Through a thermal nociception screen using the nocifensive rolling response of Drosophila larvae, we found that a deletion mutant line of the dsk gene showed thermal hypersensitivity with a significantly shorter latency in their response to a 42 °C probe than the controls, suggesting that DSK plays a role in negatively regulating nociception (Fig. 1A and B). A genomic fragment containing the wild-type dsk gene, and no other neuropeptide genes, significantly rescued the thermal hypersensitivity of the dsk mutants (Fig. 1B), confirming that dsk is responsible for the thermal hypersensitivity.

DSK has been shown to activate two G-protein coupled receptors, CCKLR-17D1 and CCKLR-17D3 (45, 46), which are orthologous to the mammalian CCK receptors, CCKAR (also known as CCK₁) and CCKBR (also known as CCK₂) (16–18). To test whether these receptors mediate DSK signaling in nociception, we generated deletion mutants for CCKLR-17D1 and CCKLR-17D3 using CRISPR/Cas9 genome editing and tested them for thermal nociception (Fig. 1C-F). When stimulated with a 42 °C probe, three independent deletion lines of the CCKLR-17D1 and CCKLR-17D3 exhibited thermal hypersensitivity (Fig. 1D and F), further supporting the role of DSK signaling in the negative regulation of thermal nociception. To examine whether the phenotypes of CCKLR-17D1 and CCKLR-17D3 mutants are classified as hyperalgesia (hypersensitivity to normally noxious stimuli) or allodynia (abnormal hypersensitivity to normally innocuous stimuli), we tested the receptor mutants with a 38 °C probe, which is close to the threshold of larval thermal nociception (39 °C) (24). We found that the responses of the DSK receptor mutants were indistinguishable from the controls, indicating that the DSK receptor mutants are hypersensitive to suprathreshold thermal stimuli, thus hyperalgesic (Fig. S1).

We attempted to identify DSK-expressing cells in the larval CNS that are responsible for regulating nociception. Unlike mammalian CCK, which is expressed in the CNS and gastrointestinal system, Drosophila DSK is expressed in the CNS but not in the gut (19, 47–50). Previous immunohistochemical studies have reported putative DSK-expressing cells in the larval CNS (47, 49). However, since the specificity of the DSK antibodies has not been validated with null mutants in the former studies, it has been a concern that the reported DSK-expressing cells could include non-DSK cells that express the other neuropeptides sharing the C-terminal RFa motif with DSK (51). Consistent with this concern, we found that an antibody against crustacean FLRFa, an FMRFa-like neuropeptide with the C-terminal RFa motif, gives rise to a comparable staining pattern to that of the previously reported DSK antibodies, visualizing cells designated as insulin-producing cells (IPCs; referred to as SP3 in Nichols and Lim, (1996) (47)), MP1, SP1, SP2, Sv, SE2, Tv1-3, T2dm, and A8 in the larval CNS (Fig. 2A) (47, 49).

To identify bona fide DSK-expressing cells responsible for regulating nociception, we performed anti-FLRFa staining in multiple dsk null alleles and found that the staining signals persist in all but two pairs of neurons, MP1 and Sv, in the mutant CNS (Fig. 2B-D and S2A-D), suggesting that these two pairs of neurons are the only neurons in the larval CNS that express DSK. The genomic rescue fragment that rescued the thermal hypersensitivity of dsk mutants (Fig. 1B) also restored the anti-FLRFa signals in MP1 and Sv in a dsk mutant background (Fig. 2E), suggesting that the dsk gene is responsible for the anti-FLRFa signals in these neurons as well as the thermal nociceptive responses of larvae. This expression pattern was further corroborated by the transgenic reporter line DSK-GAL4 and the 2A-GAL4 knock-in reporter line DSK-2A-GAL4 (52), both of which we found were expressed only in MP1 and Sv neurons among the anti-FLRFa-positive neurons (Fig. 2A, C, and F; Fig. S2B-F). We also found that anti-FLRFa, DSK-GAL4, and DSK-2A-GAL4 do not visualize larval peripheral neurons including C4da nociceptors (Fig. 2A and F; Fig. S2G). Taken together, these results identified two sets of brain neurons, MP1 and Sv, as the DSK-expressing cells that are involved in regulating nociception in larvae.

We sought the potential target cells of DSK signaling for regulating nociception. Since DSK receptor mutants were thermally hypersensitive consistently to dsk mutants (Fig. 1B, D, and F), neurons in the larval nociceptive circuit that express DSK receptors were promising candidates. To visualize the cells expressing DSK receptors, we generated T2A-GAL4 knock-ins in CCKLR genes. Both CCKLR-17D1-T2A-GAL4 and CCKLR-17D3-T2A-GAL4 were widely expressed in the larval CNS, predominantly in neuronal cells (Fig. 3A and B; Fig. S3A-D). By performing double-labeling experiments, we found that CCKLR-17D1-T2A-GAL4 and CCKLR-17D3-T2A-GAL4 are not expressed in the nociceptors at the periphery (Fig. 3C and D; Fig. S3E and F), or in nociceptive interneurons including Basin1-4 (53), A08n (36, 41), and DnB neurons (35) in the larval VNC (Fig. S3G-L). However, we found that they are expressed in Goro neurons (Fig. 3E and F), which are the fourth-order nociceptive interneurons located in the larval VNC (53).

To test whether the DSK receptors in Goro neurons are functionally important for regulating nociception, we performed RNAi and rescue experiments using R69E06-GAL4 that marks Goro neurons (53). RNAi knockdown of CCKLR-17D1, but not CCKLR-17D3, in R69E06-GAL4 neurons induced thermal hypersensitivity (Fig. 4A and Fig. S4A). R69E06-GAL4 is also expressed in multiple neurons in the larval brain other than Goro neurons in the VNC (53). However, the thermal hypersensitivity was not observed when the expression of CCKLR-17D1 RNAi was excluded from Goro neurons by tsh-GAL80, pointing to the requirement of CCKLR-17D1 in Goro neurons (Fig. S4B-D). Consistent with these RNAi results, expressing wild-type CCKLR-17D1, but not CCKLR-17D3, with R69E06-GAL4 rescued the hypersensitivity of the respective mutants (Fig. 4B and C). Furthermore, the morphology of Goro neurons was not affected by CCKLR-17D1 knockdown (Fig. S4E). Therefore, we conclude that CCKLR-17D1 functions in Goro neurons to negatively regulate thermal nociception, but the function of CCKLR-17D3 in nociceptive regulations resides elsewhere.

Activation of Goro neurons elicits nocifensive rolling in larvae (53). Hence, if CCKLR-17D1 functions in Goro neurons to negatively regulate nociception, the lack of CCKLR-17D1 should sensitize these neurons to noxious heat. We directly addressed this hypothesis by using a Ca²⁺ imaging technique we had previously developed (32, 35), whereby we monitored GCaMP6m signals in Goro neurons while locally applying thermal ramp stimuli to the larval body wall. In our thermal ramp stimulation protocol, the temperature around the larval CNS hardly reached 28 °C during the heat ramp stimulations (27.0 ± 0.2 °C at the peak, n = 12), suggesting that the larval nociceptive system in the CNS that functions as an internal sensor for fast temperature increase (54) was unlikely to be activated.

Goro neurons in CCKLR-17D1^Δ1 mutants showed a significantly steeper GCaMP6m signal increase from 40 to 50 °C in comparison with the wild-type controls (Fig. 5A and B, Movie S1 and S2). Since the baseline fluorescence levels of GCaMP6m were not significantly different in the wild-type and CCKLR-17D1^Δ1 mutants (Fig. 5C), these data demonstrate that Goro neurons in CCKLR-17D1^Δ1 mutants are specifically sensitized to a noxious range of heat. Suppressing CCKLR-17D1 by RNAi in Goro neurons also induced significantly sensitized responses of Goro to noxious temperatures of 44–49 °C (Fig. 5D-F, Movie S3 and S4). In contrast, Goro neurons in CCKLR-17D3^Δ1 mutants exhibited GCaMP6m signals that were mildly elevated but largely parallel compared with that of the controls (Fig. S5, Movie S5 and S6), providing further evidence for the major functioning of CCKLR-17D3 in nociceptive regulation outside Goro neurons. Considered together, these data demonstrate that CCKLR-17D1 functions to negatively regulate the activity of Goro neurons, thereby attenuating behavioral nociceptive responses.

If CCKLR-17D1 is involved in regulating the activity of Goro neurons, how can DSK be conveyed from the brain to the VNC? We noticed that some of the DSK-GAL4 positive brain neurons sent descending neural processes to the VNC (Fig. 6A), which were all anti-FLRFa positive (Fig. 6B). Furthermore, the anti-FLRFa signals in the descending projections were completely absent in the dsk mutants (Fig. 6C), suggesting that these descending projections likely originated from the DSK-expressing brain cells, namely MP1 and/or Sv. In analyzing DSK-2A-GAL4, we found that MP1 neurons in fact sent the descending projections to the VNC (Fig. 6D). To further understand the projection patterns of MP1 and Sv neurons in detail, we performed single-cell labeling using an FLP-out technique and revealed that MP1 neurons sent descending projections contralaterally to the VNC (Fig. 6E), while Sv neurons projected within the brain (Fig. 6F). In comparison with the longitudinal processes to the VNC, MP1 neural processes in the brain possessed few anti-FLRFa positive puncta, which represent DSK in these neurons (Fig. 6G). Furthermore, when the somatodendritic marker UAS-Denmark (55) and the synaptic vesicle marker UAS-syt::eGFP were expressed in MP1 neurons, Denmark preferentially localized in the neural processes within the brain while syt::eGFP strongly accumulated in the processes descending to the VNC (Fig. 6H). Collectively, these data demonstrate that MP1 neurons project DSK-positive descending axons to the VNC from the brain.

Next, we investigated whether some of these DSK-expressing neurons are responsive to noxious thermal stimuli. In order to examine the responsiveness of multiple neurons in intact larvae, we took the snapshot approach using CaMPARI, a genetically-encoded Ca²⁺ sensor whose fluorescence irreversibly changes from the green to the red region of the spectrum in response to irradiation with 405 nm UV light in a Ca²⁺ dependent manner, and is thus useful to monitor the activities of multiple neurons simultaneously in intact animals (56). When the larvae expressing CaMPARI2 (an improved version of CaMPARI (57)) in Goro neurons were stimulated by local thermal ramp stimuli under a 405 nm UV light, a significantly increased CaMPARI2 photoconversion was detected compared to the control group that had only been irradiated with 405nm UV (Fig. 7A and B), confirming that by using CaMPARI2 in our experimental setup we managed to successfully capture the increased activity of Goro neurons in response to noxious heat in intact larvae. We then tested the responsiveness of DSK-2A-GAL4 neurons using the same stimulation and photoconversion protocol and found that none of the MP1 or Sv neurons, nor IPCs showed increased CaMPARI2 photoconversion in response to local thermal ramp stimuli (Fig. 7A, C-E). Interestingly, MP1 neurons exhibited high CaMPARI2 photoconversion regardless of the presence of local heat ramp stimuli, which was even comparable to that observed in Goro neurons activated by noxious heat stimuli (Fig. 7A and C). These data suggest that none of the DSK-2A-GAL4 neurons are clearly responsive to noxious heat, but the markedly high CaMPARI2 photoconversion observed in MP1 neurons raises the possibility that these neurons may be active regardless of the presence of noxious heat stimuli.

To gain more insights into the connectivity between DSK-expressing brain neurons and Goro neurons, we further examined the anatomical relationship between MP1 axons and Goro neurons in the VNC. Performing double-labeling experiments, we found that MP1 axons with anti-FLRFa signals (representing DSK in MP1 axons) partially overlapped with Goro neurites in the thoracic segments of the larval VNC (Fig. 8A). GFP reconstitution was reproducibly detected between MP1 and Goro neurons using the CD4-GRASP (GFP Reconstitution Across Synaptic Partners) system (58, 59), thus confirming that MP1 axons and Goro neurons are indeed in close proximity (Fig. 8B). Since the CD4-GRASP is known to detect general cell-cell contacts (60, 61), we further investigated whether MP1 and Goro neurons are synaptically connected by using the nSyb-GRASP technique, which specifically enables synapse detection by localizing one of the split-GFP fragments in presynaptic sites (62). However, no signals of reconstituted GFP were detected between MP1 and Goro with nSyb-GRASP, although we found proximate localizations of nSyb-GFP_1-10 in the MP1 axons with the Goro neurites expressing CD4::GFP₁₁ (Fig. 8C). We also employed the trans-Tango system (63) as another tool to detect synaptic connectivity. However, when the trans-Tango ligand was expressed by DSK-2A-GAL4, synaptic connectivity between MP1 and Goro was not indicated since the postsynaptic activation of trans-Tango signals was not observed in Goro neurons, although some trans-Tango-positive neurons were observed around Goro neurons (Fig. 8D).

Although we could not find positive evidence for synaptic connectivity between MP1 axons and Goro neurons, we further tested the hypothesis that DSK-expressing neurons in the brain may have functional connectivity to Goro neurons. To achieve this, we first attempted artificial activation experiments of DSK-2A-GAL4 neurons using UAS-dTRPA1 to determine whether activating DSK-expressing neurons actually attenuates larval nociception. dTRPA1 is a cation channel gated by warm temperature (> 29 °C) that has been used as a tool to artificially activate neurons of interest in Drosophila (64). When the larvae expressing dTRPA1 in C4da nociceptors with ppk1.9-GAL4 were placed in a chamber at 35°C, dTRPA1-induced thermogenetic activation of C4da nociceptors caused nocifensive rolling responses within two seconds in the majority of animals, as reported previously (65) (Fig. 9A and B). In contrast, when the larvae expressing dTRPA1 simultaneously in ppk1.9-GAL4 and DSK-2A-GAL4 were placed in a chamber at 35°C, the additional activation of DSK-2A-GAL4 neurons to C4da nociceptors resulted in a markedly reduced percentage of larvae showing rolling responses within two seconds and in significantly lengthened latencies to exhibit a rolling response (Fig. 9A and B). None of the animals expressing dTRPA1 with DSK-2A-GAL4 alone showed a rolling response within 10 seconds (Fig. 9A).

The above data suggest that activation of DSK-2A-GAL4 neurons inhibits larval nociception. We then investigated whether activating DSK-2A-GAL4 neurons could attenuate the activity of Goro neurons, by combining the artificial activation experiments described above with CaMPARI2 snapshot imaging of Goro neurons. Indeed, CaMPARI2 photoconversion in Goro neurons induced by the thermogenetic activations of C4da nociceptors was significantly decreased by 35% with the simultaneous activation of DSK-2A-GAL4 neurons (Fig. 9C and D). Given that MP1 neurons were the only cells that were labeled by DSK-2A-GAL4 with 100% reproducibility, while the expression of DSK-2A-GAL4 in IPCs and Sv neurons was minor and stochastic (Fig. 2F), the observed inhibition of behavioral nociception and Goro neurons caused by activating DSK-2A-GAL4 neurons is mostly attributable to the activation of MP1 neurons. Thus, these data support the functional connectivity between DSKergic MP1 neurons in the brain and Goro neurons.

DSK has been implicated in multiple physiological and developmental processes in Drosophila (19, 21–23). 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 (45, 66), 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 (Fig. S4E). Furthermore, the simultaneous thermogenetic activations of DSK-2A-GAL4 neurons and C4da nociceptors inhibited larval nociception and the activity of Goro neurons (Fig. 9). 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 our observation that CCKLR-17D1 knockdown in Goro produced the thermal hypersensitivity and exaggerated Ca²⁺ responses (Fig 4 and 5). 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.

A previous study has reported the expression of DSK in a population of larval IPCs and its functions in responding to starving conditions (49). However, we observed that anti-FLRFa signals in IPCs persisted in dsk null mutants and that anti-FLRFa signals in IPCs hardly overlap with either DSK-GAL4 or DSK-2A-GAL4 expressions (Fig. 2 and S2). Although we used a different antibody from that in the study mentioned above, the number of IPCs visualized by anti-FLRFa was comparable to that of cells visualized by anti-DSK (Fig. S2) (49). Regarding the functions of IPCs in nociception, Im et al. showed that silencing dilp2-GAL4 positive IPCs has no significant effect on the baseline nociceptive responses (67). In our CaMPARI2 imaging, IPCs marked by DSK-2A-GAL4 showed very low baseline activity and no responsiveness to noxious heat (Fig. 7). 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.

Although our 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. Our 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.

Second, how this DSKergic system is activated to regulate nociception in vivo needs further investigation. We observed very high CaMPARI2 photoconversion in MP1 neurons without noxious heat stimuli, which did not further increase in response to noxious heat application (Fig. 7). 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 our data showing that dsk and CCLR-17D1 mutations, as well as CCLR-17D1 RNAi in Goro neurons, all caused thermal hypersensitivity under normal conditions (Fig. 1 and 4). 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.

Third, the transmission mechanisms of DSK from the brain to Goro neurons need further clarification. Our nSyb-GRASP and trans-Tango experiments consistently showed negative results, indicating no synaptic connectivity between MP1 descending axons and Goro neurons (Fig. 8). 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 (68). Many of the CCKLR-expressing neurons in the VNC are distantly located from the descending MP1 axons (Fig. 3 and 6). 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.

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 (Fig. 1D and F), our RNAi and rescue experiments failed to locate the function of CCKLR-17D3 to Goro neurons (Fig. 4A and C). Our GCaMP Ca²⁺ imaging also revealed that Goro neurons lacking CCKLR-17D3 showed modestly sensitized responses to noxious heat (Fig. S5). These results suggest the major functioning of CCKLR-17D3 in Goro-independent nociceptive pathways. We also observed that DSK receptor mutants exhibited more severe thermal phenotypes than dsk mutants (Fig. 1), 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.

The CCK system is thought to be one of the most ancient neuropeptide systems, suggested to have multiple common physiological functions across taxa (16–20). In this study, we demonstrate 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.

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 (10–14, 69–73). 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.

Non-mammalian model systems have been increasingly recognized as powerful tools to identify novel pain-related molecular pathways (2, 74–77). 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 (2, 78). Descending nociceptive control mechanisms are crucial for central pain modulation and have been implicated in the development of chronic pain states in humans (5, 6). 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.