception from the brain in a non-mammalian species that is mediated by the evolutionarily conserved CCK system, raising the possibility that the descending inhibition is an ancient mechanism to regulate nociception (Oikawa, 2023).

This study has demonstrated that (1) DSK and its receptors CCKLR-17D1 and CCKLR-17D3 are involved in negatively regulating thermal nociception. (2) Two sets of brain neurons, MP1 and Sv, express DSK in the larval nervous system. (3) One of the DSK receptors (CCKLR-17D1) functions in Goro neurons in the VNC to negatively regulate thermal nociception, and (4) Thermogenetic activation of DSK-2A-GAL4 neurons attenuates both larval nociception and the activity of Goro neurons. Based on these results, it is proposed that the DSKergic neurons regulating the activity of Goro neurons constitute a descending inhibitory pathway of nociception from the brain to the VNC in larval Drosophila. These findings represent the first evidence of a descending mechanism modulating nociception from the brain in a non-mammalian species (Oikawa, 2023).

DSK has been implicated in multiple physiological and developmental processes in Drosophila. Previous studies have shown that dsk and CCKLR-17D1 mutants exhibit significant reductions of synaptic growth and excitability in larval neuromuscular junctions (NMJ) and larval locomotion under bright light, suggesting the importance of the DSK/CCKLR-17D1 signaling pathway in the developmental processes of motoneurons. However, in this study, no major developmental defects were observed in Goro neurons with CCKLR-17D1 RNAi. Furthermore, the simultaneous thermogenetic activations of DSK-2A-GAL4 neurons and C4da nociceptors inhibited larval nociception and the activity of Goro neurons. Given the pronociceptive role of Goro neurons, their reduced synaptic or neuronal activity should cause nociceptive insensitivity or reduced Ca2+ responses, which is contradicting to the observation that CCKLR-17D1 knockdown in Goro produced the thermal hypersensitivity and exaggerated Ca2+ responses. Thus, these data consistently support a physiological role for DSKergic descending signals in the modulation of the activity of Goro neurons rather than a developmental role, highlighting the functional differences of the DSK/CCKLR-17D1 pathway between the NMJ and the nociceptive system in the CNS (Oikawa, 2023).

A previous study has reported the expression of DSK in a population of larval insulin-producing cells (IPCs) and its functions in responding to starving conditions. However, this study observed that anti-FLRFa signals in IPCs persisted in dsk null mutants and that anti-FLRFa signals in IPCs, detected with an antibody against a crustacean neurohormone, hardly overlap with either DSK-GAL4 or DSK-2A-GAL4 expressions. Although a different antibody from that used in the study mentioned above, the number of IPCs visualized by anti-FLRFa was comparable to that of cells visualized by anti-DSK. Regarding the functions of IPCs in nociception it has been shown that silencing dilp2-GAL4 positive IPCs has no significant effect on the baseline nociceptive responses. In CaMPARI2 imaging, IPCs marked by DSK-2A-GAL4 showed very low baseline activity and no responsiveness to noxious heat. Thus, the current and previous studies strongly suggest that IPCs are likely irrelevant for DSK-mediated nociceptive regulation, at least under normal conditions, although it is still possible for DSK to be expressed stochastically in a limited population of IPCs (Oikawa, 2023).

Although the data suggest the existence of a DSKergic descending inhibitory system from the brain to the Goro neurons in the VNC, there are multiple remaining questions for future studies on the mechanisms by which DSK-expressing neurons in the brain regulate Goro neurons: First, whether both MP1 and Sv neurons are involved in nociceptive inhibition remains unclear. The findings consistently point to MP1 neurons over Sv neurons as a major source of DSK regulating Goro neurons. However, given the potential of DSK for distant actions through diffusion, the possibility of Sv neurons communicating with Goro neurons cannot be eliminated. Further analysis using finer genetic/neuronal tools will be necessary to tease the functions of MP1 and Sv neurons apart and understand the functional mechanism of the DSKergic system in the regulation of larval nociception (Oikawa, 2023).

Second, how this DSKergic system is activated to regulate nociception in vivo needs further investigation. Very high CaMPARI2 photoconversion was observed in MP1 neurons without noxious heat stimuli, which did not further increase in response to noxious heat application. These data raise the hypothesis that MP1 neurons may function as a tonically active brake for nociceptive circuits, rather than a simple negative feedback system that is activated by nociceptive stimuli as input. The tonically active model of MP1 function is also consistent with data showing that dsk and CCKLR-17D1 mutations, as well as CCKLR-17D1 RNAi in Goro neurons, all caused thermal hypersensitivity under normal conditions. Up to this point, no external or internal signals have been directly shown to be the input into larval DSKergic neurons. However, since DSK has been implicated in the regulation of several physiological processes such as hunger and stress, sensory or molecular signals that are involved in these physiological processes have the potential to serve as upstream cues that activate DSKergic neurons. Alternatively, it is also possible that MP1 neurons may have spontaneous activity. Further physiological studies on the activity and responsiveness of DSKergic neurons would be required to elucidate how the DSKergic system functions to regulate nociception in larvae under normal conditions (Oikawa, 2023).

Third, the transmission mechanisms of DSK from the brain to Goro neurons need further clarification.nSyb-GRASP and trans-Tango experiments consistently showed negative results, indicating no synaptic connectivity between MP1 descending axons and Goro neurons. Thus, the interaction between DSK from DSKergic neurons in the brain and Goro neurons in the VNC may be mediated non-synaptically through volume transmission, as described in many neuropeptidergic systems. Many of the CCKLR-expressing neurons in the VNC are distantly located from the descending MP1 axons. Since it is unlikely that all these CCKLR-expressing neurons are synaptically connected to MP1 axons, neuronal communications through volume transmission can be fairly assumed for DSKergic systems in the larval VNC. More detailed analyses at an electron microscopic level of the circuitry connectivity between MP1 and Goro neurons and of the localization of DSK-containing vesicles in DSKergic neurons as well as DSK receptors in Goro neurons would be necessary to further clarify the transmission mechanisms of DSK from the brain to Goro neurons (Oikawa, 2023).

The data presented in this study also suggest that DSK signaling could regulate larval nociception through multiple pathways other than the DSK/CCKLR-17D1 system. For example, while CCKLR-17D3 mutants exhibited as severe thermal hypersensitivity as CCKLR-17D1 mutants, RNAi and rescue experiments failed to locate the function of CCKLR-17D3 to Goro neurons. GCaMP Ca2+ imaging also revealed that Goro neurons lacking CCKLR-17D3 showed modestly sensitized responses to noxious heat. These results suggest the major functioning of CCKLR-17D3 in Goro-independent nociceptive pathways. It was also observed that DSK receptor mutants exhibited more severe thermal phenotypes than dsk mutants , which may indicate the presence of unidentified DSK- or CCKLR-dependent signaling pathways in nociception-related cells. Further research is apparently necessary to reveal the whole picture of nociceptive regulations mediated by DSK and its receptors in larval Drosophila (Oikawa, 2023).

The CCK system is thought to be one of the most ancient neuropeptide systems, suggested to have multiple common physiological functions across taxa. In this study, it was demonstrated that CCKergic signaling in Drosophila participates in nociceptive modulation through a descending inhibitory pathway similarly to the mammalian CCK system, adding new evidence about the conserved physiological roles of CCK (Oikawa, 2023).

Unlike the peripheral nociceptive systems, it is still challenging to align the Drosophila and mammalian CCKergic descending pathways due to low homologies of the CNS structures between Drosophila and mammals, wide-spread CCK expression in the mammalian CNS, and the multiple roles of the CCKergic systems in mammalian nociceptive controls. However, the common usage of an orthologous molecular pathway in descending controls of nociception between the two evolutionarily distant clades raises a fascinating new hypothesis that the descending control from the brain may also be an ancient, conserved mechanism of nociception, which has emerged in the common ancestor of protostomes and deuterostomes. It will be of interest for future research to investigate whether the role of CCK signaling in descending nociceptive controls is also present in other species (Oikawa, 2023).

Non-mammalian model systems have been increasingly recognized as powerful tools to identify novel pain-related molecular pathways. However, the utilization of these models has so far been mostly limited to the research on peripheral pain pathophysiology, and few studies have used them to investigate central pain pathophysiology. Descending nociceptive control mechanisms are crucial for central pain modulation and have been implicated in the development of chronic pain states in humans. Thus, the current study opens the door to a new approach to using powerful neurogenetic tools and the simpler nervous system of Drosophila for elucidating the functional principles of descending nociceptive systems, which may potentially contribute to our understanding of the mechanisms underlying central pain modulation and pain pathology due to dysfunctions of descending modulatory pathways (Oikawa, 2023).

Drosulfakinin signaling in fruitless circuitry antagonizes P neurons to regulate sexual arousal in Drosophila

Animals perform or terminate particular behaviors by integrating external cues and internal states through neural circuits. Identifying neural substrates and their molecular modulators promoting or inhibiting animal behaviors are key steps to understand how neural circuits control behaviors. This study identified the Cholecystokinin-like peptide Drosulfakinin (DSK) that functions at single-neuron resolution to suppress male sexual behavior in Drosophila. Dsk neurons physiologically interact with male-specific P neurons, part of a command center for male sexual behaviors, and function oppositely to regulate multiple arousal-related behaviors including sex, sleep and spontaneous walking. This study further found that the DSK- peptide functions through its receptor CCKLR-17D to suppress sexual behaviors in flies. Such a neuropeptide circuit largely overlaps with the fruitless -expressing neural circuit that governs most aspects of male sexual behaviors. Thus DSK/CCKLR signaling in the sex circuitry functions antagonistically with P neurons to balance arousal levels and modulate sexual behaviors (Wu, 2019).

Male courtship in Drosophila melanogaster is one of the best-understood innate behaviors, and largely controlled by the fruitless (fru) gene and doublesex (dsx) gene, which encode sex-specific transcription factors (FRUM and DSXM in males and DSXF in females). FRUM is responsible for most aspects of male courtship, and DSXM is important for the experience-dependent acquisition of courtship in the absence of FRUM, and courtship intensity and sine song production in the presence of FRUM. FRUM is expressed in a dispersed subset of ca. 2000 neurons including sensory neurons, interneurons, and motor neurons that are potentially interconnected to form a sex circuitry controlling sexual behaviors. In contrast, DSXM is expressed in ca. 700 neurons in males, the majority of which also express FRUM, and are crucial for male courtship. Recently, substantial progress has been made into how external sensory cues are perceived and integrated by fruM and/or dsxM neurons to initiate male courtship, in particular, how a subset of male-specific fruM- and dsxM-expressing P1 neurons integrate olfactory and gustatory cues from female or male targets to initiate or terminate courtship. Such a neuronal pathway is also conserved in other Drosophila species (Wu, 2019).

Behavioral decisions depend on both excitatory and inhibitory modulations. P1 neurons represent an excitatory center that integrates multiple (both excitatory and inhibitory) sensory cues and initiates courtship. However, whether there is an inhibitory counterpart that operates against P1 neurons to balance sexual activity is still unknown. Indeed, males do not absolutely court virgin females even if these females may provide the same visual, olfactory, and gustatory cues, depending on the male's internal states and past experiences. It has been previously shown that neuropeptide SIFamide acts on fruM-positive neurons and inhibits male-male but not male-female courtship, and SIFamide neurons also integrate multiple peptidergic neurons to orchestrate feeding behaviors, but whether SIFamide inhibits internal arousal states for sexual behaviors is not clear. Recently, it was found that sleep and sex circuitries interact mutually and demonstrate how DN1 neurons in the sleep circuitry and P1 neurons in the courtship circuitry function together to coordinate behavioral choices between sleep and sex. However, very little is known about the inhibitory pathway(s) that may represent internal arousal states and inhibit courtship toward females (Wu, 2019).

This study sets out to identify courtship inhibitory neurons that express neuropeptides in Drosophila, as neuropeptides play key roles in adjusting animal behaviors based on environmental cues and internal needs. This study identifies that the neuropeptide Drosulfakinin (DSK), the fly ortholog of Cholecystokinin (CCK) in mammals, functions through its receptor CCKLR-17D3 in the fruM-expressing sex circuitry to inhibit male courtship toward females. It is further demonstrate dthat Dsk neurons and P1 neurons interact and oppositely regulate male sexual behaviors (Wu, 2019).

The results identify, at single-neuron resolution, four pairs of fruM-expressing Dsk neurons (MP1 and MP3) that suppress male and female sexual behaviors. The suppression of male and female sexual behaviors depends on the secretion of the neuropeptide DSK-2, which then acts on one of its receptors CCKLR-17D3 that is expressed in many fruM neurons including P1 neurons and the mushroom bodies. Dsk neurons function antagonistically with courtship promoting P1 neurons to co-regulate male courtship, as well as sleep and spontaneous walking (Wu, 2019).

Cholecystokinin (CCK) signaling appears well conserved over evolution and modulates multiple behaviors. In Drosophila, the CCK-like sulfakinin (DSK) is multifunctional and has been reported to be involved in regulating aspects of food ingestion and satiety, aggression, as well as escape-related locomotion and synaptic plasticity during neuromuscular junction development. In mammals, CCK generated from the intestine acts on its receptors in the nucleus of the solitary tract of the brain to transmit satiety signaling and thus inhibit feeding. Furthermore, CCK signaling in the nucleus accumbens modulates dopaminergic influences on male sexual behaviors in rats. CCK is also involved in nociception, learning and memory, aggression and depressive-like behaviors (Wu, 2019).

Despite its significant and conserved roles in modulating multiple innate and learned behaviors, how CCK or DSK signaling responds to environment and/or internal changes and acts on specific neurons expressing its receptors to modulate multiple behaviors, is still rarely known. The finding that DSK/CCKLR signaling functions in the fruM- and/or dsx-expressing sex circuitry to inhibit male courtship is an effort to use Drosophila as a model to investigate how this conserved signaling modulates animal behaviors (Wu, 2019).

The results uncovered a functional circuitry from many dsx neurons (including courtship-promoting P1 neurons) to four pairs of Dsk MP neurons via direct synaptic transmission, and these MP neurons then modulate CCKLR-17D3 neurons including many fruM and/or dsx neurons via secretion of DSK peptides. It is of particular interest to reveal how the four pairs of MP neurons integrate sensory information (in any), physiological states and past experiences in the future to better understand how this neuropeptide signaling modulate arousal states. It is still not known if these MP neurons receive sensory inputs, but since they receive inputs from many dsx neurons, including P1 neurons that integrate multiple chemosensory information, these sensory inputs will at least relay to Dsk MP neurons via P1. Whether there are other pathways from sensory inputs to MP neurons awaits further study. It is also noted that multiple physiological changes including feeding states, aging and sleep deprivation, as well as past housing conditions affect expression of DSK/CCKLR-17D3, but how they affect DSK signaling and behaviors is still unclear. This study showed that male-male group-housing increases DSK expression and thereby reduces male courtship at least under a restricted condition, and previous findings also revealed opposite effects of group-housing on the excitability of P1 neurons in males that have fruM function or lack fruM function. Such male-male housing experience may mildly reduce sexual arousal in a persistent manner, perhaps by increasing DSK expression, but how such housing condition affects physiological roles of Dsk MP neurons and P1 neurons awaits further functional imaging studies on a potential P1-Dsk-P1 functional loop (and a much complex dsx-Dsk-17D3 pathway) with sensory stimulation under different physiological states. In terms of the time scale that DSK functions to inhibit male courtship, the results indicate an immediate behavioral effect upon Dsk neuronal activation. It is also noted that activation of Dsk neurons inhibits male courtship and lasts for minutes, and previous findings also showed that activation of P1 neurons promoted wing extension and aggression and lasts for minutes. These persistent behavioral effects may represent persistent arousal states regulated by Dsk and P1 neurons in this study; however, how this persistency is generated both in the circuit level and behavioral level still needs further investigation (Wu, 2019).

Dsk mutants do not have obvious courtship abnormality under the courtship assays. There are at least two possibilities: (1) DSK may function only in specific conditions (e.g., group-housing) that increase its expression to inhibit courtship; and (2) There are redundant inhibitory signals for courtship, such as another neuropeptide SIFamide that acts on fruM neurons, although they specifically inhibit male-male courtship. Further studies on how DSK/CCKLR signaling is activated under certain conditions, as well as how DSK, SIFamide and other inhibitory signals (if any) jointly modulate male courtship are needed to fully understand this. Nevertheless, that courtship inhibition by activation of Dsk neurons depends on DSK/CCKLR-17D3 signaling, and increasing such signaling through Cas9 activators in an otherwise wild-type male efficiently inhibited courtship, unambiguously reveal the role of DSK/CCKLR-17D3 signaling in suppressing sexual behaviors (Wu, 2019).

As DSK signaling modulates multiple behaviors, one may argue that its role in male courtship is not specific, e.g., activation of Dsk neurons may drive a competing behavior that phenotypically shunting male courtship. Although such possibility cannot be excluded, a number of evidences are listed to support DSK's role with specificity in courtship inhibition: (1) DSK functions in four pairs of fruM-expressing neurons to inhibit courtship; (2) males with activated Dsk neurons rarely court virgin females, while they follow rotating visual objects normally; (3) Dsk neurons receive synaptic transmission from courtship promoting P1 neurons (and many other dsx-expressing neurons) in an experience-dependent manner; (4) Dsk and P1 neurons antagonistically modulate sexual behaviors and wakefulness; and (5) DSK receptor CCKLR-17D3 inhibits male courtship and expresses in many fruM and/or dsx neurons including P1 neurons. It is noted that CCKLR-17D3 is expressed broadly in the CNS including not only P1 neurons, but also mushroom bodies that regulate a range of behaviors including learning, locomotion and sleep. That DSK signaling is multifunctional is possibly due to broad expression of its receptors, and further studies on dissection of CCKLR function in subsets of neurons will help to understand how DSK/CCKLR signaling modulates multiple behaviors (Wu, 2019).

The decision for male flies to court or not depends on not only environmental cues such as availability and suitability of potential mates (males, virgin females, or mated females), but also their internal states (e.g., thirsty or sleepy). It is proposed that there are at least four factors affecting such a decision: (1) external cues that inhibit courtship, referred to as Ex-In factor, such as the male-specific pheromone cVA4; (2) external cues that are excitatory for courtship, referred to as Ex-Ex factor, such as courtship song; (3) internal states that inhibit courtship, referred to as In-In factor; and (4) internal states that are excitatory for courtship, referred to as In-Ex factor. These factors dynamically change and jointly determine males' decision to court or not (Wu, 2019).

Substantial progress has been made on how Ex-In and Ex-Ex factors jointly modulate the activity of male-specific P1 neurons, which is crucial for courtship initiation. In contrast, much less is understood on In-In and In-Ex factors. Recently, Zhang (2016; 2018) found that dopaminergic modulation of P1 neurons drives male courtship not only by desensitizing P1 to inhibition, but also by promoting recurrent P1 stimulation, thus may act as an In-Ex factor for male courtship. Note that all the three factors mentioned above converge on P1 neurons, making them a decision-making center for male courtship. The DSK/CCKLR signaling identified in this study is of particular interest, as it is likely to act as an In-In factor for male courtship, and above all, it does not simply act on P1 neurons like three other factors, but instead forms a potential functional loop with P1 neurons and antagonizes P1 function in modulating male courtship and wakefulness. That Dsk neurons receive synaptic transmission from P1 neurons and other dsx -expressing neurons in an experience-dependent manner further highlights a central role that the DSK/CCKLR signaling plays. These factors, excitatory vs. inhibitory, external vs. internal, jointly control appropriate performance of sexual behaviors, and further studies will reveal how P1 and other dsx-expressing neurons physiologically interact with Dsk neurons to balance behavioral output (Wu, 2019).

A prominent feature of the neuronal control of male and female sexual behaviors in Drosophila is that, despite large similarity in sensory systems, central integrative neurons are sex-specific in the two sexes, with dsx -expressing pC1 neurons integrating olfactory and auditory cues and promoting receptivity to courting males, and fruM-expressing P1 neurons (largely overlapped with dsx-expressing pC1) integrating olfactory, gustatory, and auditory cues and promoting courtship to females. In contrast, the four pairs of Dsk-expressing MP neurons investigated in this study are sexually monomorphic and inhibit both male courtship and female receptivity. Thus DSK/CCKLR signaling may inhibit sexual behaviors in response to physiological changes that are common to both sexes, while sex-promoting central neurons integrating distinct sensory cues are sexually dimorphic. Interestingly, these Dsk neurons common to both sexes receive synaptic transmission from sexually dimorphic dsx neurons in both males and females, providing a simple solution to link sex-specific excitatory and sexually non-specific inhibitory control of sexual behaviors in males and females (Wu, 2019).

The neuropeptide Drosulfakinin regulates social isolation-induced aggression in Drosophila

Social isolation strongly modulates behavior across the animal kingdom. This study utilized the fruit fly Drosophila melanogaster to study social isolation-driven changes in animal behavior and gene expression in the brain. RNA-seq identified several head-expressed genes strongly responding to social isolation or enrichment. Of particular interest, social isolation downregulated expression of the gene encoding the neuropeptide Drosulfakinin (Dsk), the homologue of vertebrate cholecystokinin (CCK), which is critical for many mammalian social behaviors. Dsk knockdown significantly increased social isolation-induced aggression. Genetic activation or silencing of Dsk neurons each similarly increased isolation-driven aggression. The results suggest a U-shaped dependence of social isolation-induced aggressive behavior on Dsk signaling, similar to the actions of many neuromodulators in other contexts (Agrawal, 2020).

This study has shown that knockdown of the neuropeptide Dsk or its receptor CCKLR-17D1 in the pars intercerebralis (PI) increases social isolation-driven aggression of male flies. Moreover, Dsk appears to act in a U-shaped fashion, with both knockdown (the current results) and overexpression (Williams, 2014) increasing aggression. Dsk neuronal activity follows a similar trend, with both activation and silencing increasing aggression. Williams (2014) overexpressed the Dsk transcript in the PI region, which resulted in increased aggression; furthermore, activation of PI neurons was also shown to increase aggression in a separate study. Taken together, this suggests that the primary role of these neurons in this context is indeed production and secretion of Dsk (Soderberg, 2012). Transcription factors in the fly PI neurons regulating aggression were recently identified (Davis, 2014), and it was shown that activation of PI neurons increases aggression. However, the downstream neuropeptides were not known. The current findings identify Dsk as a key neuropeptide expressed in the PI region that regulates aggression. Further work will be required to delineate the aggression-modulating functions, if any, of other neuropeptides also secreted from the PI region (Agrawal, 2020).

A recent neural activation screen (Asahina, 2014) explored the role of neuropeptides in aggression in Drosophila, but investigated only group-housed flies. Intriguingly, Asahina (2014) identified tachykinin signaling in the lateral protocerebrum and did not find increased aggression in group-housed (GH) flies upon activation of Dsk neurons. Thus, male-male aggression in GH and solitary-housed (SH) flies appears to be controlled by different neuropeptides in different brain regions. The absence of Dsk neurons from the screen results in GH flies (Asahina, 2014), combined with the results showing suppressed aggression in GH flies regardless of Dsk transcription or neural activity, suggests a mechanism that overrides Dsk function (Agrawal, 2020).

Downregulation of the Dsk receptor CCKLR-17D1 in Dsk/Dilp2 neurons also increased aggression, consistent with the observation that some neuropeptidergic neurons, e.g. those for neuropeptide F, neuropeptide Y and FMRFamide, have receptors to modulate their signaling in an autocrine manner. However, pan-neuronal downregulation of CCKLR-17D1 receptor did not affect aggression, suggesting potential antagonistic effects outside Dsk/Dilp2 neurons (Agrawal, 2020).

To address potential developmental effects of Dsk signaling, it would be useful to temporally restrict neural perturbation. However,efforts to conditionally silence Dsk+ neurons only in the adult using temperature-sensitive UAS-Kir2.1-GAL80ts were inconclusive, because prolonged exposure of flies (including controls) to the permissive temperature (30°C) affected their basal locomotion and aggression. To address potential off-target targets of the TRiP Dsk RNAi line, another RNAi line against Dsk (VDRC 14201) was tested but no significant reduction in Dsk levels were observed. It would be useful to test other Dsk loss-of-function alleles in future. However, the current conclusions about the involvement of Dsk in isolation-mediated aggression are supported by the similar effects from knockdown of its receptor CCKLR-17D1, as well as silencing and activation of Dsk-secreting neurons (Agrawal, 2020).

The U-shaped ('hormetic') response of the aggression phenotype to both Dsk levels and Dsk+ neuronal activity is similar to such responses seen for NPF and dopamine neurons in Drosophila aggression. Such effects are not unexpected, given the ubiquity of such hormetic responses in neuromodulator signaling pathways and receptors in general. At the level of individual G-protein coupled receptors, such U-shaped responses (low-dose agonism, high-dose antagonism) arise directly from equations considering receptor expression level and the effects of receptor activation on downstream signaling pathways. At the circuit level, it is thought that such U-shaped responses help to maintain neuronal activity patterns, and the resulting behaviors, near homeostatic optima, with deviations resulting in negative feedback (Agrawal, 2020).

There have been a number of prior studies on the genetic basis of aggression in Drosophila, many of them performed with DNA microarrays rather than with RNA-seq, that record counts for specific transcripts of interest. These studies counted all transcripts within cells. Four such studies have been performed in recent years, each identifying a large number of putative aggression-related genes. Given that the involvement of Dsk in aggression is quite context-specific. Asahina (2014) explicitly ruled out involvement of Dsk in aggression of group-housed flies. Therefore, it is perhaps unsurprising that it was not found in several of the screens. In fact, the only one of these four studies to uncover Dsk was the one that utilized socially isolated flies, strengthening the notion that Dsk specifically links social isolation to aggression. It was this link with social behavior that drew attention to Dsk, and indeed the current experiments bear out that this function is mediated through activity in the brain. The PI region has been shown to be the seat of regulation of many other social and sexually dimorphic behaviors (Agrawal, 2020).

In mammals, the Dsk homologue cholecystokinin (CCK) and its receptors regulate aggression, anxiety and social-defeat responses. For instance, intravenous injection of the smallest isoform, CCK-4, in humans reliably induces panic attack and is often used to screen anxiolytic drug candidates. However, in other contexts, such as in mating and juvenile play, CCK encodes strong positive valence. CCK colocalizes with dopamine in the ventral striatum, and microinjection of CCK into the rat nucleus accumbens phenocopies the effects of dopamine agonists, increasing attention and reward-related behaviors, further supporting its role in positive valence encoding. CCK actions differ across brain regions, in a context-dependent manner. For instance, time pinned (negative valence) during rough-and-tumble play correlated with increased CCK levels in the posterior cortex and decreased levels in hypothalamus. However, lower hypothalamic CCK also correlated with positive-valence play aspects including dorsal contacts and 50 kHz ultrasonic vocalizations. Thus, CCK can encode both positive- and negative-valence aspects of complex behaviors differentially across the brain. As with many neuromodulators, CCK appears to act in a U-shaped fashion, with increases and decreases of signaling from baseline levels often producing similar phenotypes (Agrawal, 2020).

Taken together, the results suggest an evolutionarily conserved role for neuropeptide signaling through the Drosulfakinin pathway (homologue of cholecystokinin) in promoting aggression. Intriguingly, this pathway only seems active in socially isolated flies; in socially enriched flies, aggression is controlled by tachykinin (a.k.a. Substance P) signaling. The PI region, in which the Dsk/Dilp2 neurons reside, has considerable similarities with the hypothalamus, a brain region crucial for regulating aggression in mammals, with the most relevant activity localized to the ventrolateral subdivision of the ventromedial hypothalamus, where CCK neurons reside. Thus, the predominant aggression-regulating mechanism in rodents bears strong homology to the fly pathway regulating aggression of socially deprived, but not socially enriched, individuals (Agrawal, 2020).

Drosulfakinin signaling modulates female sexual receptivity in Drosophila

Female sexual behavior as an innate behavior is of prominent biological importance for survival and reproduction. However, molecular and circuit mechanisms underlying female sexual behavior is not well understood. This study identified the Cholecystokinin-like peptide Drosulfakinin (DSK) to promote female sexual behavior in Drosophila. Loss of DSK function reduces female receptivity while overexpressing DSK enhances female receptivity. Two pairs of Dsk-expressing neurons in the central brain were found to promote female receptivity. DSK peptide acts through one of its receptors, CCKLR-17D3, to modulate female receptivity. Manipulation of CCKLR-17D3 and its expressing neurons alters female receptivity. It was further revealed that the two pairs of Dsk-expressing neurons receive input signal from pC1 neurons that integrate sex-related cues and mating status. These results demonstrate how a neuropeptide pathway interacts with a central neural node in the female sex circuitry to modulate sexual receptivity (Wang, 2022).

This study systematically investigated DSK-mediated neuromodulation of female sexual receptivity. At the molecular level, it was revealed that DSK neuropeptide and its receptor CCKLR-17D3 are crucial for modulating female sexual receptivity. At the neuronal circuit level, it was identified that DSK neurons are the immediate downstream targets of sex-promoting pC1 neurons in controlling female sexual receptivity. Moreover, intersectional tools were employed to subdivide DSK neurons into medial DSK neurons (DSK-MP1) and lateral DSK neurons (DSK-MP3) and uncovered that DSK-MP1 neurons rather than DSK-MP3 neurons play essential roles in modulating female receptivity. Collectively, these findings illuminate a pC1-DSK-MP1-CCKLR-17D3 pathway that modulates female sexual behaviors in Drosophila (Wang, 2022).

The female sexual behavior is a complex innate behavior. The decision for the female to accept a courting male or not depends on not only sensory stimulation but also internal states. If the female is willing to mate, she slows down, pauses, and opens her vaginal plates to accept a courting male, if not, she extrudes her ovipositor to deter a courting male or flies away. Tbe results of this study show that DSK signaling is crucial for virgin female receptivity but has no effect on ovipositor extrusion behavior. How exactly does the DSK signaling regulate virgin female receptivity is still not clear. One possibility is that DSK signaling regulates pausing behavior in response to male courtship, as DSK receptor CCKLR-17D3 expresses in the central complex that has been found to be crucial for locomotor behaviors. However, no change was observed in locomotor activity in DSK or CCKLR-17D3 mutant females. Note only locomotor behavior was studied in single females but not in females paired with courting males due to technical limit for analysis, and it is possible that the DSK signaling does not affect general locomotor behavior but regulates courtship-stimulated pausing behavior (Wang, 2022).

The four pairs of DSK neurons are classified into two types (DSK-MP1 and DSK-MP3) based on the location of the cell bodies, and DSK-MP1 neurons extend descending fibers to ventral nerve cord. In this study, it was also found that activating DSK-MP1 neurons enhance female receptivity whereas inactivating DSK-MP1 neurons reduce female receptivity. Silencing adult Abd-B neurons and SAG neurons located in the abdominal ganglion inhibits female sexual receptivity. It has been found that Abd-B neurons control female pausing behavior, and it would be interesting to further investigate whether DSK-MP1 neurons relay information from Abd-B neurons to regulate pausing and receptivity in females. It is also noted that DSK-MP1 neurons extend projections to suboesophageal ganglion (SOG), and the SOG is the terminus of ascending projections from a subset of female reproductive tract sensory neurons labeled by pickpocket (ppk), fruitless (fru), and doublesex (dsx). It is also possible that DSK-MP1 neurons may integrate information directly from these sensory neurons to regulate female receptivity. Another study further classified the DSK-MP1 neurons into two types (MP1a and MP1b) based on the morphology of their neuritis. Future studies would further build genetic tools to uncover the function of each subset of DSK neurons in regulating distinct innate behaviors, such as male courtship, aggression, and female sexual behavior (Wang, 2022).

Previous studies have revealed that pC1 neurons extend projections to lateral protocerebral complex (LPC) and this neural cluster responds to courtship song and cVA. Moreover, recent works have shown that DSK-MP1 neurons project to the region of LPC. This study used GRASP, trans-Tango, and patch-clamp techniques and revealed that DSK-MP1 neurons are direct downstream target of R71G01-GAL4 neurons that include pC1. EM reconstruction revealed that pC1 neurons have intense synaptic input on MP1b but not MP1a neurons, suggesting a crucial role of the single pair of MP1b neurons in female receptivity. Based on these findings, it is propose that: (1) pC1 neurons act as a central node for female sexual receptivity by integrating sex-related sensory cues (courtship song and cVA) and mating status; and (2) DSK-MP1 neurons may integrate internal states and pC1-encoded information to modulate female sexual behavior. Thus, it is of prime importance to further investigate how a neuropeptide pathway modulate a core neural node in the sex circuitry to fine-tune the female's willingness for sexual behavior in the future (Wang, 2022).

The 5-amino acid N-terminal extension of non-sulfated drosulfakinin II is a unique target to generate novel agonists

The ability to design agonists that target peptide signaling is a strategy to delineate underlying mechanisms and influence biology. A sequence that uniquely characterizes a peptide provides a distinct site to generate novel agonists. Drosophila melanogaster sulfakinin encodes non-sulfated drosulfakinin I (nsDSK I; FDDYGHMRF-NH2) and nsDSK II (GGDDQFDDYGHMRF-NH2). Drosulfakinin is typical of sulfakinin precursors, which are conserved throughout invertebrates. Non-sulfated DSK II is structurally related to DSK I, however, it contains a unique 5-residue N-terminal extension; drosulfakinins signal through G-protein coupled receptors, DSK-R1 (Cholecystokinin-like receptor at 17D3) and DSK-R2 (Cholecystokinin-like receptor at 17D1) (Leander, 2016).

Drosulfakinin II distinctly influences adult and larval gut motility and larval locomotion; yet, its structure-activity relationship was unreported. It was hypothesized that substitution of an N-terminal extension residue may alter nsDSK II activity. By targeting the extension analogs were identified that mimicking nsDSK II, yet, surprisingly, novel agonists with were also discovered that increased (super) and opposite (protean) effects. It was determined [A3] nsDSK II increased larval gut contractility rather than, like nsDSK II, decrease it. [N4] nsDSK II impacted larval locomotion, although nsDSK II was inactive. In adult gut, [A1] nsDSK II, [A2] nsDSKII, and [A3] nsDSK II mimicked nsDSK II, and [A4] nsDSK II and [A5] nsDSK II were more potent; [N3] nsDSK II and [N4] nsDSK II mimicked nsDSK II. This study reports nsDSK II signals through DSK-R2 to influence gut motility and locomotion, identifying a novel role for the N-terminal extension in sulfakinin biology and receptor activation; it also led to the discovery of nsDSK II structural analogs that act as super and protean agonists (Leander, 2016).

Obesity-linked homologues TfAP-2 and Twz establish meal Frequency in Drosophila melanogaster

In all animals managing the size of individual meals and frequency of feeding is crucial for metabolic homeostasis. The current study demonstrates that the noradrenalin analogue octopamine and the cholecystokinin (CCK) homologue Drosulfakinin (Dsk) function downstream of TfAP-2 and Tiwaz (Twz) to control the number of meals in adult flies. Loss of TfAP-2 or Twz in octopaminergic neurons increased the size of individual meals, while overexpression of TfAP-2 significantly decreased meal size and increased feeding frequency. Of note, this study reveals that TfAP-2 and Twz regulate octopamine signaling to initiate feeding; then octopamine, in a negative feedback loop, induces expression of Dsk to inhibit consummatory behavior. Intriguingly, it was found that the mouse TfAP-2 and Twz homologues, AP-2beta and Kctd15, co-localize in areas of the brain known to regulate feeding behavior and reward, and a proximity ligation assay (PLA) demonstrated that AP-2beta and Kctd15 interact directly in a mouse hypothalamus-derived cell line. Finally, it was show that in this mouse hypothalamic cell line AP-2beta and Kctd15 directly interact with Ube2i, a mouse sumoylation enzyme, and that AP-2beta may itself be sumoylated. This study reveals how two obesity-linked homologues regulate metabolic homeostasis by modulating consummatory behavior (Williams, 2014).

Cholecystokinin-like peptide (DSK) in Drosophila, not only for satiety signaling

Cholecystokinin (CCK) signaling appears well conserved over evolution. In Drosophila, the CCK-like sulfakinins (DSKs) regulate aspects of gut function, satiety and food ingestion, hyperactivity and aggression, as well as escape-related locomotion and synaptic plasticity during neuromuscular junction development. Activity in the DSK-producing neurons is regulated by octopamine. Mechanisms behind CCK function in satiety, aggression, and locomotion are discussed in some detail and highlight similarities to mammalian CCK signaling (Nassel, 2014).

Knockdown of DSK by RNAi targeted to DSK-producing neurons decreased satiety signaling in flies and hence intake of food increased, even when it was less palatable with no sugar or bitter with caffeine added. It was furthermore shown that knockdown of DSK only in the IPCs was sufficient to produce the same phenotype, suggesting that the hormonal action of DSKs is important as a satiety signal. Similar results were obtained from third instar larvae. In flies, inactivation of the IPCs or all the DSK-producing neurons by targeted expression of a hyperpolarizing potassium channel (Ork1) generated the same phenotype on food intake, indicating that activity in the IPCs is required to induce satiety. Flies deficient in DSKs displayed increased resistance to starvation compared to control flies, probably as a consequence of the dysregulated satiety signaling and resulting increase in food intake. Interestingly, knocking down DSKs either in IPCs or in all DSK-producing cells led to compensatory increases of Dilp2, 3, and 5 transcripts in the brain of flies fed ad libitum, but had no effect on flies starved for 24 h. Another study revealed that the Drosophila obesity-linked homologs Transcription factor AP-2 (TfAP-2) and Tiwaz (Twz) regulate octopamine signaling to initiate feeding and then octopamine, in a negative feedback loop, induces expression of Dsk to inhibit consummatory behavior (Williams, 2014). Combined, these findings suggest that DSKs released from IPCs are sufficient to induce satiety in larval and adult Drosophila, but the mechanisms remain elusive. The DSK receptor localization and targets of the peptide are yet to be identified, and it remains possible that the action could be either central or peripheral (Nassel, 2014).

There are several sets of neurons in the brain known to regulate feeding. Among these are the so-called hugin neurons that produce a neuropeptide of pyrokinin type, whose branches are known to superimpose those of the IPCs. Functional interactions between the brain IPCs and the hugin neurons were demonstrated recently. The IPCs could signal to the hugin neurons by both DILPs and DSKs and thereby regulate the activity in these neurons that are at the interface between gustatory inputs and feeding regulation. There are several other candidate targets among central neurons. Neurons in circuits that use the following neurotransmitters and neuropeptides have been implicated in the regulation of foraging and feeding in addition to DILPs and DSKs: dopamine (DA), neuropeptide F, short neuropeptide F, allatostatin A, leucokinin, and hugin (Nassel, 2014).

Drosulfakinin activates CCKLR-17D1 and promotes larval locomotion and escape response in Drosophila

Neuropeptides are ubiquitous in both mammals and invertebrates and play essential roles in regulation and modulation of many developmental and physiological processes through activation of G-protein-coupled-receptors (GPCRs). However, the mechanisms by which many of the neuropeptides regulate specific neural function and behaviors remain undefined. This study investigated the functions of Drosulfakinin (DSK), the Drosophila homolog of vertebrate neuropeptide cholecystokinin (CCK), which is the most abundant neuropeptide in the central nervous system. Biochemical evidence is provided that sulfated DSK-1 and DSK-2 activate the CCKLR-17D1 receptor in a cell culture assay. This study further examine the role of DSK and CCKLR-17D1 in the regulation of larval locomotion, both in a semi-intact larval preparation and in intact larvae under intense light exposure. The results suggest that DSK/CCKLR-17D1 signaling promote larval body wall muscle contraction and is necessary for mediating locomotor behavior in stress-induced escape response (Chen, 2012a).

A neuropeptide signaling pathway regulates synaptic growth in Drosophila

Neuropeptide signaling is integral to many aspects of neural communication, particularly modulation of membrane excitability and synaptic transmission. However, neuropeptides have not been clearly implicated in synaptic growth and development. This study demonstrates that cholecystokinin-like receptor (CCK-like receptor at 17D1; CCKLR), and Drosulfakinin (DSK), its predicted ligand, are strong positive growth regulators of the Drosophila melanogaster larval neuromuscular junction (NMJ). Mutations of CCKLR (CCKLR-17D1 but not CCKLR-17D3) or dsk produce severe NMJ undergrowth, whereas overexpression of CCKLR causes overgrowth. Presynaptic expression of CCKLR is necessary and sufficient for regulating NMJ growth. CCKLR and dsk mutants also reduce synaptic function in parallel with decreased NMJ size. Analysis of double mutants revealed that DSK/CCKLR regulation of NMJ growth occurs through the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA)-cAMP response element binding protein (CREB) pathway. These results demonstrate a novel role for neuropeptide signaling in synaptic development. Moreover, because the cAMP-PKA-CREB pathway is required for structural synaptic plasticity in learning and memory, DSK/CCKLR signaling may also contribute to these mechanisms (Chen, 2012b).

Proper synaptic growth is essential for normal development of the nervous system and its function in mediating complex behaviors such as learning and memory. The Drosophila larval neuromuscular junction (NMJ) has become a powerful system for studying the molecular mechanisms underlying synaptic development and plasticity, and many of the key synaptic proteins are evolutionarily conserved (Chen, 2012b).

Genetic and molecular analysis in Drosophila has uncovered numerous molecules and pathways that regulate NMJ growth, including proteins required for cell adhesion, endocytosis, cytoskeletal organization, and signal transduction via TGF-β, Wingless, JNK, cAMP, and other signaling molecules. For example, previous studies revealed that increased cAMP levels led to a down-regulation of the cell adhesion protein FasII at synapses, and the activation of the cAMP response element binding protein (CREB) transcription factor to achieve long-lasting changes in synaptic structure and function. Despite the identification and characterization of these various positive and negative regulators, understanding of the networks that govern synaptic growth is still incomplete, with many of the key components and mechanisms yet to be uncovered and analyzed (Chen, 2012b).

To search for new regulators of synaptic growth, a forward genetic screen was conducted for mutants exhibiting altered NMJ morphology. In this screen, a new mutation was discovered that exhibits strikingly undergrown NMJs, which indicates disruption of a positive regulator of NMJ growth. The affected protein was identified as cholecystokinin (CCK)-like receptor (CCKLR), a putative neuropeptide receptor that belongs to the family of G-protein coupled receptors (GPCRs) sharing a uniform topology with seven transmembrane domains. When activated by their ligands, neuropeptide GPCRs affect levels of second messengers such as cAMP, diacylglycerol, inositol trisphosphate, and intracellular calcium. Through activation of their cognate receptors, secreted neuropeptides mediate communication among various sets of neurons as well as other cell types to regulate several physiological activities, including feeding and growth, molting, cuticle tanning, circadian rhythm, sleep, and learning and memory. In general, neuropeptides act by modulating neuronal activity through both short-term and long-term effects. Short-term effects include modifications of ion channel activity and alterations in release of or response to neurotransmitters. Long-term effects include changes in gene expression through activation of transcription factors and protein synthesis. In contrast with the well-known effects of neuropeptide signaling on neuronal activity and the strength of synaptic transmission, regulation of synaptic growth and development by neuropeptides has not previously been clearly established (Chen, 2012b).

This study demonstrates that CCKLR is required presynaptically to promote NMJ growth. Moreover, mutations of drosulfakinin (dsk), which encode the predicted ligand of CCKLR, cause similar NMJ undergrowth and interact genetically with CCKLR mutations, indicating that DSK and CCKLR are components of a common signaling mechanism that regulates NMJ growth. In addition to the morphological phenotypes caused by mutations of CCKLR and dsk, mutant larvae also exhibit deficits in synaptic function. Through phenotypic analysis of double mutant combinations, it was shown that DSK/CCKLR signals through the cAMP-PKA-CREB pathway to regulate NMJ growth. The results suggest a novel role for neuropeptide signaling in regulation of synaptic development. Moreover, because the cAMP-PKA-CREB pathway is required for structural plasticity of synapses in learning and memory, DSK/CCKLR signaling may also contribute to these mechanisms (Chen, 2012b).

Neuropeptides, whose effects have been extensively studied at NMJs, are usually described as neuromodulators because they modify the strength of synaptic transmission. For example, proctolin can potentiate the action of glutamate at certain NMJs in insects. However, involvement of neuropeptides in regulating neural development has not been well characterized. Recently, a C-type natriuretic peptide acting through a cGMP signaling cascade was found to be required for sensory axon bifurcation in mice, which suggests that neuropeptides may have a broader role in development than previously appreciated. The current studies demonstrate that DSK and its receptor, CCKLR, are strong positive regulators of NMJ growth in Drosophila (Chen, 2012b).

DSK belongs to the family of FMRFamide-related peptides (FaRPs), which is very broadly distributed across invertebrate and vertebrate phyla. Originally identified in clams, FaRPs affect heart rate, blood pressure, gut motility, feeding behavior, and reproduction in invertebrates. They have been shown to enhance synaptic efficacy at NMJs in locust and to modulate presynaptic Ca²⁺ channel activity in crustaceans. In Drosophila, various neuropeptides derived from the FMRFa gene can modulate the strength of muscle contraction when perfused onto standard larval nerve-muscle preparations. To these previously described functions of FaRPs, this study adds a new role as a positive regulator of NMJ development (Chen, 2012b).

Transgenic rescue experiments, RNAi expression, and overexpression of WT CCKLR demonstrate that CCKLR functions presynaptically in motor neurons to promote NMJ growth. Downstream components of this pathway were identified on the basis of known biochemistry of GPCRs and phenotypic interactions in double mutant combinations. GPCRs typically function by activating second messenger pathways via G proteins. Because loss-of-function mutations in dgs (which encodes the Gsα subunit in Drosophila) cause NMJ undergrowth, it is hypothesized that CCKLR signals through Gsα. Consistent with this idea, it was found that presynaptic constitutively active dgs overexpression rescues the NMJ undergrowth phenotype of CCKLR mutants. Conversely, dominant dose-dependent interactions were observed between CCKLR-null mutations and mutations of rut, which encodes an AC; or PKA-C1, which encodes a cAMP-dependent protein kinase, resulting in significant reductions in NMJ growth. These data place CCKLR together with the other genes in a common cAMP-dependent signaling pathway that regulates NMJ growth (Chen, 2012b).

It is known that the AC encoded by rut is activated by Gsα, and on the basis of the results, it is proposed that Gsα is downstream of CCKLR signaling. However, the NMJ undergrowth in rut¹, which is a presumptive null mutant, is not as severe as that of a CCKLR-null mutant. This is likely due to the fact that the Drosophila genome contains up to seven different AC-encoding genes, all of which are stimulated by Gsα. Presumably, one or more additional AC-encoding genes share some functional overlap with rut in regulation of NMJ growth. This idea is in good agreement with the results of Wolfgang (2004), who found that the NMJ undergrowth phenotype of rut¹ is weaker than that of dgs mutants, which they also interpreted as an indication that multiple ACs are activated by the Gsα encoded by dgs (Chen, 2012b).

The primary effector of this pathway is CREB2, a transcriptional regulatory protein that is activated upon phosphorylation by PKA. Consistent with the idea that CCKLR ultimately acts via activation of CREB, loss-of-function mutations of dCreb2 or neuronal overexpression of a dominant-negative dCreb2 transgene cause NMJ undergrowth similar to that of CCKLR-null mutants. Additionally, loss of one copy of dCreb2 in a CCKLR heterozygous background also causes NMJ undergrowth, and overexpression of WT dCreb2 fully rescues the NMJ undergrowth phenotype of CCKLR null, even leading to NMJ overgrowth. Thus, regulation of NMJ growth through the CCKLR signaling pathway is clearly mediated by dCreb2, whose activity is itself necessary and sufficient for regulating NMJ growth. This conclusion differs from another study that suggested that dCreb2 is required for NMJ function but not NMJ growth. One possible explanation for this discrepancy is that a weaker, inducible heat shock-driven transgene was used to express dCreb2 in the earlier work, whereas strong constitutive neuronal drivers were used this study. In any case, the current results demonstrate that in addition to its known role in NMJ function, CREB2 is also a strong positive regulator of NMJ growth and is likely to play a greater role in structural plasticity of synapses in learning and memory in Drosophila than previously suggested. This conclusion is consistent with a recent study indicating that sprouting of type II larval NMJs in response to starvation is stimulated by a cAMP/CREB-dependent pathway via activation of an octopamine GPCR (Chen, 2012b).

In addition to being undergrown, NMJs in CCKLR mutant larvae also exhibit a functional deficit. This is perhaps less straightforward than it might seem. Previous analyses of mutations affecting growth of the larval NMJ in Drosophila have shown that there is no simple correlation between the size and complexity of the NMJ and the amplitude of EJPs or amount of neurotransmitter release. These discrepancies arise because of various homeostatic compensatory mechanisms and because some of the affected signaling pathways alter synaptic growth and synaptic function in different ways via distinct downstream targets. For example, a mutation in highwire, which has the most extreme NMJ overgrowth phenotype described, is associated with a decrease in synaptic transmission. Wallenda mutations have been shown to fully suppress the overgrowth phenotype, but have no effect on the deficit in synaptic transmission (Chen, 2012b).

In the case of CCKLR mutants, however, there appears to be a very good correspondence between the morphological phenotype and the electrophysiological phenotype: the reduction in the total number of active zones in CCKLR larvae as measured morphologically correlates very well with the reduction in quantal content that was observe. In addition, no difference in CCKLR mutants was detected in calcium sensitivity of transmitter release or in the size or frequency of spontaneous release events. Thus, the synaptic growth phenotype of CCKLR mutant NMJs is sufficient to account for the functional phenotype. However, the possibility cannot be ruled out that DSK/CCKLR signaling also exerts some modulatory effect on NMJ function that is distinct from its effect on NMJ development (Chen, 2012b).

DSK is identified as the Drosophila orthologue of CCK, the ligand of CCKLR in vertebrates, on the basis of sequence analysis. The genetic analysis strongly supports the conclusion that DSK is the ligand of CCKLR at the larval NMJ. First, mutations of dsk and expression of dsk RNAi result in NMJ undergrowth phenotypes similar to that of CCKLR mutants. Second, loss of one copy of both dsk and CCKLR in double heterozygotes results in NMJ undergrowth. Third, heterozygosity for dsk does not further enhance the phenotype of a CCKLR-null mutant as expected if DSK regulates NMJ growth through its action on CCKLR. Fourth, overexpression of UAS-dsk does not rescue the undergrowth phenotype of a CCKLR-null mutant, but CCKLR overexpression can rescue NMJ undergrowth of a dsk hypomorphic mutant (Chen, 2012b).

The discovery of an entirely novel role for neuropeptide signaling in NMJ growth raises several questions about how this signaling is regulated and the biological significance of this mechanism. Although answers to these questions will require much additional work, an immediate question is whether a paracrine or autocrine mechanism is involved. In the case of octopamine-mediated synaptic sprouting in response to starvation, both autocrine and paracrine signaling are involved in the sprouting of type II and type I NMJs, respectively. In an early immunohistochemical investigation, it was reported that DSK was detected in medial neurosecretory cells in the larval CNS that extended projections anteriorly into the brain and posteriorly to the ventral ganglion. As it was not possible to obtain the original DSK antiserum and raising a new antiserum was not successful, the previous report has not been extended or confirmed. Instead, tissue-specific RNAi experiments were performed to examine the spatial requirement for DSK. Pan-neuronal dsk RNAi expression indicates that DSK expression in neurons is required to promote NMJ growth. In addition, C739-Gal4-driven dsk RNAi also causes NMJ undergrowth, whereas OK-Gal4-driven dsk RNAi in motor neurons does not. The expression pattern of C739-Gal4 overlaps with the DSK-positive cells previously identified by immunohistochemistry, which suggests that DSK produced by those neurosecretory cells is required for normal NMJ growth. Thus, from available data, it seems most likely that DSK is acting in paracrine fashion to regulate NMJ growth. However, further investigation will be necessary to determine the exact source of the DSK that promotes NMJ growth to fully understand how this neuropeptide regulates NMJ development (Chen, 2012b).

Studies have demonstrated a role for CREB in long-term synaptic plasticity-structural changes in synaptic morphology that underlie the formation of long-term memories. This study shows that in addition to CREB's role in structural modification of synapses in response to experience after development is complete, it is also a key regulator of growth and morphology during development of the larval NMJ. Moreover, although CREB is the transcriptional effector for many GPCRs, the fact that NMJs in CCKLR mutants are as undergrown as those of CREB mutants suggests that DSK/CCKLR signaling is a major input to CREB during NMJ growth. Many of the genes encoding intermediate components of the pathway such as dnc, rut, and PKA also have effects on NMJ growth and development as well as on synaptic plasticity and learning and memory, further emphasizing an overlap between the mechanisms that regulate synaptic growth during development and those that regulate postdevelopmental structural synaptic plasticity. These results raise the possibility that DSK/CCKLR signaling also plays a role in long-term synaptic plasticity and learning as well as in synaptic development (Chen, 2012b).

Drosulfakinin activates CCKLR-17D1 and promotes larval locomotion and escape response in Drosophila

Neuropeptides are ubiquitous in both mammals and invertebrates and play essential roles in regulation and modulation of many developmental and physiological processes through activation of G-protein-coupled-receptors (GPCRs). However, the mechanisms by which many of the neuropeptides regulate specific neural function and behaviors remain undefined. This study investigated the functions of Drosulfakinin (DSK), the Drosophila homolog of vertebrate neuropeptide cholecystokinin (CCK), which is the most abundant neuropeptide in the central nervous system. Biochemical evidence is provided that sulfated DSK-1 and DSK-2 activate the CCKLR-17D1 receptor in a cell culture assay. The role of DSK and CCKLR-17D1 was investigated in the regulation of larval locomotion, both in a semi-intact larval preparation and in intact larvae under intense light exposure. The results suggest that DSK/CCKLR-17D1 signaling promote larval body wall muscle contraction and is necessary for mediating locomotor behavior in stress-induced escape response (Chen, 2012a).

Insulin-producing cells in the Drosophila brain also express satiety-inducing Cholecystokinin-like peptide, Drosulfakinin

Regulation of meal size and assessing the nutritional value of food are two important aspects of feeding behavior. The mechanisms that regulate these two aspects have not been fully elucidated in Drosophila. Diminished signaling with insulin-like peptides Drosophila insulin-like peptides (DILPs) affects food intake in flies, but it is not clear what signal(s) mediates satiety. This study investigate the role of DILPs and drosulfakinins (DSKs), cholecystokinin-like peptides, as satiety signals in Drosophila. DSKs and DILPs are co-expressed in insulin-producing cells (IPCs) of the brain. Next, the effects were analyzed of diminishing DSKs or DILPs employing the Gal4-UAS system by (1) diminishing DSK-levels without directly affecting DILP levels by targeted Dsk-RNAi, either in all DSK-producing cells (DPCs) or only in the IPCs or (2) expressing a hyperpolarizing potassium channel to inactivate either all the DPCs or only the IPCs, affecting release of both peptides. The transgenic flies were assayed for feeding and food choice, resistance to starvation, and for levels of Dilp and Dsk transcripts in brains of fed and starved animals. Diminishment of DSK in the IPCs alone is sufficient to cause defective regulation of food intake and food choice, indicating that DSK functions as a hormonal satiety signal in Drosophila. Quantification of Dsk and Dilp transcript levels reveals that knockdown of either peptide type affects the transcript levels of the other, suggesting a possible feedback regulation between the two signaling pathways. In summary, DSK and DILPs released from the IPCs regulate feeding, food choice and metabolic homeostasis in Drosophila in a coordinated fashion (Soderberg, 2012).

Plasticity in the effects of sulfated and nonsulfated sulfakinin on heart contractions. Front Biosci (Landmark Ed)

Neuropeptides regulate the frequency of heart contractions. Drosophila melanogaster sulfakinin (drosulfakinin) encodes FDDYGHMRFamide, DSK I, and GGDDQFDDYGHMRFamide, DSK II. Invertebrate sulfakinins are structurally and functionally related to vertebrate cholecystokinins. Naturally-occurring drosulfakinins contain a sulfated or nonsulfated tyrosine and are designated sDSK I, sDSK II, nsDSK I, and nsDSK II. A novel neural-cardiovascular preparation was developed and mechanisms were investigated regulating the effect of sulfakinins on D. melanogaster heart. The preparation was extablished in larva, pupa, and adult to examine plasticity in neural regulation of cardiovascular parameters. sDSK I was found to increase the frequency of larval, pupal, and adult heart contractions; nsDSK I only increased the frequency of larval contractions, not pupal or adult. It was also discovered that sDSK II and nsDSK II increased the frequency of larval and adult contractions, not pupal. This is the first report of nonsulfated sulfakinin activity on heart, and sulfakinin activity examined in 3 developmental stages within the same animal species. These data demonstrate a role for plasticity in the effects of sulfakinins on heart contractions, and suggest multiple mechanisms are involved (Nichols, 2009).

Search PubMed for articles about Drosophila Drosulfakinin

Agrawal, P., Kao, D., Chung, P. and Looger, L. L. (2020). The neuropeptide Drosulfakinin regulates social isolation-induced aggression in Drosophila. J Exp Biol. PubMed ID: 31900346

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(1-2): 221-235. PubMed ID: 24439378

Chen, X., Peterson, J., Nachman, R. J. and Ganetzky, B. (2012a). Drosulfakinin activates CCKLR-17D1 and promotes larval locomotion and escape response in Drosophila. Fly (Austin) 6(4): 290-297. PubMed ID: 22885328

Chen, X. and Ganetzky, B. (2012b). A neuropeptide signaling pathway regulates synaptic growth in Drosophila. J Cell Biol 196(4): 529-543. PubMed ID: 22331845

Davis, S. M., Thomas, A. L., Nomie, K. J., Huang, L. and Dierick, H. A. (2014). Tailless and Atrophin control Drosophila aggression by regulating neuropeptide signalling in the pars intercerebralis. Nat Commun 5: 3177. PubMed ID: 24495972

Duistermars, B. J., Pfeiffer, B. D., Hoopfer, E. D. and Anderson, D. J. (2018). A Brain Module for Scalable Control of Complex, Multi-motor Threat Displays. Neuron 100(6): 1474-1490 e1474. PubMed ID: 30415997

Hoopfer, E. D., Jung, Y., Inagaki, H. K., Rubin, G. M. and Anderson, D. J. (2015). P1 interneurons promote a persistent internal state that enhances inter-male aggression in Drosophila. Elife 4. PubMed ID: 26714106

Jung, Y., Kennedy, A., Chiu, H., Mohammad, F., Claridge-Chang, A. and Anderson, D. J. (2020). Neurons that Function within an Integrator to Promote a Persistent Behavioral State in Drosophila. Neuron 105(2): 322-333 e325. PubMed ID: 31810837

Kim, J. J. and Jung, M. W. (2018). Fear paradigms: The times they are a-changin'. Curr Opin Behav Sci 24: 38-43. PubMed ID: 30140717

Koganezawa, M., Kimura, K. and Yamamoto, D. (2016). The neural circuitry that functions as a switch for courtship versus aggression in Drosophila males. Curr Biol 26(11): 1395-1403. PubMed ID: 27185554

Koizumi, H., Mohammad, S., Ozaki, T., Muto, K., Matsuba, N., Kim, J., Pan, W., Morioka, E., Mochizuki, T. and Ikeda, M. (2020). Intracellular interplay between cholecystokinin and leptin signalling for satiety control in rats. Sci Rep 10(1): 12000. PubMed ID: 32686770

Leander, M., Heimonen, J., Brocke, T., Rasmussen, M., Bass, C., Palmer, G., Egle, J., Mispelon, M., Berry, K. and Nichols, R. (2016). The 5-amino acid N-terminal extension of non-sulfated drosulfakinin II is a unique target to generate novel agonists. Peptides 83: 49-56. PubMed ID: 27397853

Nassel, D. R. and Williams, M. J. (2014). Cholecystokinin-like peptide (DSK) in Drosophila, not only for satiety signaling. Front Endocrinol (Lausanne) 5: 219. PubMed ID: 25566191

Nichols, R., Manoogian, B., Walling, E. and Mispelon, M. (2009). Plasticity in the effects of sulfated and nonsulfated sulfakinin on heart contractions. Front Biosci (Landmark Ed) 14: 4035-4043. PubMed ID: 19273332

Oikawa, I., Kondo, S., Hashimoto, K., Yoshida, A., Hamajima, M., Tanimoto, H., Furukubo-Tokunaga, K. and Honjo, K. (2023). A descending inhibitory mechanism of nociception mediated by an evolutionarily conserved neuropeptide system in Drosophila. Elife 12. PubMed ID: 37310871

Soderberg, J. A., Carlsson, M. A. and Nassel, D. R. (2012). Insulin-producing cells in the Drosophila brain also express satiety-inducing Cholecystokinin-like peptide, Drosulfakinin. Front Endocrinol (Lausanne) 3: 109. PubMed ID: 22969751

Wang, T., Jing, B., Deng, B., Shi, K., Li, J., Ma, B., Wu, F. and Zhou, C. (2022). Drosulfakinin signaling modulates female sexual receptivity in Drosophila. Elife 11. PubMed ID: 35475782

Williams, M. J., Goergen, P., Rajendran, J., Zheleznyakova, G., Hagglund, M. G., Perland, E., Bagchi, S., Kalogeropoulou, A., Khan, Z., Fredriksson, R. and Schioth, H. B. (2014). Obesity-linked homologues TfAP-2 and Twz establish meal Frequency in Drosophila melanogaster. PLoS Genet 10: e1004499. PubMed ID: 25187989

Wu, F., Deng, B., Xiao, N., Wang, T., Li, Y., Wang, R., Shi, K., Luo, D. G., Rao, Y. and Zhou, C. (2020). A neuropeptide regulates fighting behavior in Drosophila melanogaster. Elife 9. PubMed ID: 32314736

Wu, S., Guo, C., Zhao, H., Sun, M., Chen, J., Han, C., Peng, Q., Qiao, H., Peng, P., Liu, Y., Luo, S. D. and Pan, Y. (2019). Drosulfakinin signaling in fruitless circuitry antagonizes P neurons to regulate sexual arousal in Drosophila. Nat Commun 10(1): 4770. PubMed ID: 31628317

date revised: 2 October 2020

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