Mechanical forces are detected and processed by the somatosensory system. Mechanosensation encompasses the distinct submodalities of touch, proprioception, and mechanical nociception. Touch plays an important discriminative role and contributes to social interactions (Abraira and Ginty, 2013; McGlone et al., 2014). Nociception reports incipient or potential tissue damage. It triggers protective behaviours and can give rise to pain sensations (Basbaum et al., 2009). Thus, physically similar signals can carry fundamentally different physiological information, depending on stimulus intensity. Whereas innocuous touch sensations rely on low-threshold mechanosensory neurons, noxious mechanical stimuli activate high-threshold mechanosensory neurons, i.e. nociceptors. While mechanisms to differentiate these mechanosensory submodalities are essential for survival, little is known how this is achieved at cellular and molecular levels.

The activity of nociceptors can be increased through sensitization, e.g. upon inflammation, and decreased through antinociceptive processes, leading to pain relief. In both cases, G protein-coupled receptors (GPCRs) play an important modulatory role. Receptors that couple to heterotrimeric G_q/11 or G_s proteins, like the prostaglandin EP₂ receptor, increase the excitability of nociceptors by activating phospholipase C and adenylyl cyclase pathways, respectively. In contrast, G_i/o-coupled receptors, which are gated by soluble ligands like morphine and endogenous opioid neuropeptides generally inhibit nociceptor signalling. In mammalian nociceptors, cell surface receptors that couple to G_i/o proteins are located at presynaptic sites in the dorsal horn of the spinal cord, where they reduce glutamate release, at somata in dorsal root ganglia (DRG), and at peripheral processes, where they suppress receptor potential generation (Yudin and Rohacs, 2018).

Research on mechanosensation has focussed mainly on receptors that transduce mechanical force into electrical current, and the function of such mechanosensing ion channels is currently the subject of detailed investigations. In contrast, evidence for mechano-metabotropic signal transfer and compelling models of force conversion into an intracellular second messenger response are limited, despite the vital role of metabotropic modulation in all corners of physiology (Mederos y Schnitzler et al., 2008; Hoffman et al., 2011). Adhesion-type GPCRs (aGPCRs), a large molecule family with over 30 members in humans, operate in diverse physiological settings. Correspondingly, these receptors are associated with diverse human diseases, such as developmental disorders, defects of the nervous system, allergies and cancer (Hamann et al., 2015; Scholz et al., 2016). In contrast to most members of the GPCR phylum, aGPCRs are not activated by soluble ligands. Instead, aGPCRs interact with partner molecules tethered to membranes or fixed to the extracellular matrix via their long, adhesive N-terminal domain. This arrangement positions aGPCRs as metabotropic mechanosensors, which translate a relative displacement of the receptor-bearing cell into an intracellular second messenger signal (Langenhan et al., 2016).

CIRL (ADGRL/Latrophilin, Lphn), one of the oldest members of the aGPCR family, is expressed in the neuronal dendrites and cilia of Drosophila larval chordotonal organs (ChOs), mechanosensory structures that respond to gentle touch, sound, and proprioceptive input (Kernan, 2007; Scholz et al., 2015). Here, mechanical stimulation of CIRL triggers a conformational change of the protein and activation via its tethered agonist (Stachel) (Liebscher et al., 2014; Stoveken et al., 2015). Signalling by the activated receptor reduces intracellular cAMP levels, which in turn sensitizes ChO neurons and potentiates the mechanically-evoked receptor potential (Scholz et al., 2017). In the current study, we show that CIRL also adjusts the activity of nociceptors, which respond to strong mechanical stimuli. Here, too, its function is consistent with G_i/o coupling. However, in contrast to touch-sensitive ChO neurons, nociceptors are sensitized by elevated cAMP concentrations and toned down by an antinociceptive and Stachel-independent action of CIRL. As a result of curtailing cAMP production, CIRL modulates neural processing of noxious harsh and innocuous gentle touch bidirectionally. This elegant signalling logic serves signal discrimination by helping to separate mechanosensory submodalities.

Drosophila larvae possess two major types of peripheral sensory neurons. Monociliated type one neurons, including ChOs and external sensory organs, and type two neurons with multiple dendritic (md) projections, classified as tracheal dendrite (md-td), bipolar dendrite (md-bd), and dendritic arborization (md-da). Md-da neurons are further subdivided into four classes: C1da-C4da (Ghysen et al., 1986; Bodmer and Jan, 1987; Grueber et al., 2002). Previous work demonstrated prominent expression of Cirl in ChOs, where it modulates sensory processing of innocuous mechanical stimuli (Scholz et al., 2015; Scholz et al., 2017). In addition, Cirl transcription was also noted in md neurons. Motivated by this observation, we now turned our attention to C4da neurons: polymodal nociceptors, which respond to noxious temperatures, intense light and, importantly, harsh mechanical touch (Tracey et al., 2003; Xiang et al., 2010; Zhong et al., 2010; Zhong et al., 2012; Kim et al., 2012; Kim and Johnson, 2014). Degenerin/epithelial sodium channels (DEG/ENaCs) contribute to nociceptive mechanotransduction in invertebrates and mammals (Price et al., 2001; Chatzigeorgiou et al., 2010; Zhong et al., 2010; Gorczyca et al., 2014; Guo et al., 2014; Mauthner et al., 2014). Placing a fluorescent reporter under transcriptional control of the DEG/ENaC subunit pickpocket (ppk) reliably marks C4da neurons (Grueber et al., 2003; Han et al., 2011). We therefore combined a direct GFP-ppk promoter fusion (ppk-CD4::tdGFP) with binary expression of the red photoprotein Tomato by a Cirl promoter element in the UAS/GAL4 system (dCirlp^GAL4 >UAS-CD4::tdTomato) (Brand and Perrimon, 1993; Scholz et al., 2015). This setting revealed Cirl transcription in both ppk-negative ChOs and ppk-positive C4da neurons (Figure 1). Thus, Cirl is expressed in different sensory neurons, including proprioceptors and nociceptors.

Figure 1

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Given CIRL’s role in mechanosensation, we next tested for a specific contribution of the aGPCR to mechanical nociception. Drosophila larvae display a stereotyped response to noxious mechanical insult. Importantly, this innate nocifensive behaviour, characterized by a ‘corkscrew’ body roll, can be quantified and is distinct from the animals’ reaction to innocuous touch (Figure 2A; Video 1; Tracey et al., 2003; Zhong et al., 2010). Mechanical stimulation with a 40 mN von Frey filament triggered nocifensive behaviour in 53% of control larvae. In contrast, Cirl null mutants (dCirl^KO) showed significantly increased nocifensive behaviour and responded in 75% of the trials (Figure 2B; Table 1). Knocking-down Cirl levels specifically in C4da neurons via RNA-interference (RNAi; ppk-GAL4 >UAS-dCirl^RNAi) delivered a mutant phenocopy and re-expressing Cirl in nociceptors rescued the mutant phenotype. Notably, Cirl overexpression had the opposite effect resulting in diminished nocifensive responses (Figure 2B; Table 1). These results show that CIRL curtails mechanical nociception by carrying out a cell-autonomous, dose-dependent function in C4da neurons.

Figure 2

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Video 1

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CIRL sensitizes proprioceptive ChO neurons by translating extracellular mechanical stimulation into a drop of intracellular cAMP. Lower cAMP levels, in turn, enhance the mechanically-evoked receptor potential of ChOs through a yet unresolved molecular mechanism (Scholz et al., 2017). Intriguingly, our behavioural data point towards CIRL exerting the opposite influence on nociceptive C4da neurons, whose sensitivity to mechanical stimulation is decreased by CIRL and increased in the absence of the aGPCR. A possible explanation for the antinociceptive action of CIRL is that the aGPCR also reduces cAMP in nociceptors, but that here the second messenger cascade acts on different molecular targets, i.e. specific ion channels, to produce an inverted effect. According to this model, low cAMP levels would dampen nociceptor activity. Next, we therefore asked whether increasing cAMP in C4da neurons (as may occur in dCirl^KO) promotes nocifensive behaviour. Because chronic changes of cAMP levels strongly alter neuronal development (Griffith and Budnik, 2006) we selected an optogenetic approach to trigger a rapid, nociceptor-specific increase of cAMP. The bacterial photoactivated adenylyl cyclase bPAC can be genetically expressed in selected Drosophila neurons to evoke cAMP production upon blue light stimulation (Figure 3A; Stierl et al., 2011; Scholz et al., 2017). Indeed, driving bPAC in C4da neurons (ppk-GAL4 >UAS-bPAC) led to increased nocifensive behaviour during light exposure, whereas control animals showed no light-induced effects (Figure 3B; Table 2). Notably, bPAC expression also produced some irregular behaviour in the absence of photostimulation (‘spontaneous bending’; Figure 3B), which is likely a result of the enzyme’s residual dark activity (Stierl et al., 2011). Nocifensive responses were further enhanced by bPAC in dCirl^KO larvae (dCirl^KO ppk-GAL4 >UAS-bPAC), in line with increased cAMP levels in mutant C4da neurons. Independent support for this conclusion was received by analysing larvae carrying mutant alleles of the phosphodiesterase (PDE) dunce (Davis and Kiger, 1981). Here, pronounced nocifensive behaviour accompanies chronically elevated cAMP concentrations (Figure 3C; Table 1).

Figure 3

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To further substantiate that CIRL influences nociception by acting on cAMP-dependent signalling, we inhibited the endogenous adenylyl cyclase activity by pharmacological means. Consistent with nociceptor sensitization by cAMP, dietary supplementation with the adenylyl cyclase inhibitors SQ22536 or DDA (2',3'-dideoxyadenosine) significantly decreased nocifensive behaviour of Drosophila larvae. Importantly, this treatment produced the same responses of control and dCirl^KO animals to von Frey filament stimulation (Figure 3D; Table 1). This result shows that CIRL acts in the same pathway as cAMP production by the adenylyl cyclase and supports the notion that the aGPCR exerts its antinociceptive effect by lowering cAMP levels through G_i/o -mediated inhibition of adenylyl cyclase activity.

Next, we used calcium imaging to directly test whether CIRL modulates the mechanically-evoked activity of nociceptors. To this end, we monitored calcium signals in C4da neurons labelled with GCaMP6m (Chen et al., 2013) during von Frey filament stimulation as previously reported (Hu et al., 2017; Figure 4—figure supplement 1). Consistent with the behavioural data describing a CIRL-mediated downregulation of nociceptor function, dCirl^RNAi significantly enhanced calcium responses to noxious mechanical stimulation (Figure 4A,B; Table 1). In principle, CIRL could influence the activity of C4da neurons by modulating cellular excitability or by modulating the mechanotransduction process, as is the case in touch-sensitive ChO neurons (Scholz et al., 2017). To differentiate between these possibilities, we again chose an optogenetic strategy and circumvented mechanotransduction by stimulating the nociceptors with light. Photostimulation of C4da neurons via the optimized Channelrhodopsin-2 (ChR2) variant ChR2^XXM (Nagel et al., 2003; Scholz et al., 2017) triggered nocifensive behaviour, consistent with previous work (Hwang et al., 2007). Notably, dCirl^KO larvae (dCirl^KO ppk-GAL4 >UAS-chop2^XXM) responded more strongly than controls (ppk-GAL4 >UAS-chop2^XXM) over a range of different blue light intensities (475 nm; Figure 4C; Table 3). This demonstrates that CIRL decreases the excitability of mechanical nociceptors, contrasting its role in touch-sensitive neurons where the aGPCR specifically enhances mechanotransduction (Scholz et al., 2017).

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We further asked whether signalling to different subcellular targets may correlate with different activation mechanisms of CIRL in these two types of mechanosensory neurons. Several studies have shown that aGPCR activation can occur by means of an intramolecular tethered agonist, termed Stachel (stalk) (Liebscher et al., 2014; Monk et al., 2015; Stoveken et al., 2015, for Stachel-independent signalling see Kishore et al., 2016; Salzman et al., 2017; Sando et al., 2019). This linker sequence of approximately 20 amino acids begins at the extracellular GPCR proteolysis site (GPS) and connects the GAIN (GPCR autoproteolysis inducing) domain to the 7-transmembrane unit (Figure 4D). In order to test for a role of the Stachel sequence in activating CIRL in mechanical nociceptors, we used two established mutants. Whereas the dCirl^T>A allele carries a mutation at the +1 position of the GPS within the Stachel sequence, the −2 mutation in the dCirl^H>A allele leaves the Stachel intact but reduces protein expression (Figure 4D; Scholz et al., 2017). Our analysis of nocifensive behaviour in these mutants reported normal responses for the T > A allele and a Cirl^KO phenocopy for the H > A allele (Figure 4E, Table 1). Thus, C4da neurons appear to be sensitive to reduced CIRL expression, which is consistent with very low protein levels in wild-type animals (close to the detection threshold; Figure 4—figure supplement 2). An intact tethered agonist, in turn, is dispensable for CIRL function in mechanical nociceptors, contrasting the situation in touch-sensitive ChO neurons, where mutating the Stachel sequence disrupts receptor function (Scholz et al., 2017). Taken together, these observations indicate that the activation mechanism of CIRL differs for the two mechanosensory submodalities.

Having established that CIRL downregulates nociceptor function under physiological conditions, we sought to investigate a pathological setting. The chemotherapeutic agent paclitaxel, employed to treat solid tumours such as ovarian or breast cancer, causes dose-limiting peripheral neuropathy in patients. Similarly, feeding Drosophila larvae paclitaxel-supplemented food induces axonal injury and degeneration of C4da neurons (Bhattacharya et al., 2012). Next, we therefore examined nocifensive behaviour in the context of this established neuropathy model. Consistent with chemotherapy-induced allodynia in humans, paclitaxel strongly enhanced nocifensive responses of control larvae (Figure 5A). We observed a comparable effect in dCirl^KO animals. Overexpressing Cirl, in turn, reverted the paclitaxel-induced sensitization of C4da neurons (Figure 5A; Table 1). Thus, CIRL tones down nociceptors in both physiological and neuropathic hyperexcitable states.

Figure 5

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Paclitaxel administration causes structural damage to C4da neurons (Bhattacharya et al., 2012). Following paclitaxel treatment (10 µM), we observed dendrite loss in the wild-type background and in nociceptors overexpressing Cirl (Figure 5B,C; Table 1). In fact, elevated Cirl expression itself reduced the dendritic complexity of C4da neurons. Thus, increasing CIRL protein copy number counteracts the neuropathic hyperexcitability of mechanical nociceptors independently of neuropathy-associated morphological defects. Consistent with the interpretation that modulation of nociceptor physiology by CIRL is not tightly coupled to morphological changes, dCirl^KO C4da neurons displayed only subtle structural alterations (Figure 5D).

Considering the evolutionary conservation of signalling pathways for nociception (Im and Galko, 2012), we next turned to a rodent model of traumatic neuropathic pain: unilateral chronic constriction injury (CCI) of the sciatic nerve (Reinhold et al., 2019). This model resembles paclitaxel-induced neuropathy in the development of thermal hypersensitivity and mechanical allodynia (Sisignano et al., 2016), i.e. a noxious reaction to innocuous stimuli like touch, reaching a maximum after one week (Figure 6A; Table 1). There are three CIRL proteins in mammals (CIRL1-3 also known as ADGRL1-3 or Lphn1-3) (Langenhan et al., 2016). According to RNA sequencing data from mouse DRG neurons, Cirl1 and Cirl3 are expressed in nociceptors (Thakur et al., 2014). We therefore investigated Cirl1 and Cirl3 expression in subpopulations of DRG neurons via in situ hybridization in the neuropathic context. Interestingly, Cirl1 mRNA probes described significantly reduced transcript levels in isolectin-B4 (IB4)-positive, non-peptidergic nociceptors one week after CCI (Figure 6B,D,E; Table 1). We observed a similar, though statistically insignificant trend in peptidergic nociceptors identified by calcitonin gene-related peptide (CGRP) staining and for Cirl3 probes (Figure 6B-E; Table 1). Notably, CCI appeared to neither affect Cirl1 nor Cirl3 gene expression in non-nociceptive, large myelinated neurons marked by neurofilament protein NF200. These correlative results are consistent with the Drosophila data linking low Cirl expression levels to nociceptor sensitization. It remains to be determined whether expression of Drosophila Cirl can be regulated physiologically to adapt or tune nociceptor sensitivity and whether activation of mammalian CIRL, in turn, can provide analgesia.

Figure 6

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The sensations of touch and mechanical pain represent distinct mechanosensory submodalities, which are separated at the initial sites of mechanotransduction. Despite their important roles in health and disease, an understanding of how these mechanistically different transduction processes are carried out at the molecular level is only just beginning to emerge (Delmas et al., 2011; Julius, 2013; Murthy et al., 2017; Szczot et al., 2017; Zhang et al., 2019). Similar to mammalian DRG neurons, Drosophila nociceptors are modulated by GPCRs (Hu et al., 2017; Kaneko et al., 2017; Herman et al., 2018) and can be negatively regulated by G_i/o signalling (Christianson et al., 2016; Honjo et al., 2016). In the present study, we provide evidence that CIRL, an evolutionarily conserved aGPCR, reduces nociceptor responses to mechanical insult in Drosophila larvae. This modulation operates in the opposite direction to the sensitization of touch sensitive neurons by CIRL (Figure 7; Scholz et al., 2015; Scholz et al., 2017). In both types of mechanosensors, these effects are connected to CIRL-dependent decreases of cAMP levels. The opposing cell physiological outcomes, in turn, likely arise from specific adjustments of different effector proteins through cAMP-signalling. Candidate effectors are mechanotransduction channels and ion channels, which are mechanically-insensitive but influence the rheobase, i.e. the threshold current of the sensory neuron (Boiko et al., 2017).

Figure 7

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The transient receptor potential (TRP) channel subunits NOMPC (no receptor potential, TRPN), NAN (nanchung, TRPV), and IAV (inactive, TRPV) govern mechanosensation by larval ChO neurons (Effertz et al., 2012; Lehnert et al., 2013; Zhang et al., 2013). The mechanically gated ion channel Piezo, the DEG/ENaC subunit Pickpocket, and the TRPN channel Painless, on the other hand, are required for mechanical nociception in Drosophila (Tracey et al., 2003; Zhong et al., 2010; Kim et al., 2012; Gorczyca et al., 2014; Guo et al., 2014; Mauthner et al., 2014). It is therefore conceivable that the receptor potential generated by these different mechanotransducers may be modulated in opposite directions, i.e. decreased in ChO neurons and increased in nociceptors, by cAMP/PKA (protein kinase A)-dependent channel phosphorylation. Matching our results in Drosophila, enhanced nociceptor activity in mammals has been linked to elevated cAMP levels. For example, mechanical hyperalgesia during inflammation involves cAMP-modulated HCN channels and sensitization of mammalian Piezo2 via PKA and protein kinase C (PKC)-based signalling (Emery et al., 2011; Dubin et al., 2012). Conversely, G_i/o-coupled receptors, such as opioid, somatostatin, and GABA_B receptors, counteract cAMP-dependent nociceptor sensitization (Yudin and Rohacs, 2018). In addition to this second messenger pathway, G_βγ subunits of G_i/o-coupled GPCRs can directly interact with ion channels. Thereby nociceptor signalling can be suppressed via activation of G protein regulated inwardly rectifying K⁺ channels (GIRK) or by inhibition of voltage-gated Ca²⁺ channels (Logothetis et al., 1987; Marker et al., 2005; Bourinet et al., 2014). Recent work has identified that CIRL2 and CIRL3 promote synapse formation in the mouse hippocampus (Sando et al., 2019). While Drosophila CIRL may also shape synaptic connectivity, our results indicate that CIRL modulates the mechanically-evoked activity of nociceptors independently of such an additional function.

The present findings show that CIRL decreases the activity of C4da neurons independently of mechanotransduction and that the aGPCR feeds into the same pathway as the adenylyl cyclase. Taken together, this strongly suggests that G_i/o coupling by CIRL regulates cAMP-dependent modulation of ion channels, which control nociceptor excitability. Work in cell culture has put forward a model in which Stachel-dependent and -independent aGPCR activation triggers different downstream signalling pathways (Kishore et al., 2016; Salzman et al., 2017). Here, we provide evidence in support of such a dual activation model in a physiological setting. The dispensability of an intact Stachel sequence in mechanical nociceptors and its necessity in touch-sensitive neurons argues for alternative activation modes of CIRL in these two types of mechanosensory neurons. This raises the intriguing possibility that such functional differentiation may be connected to specific downstream effects, e.g. Stachel-dependent, phasic modulation of mechanotransduction in ChO neurons versus Stachel-independent, tonic modulation of nociceptor excitability (Figure 7).

Many genes display altered expression in DRG neurons in neuropathy (Lopes et al., 2017). For example, receptors and ion channels involved in sensitization are upregulated, whereas endogenous antinociceptive mechanisms, including opioid receptors and their peptides, are downregulated in certain neuropathy models (Herradon et al., 2008; Hervera et al., 2011). Thus, neuropathy not only enhances pro-nociceptive mechanisms but also decreases endogenous antinociceptive pathways. Our analysis of rodent DRGs indicates that neuropathy-induced allodynia correlates with reduced Cirl1 expression in IB4-positive non-peptidergic nociceptors (Fang et al., 2006), a class of neurons, which have been linked to mechanical inflammatory hypersensitivity (Pinto et al., 2019). It is therefore tempting to speculate that CIRL operates via a conserved antinociceptive mechanism in both invertebrate and mammalian nociceptors to reduce cAMP concentrations. Future work will have to test this hypothesis by examining a direct causal relation between CIRL activation and nociceptor attenuation in the mammalian peripheral nervous system and to explore whether metabotropic mechanosensing by CIRL is a possible target for analgesic therapy. Limited options for treating chronic pain have contributed to the current opioid epidemic (Skolnick and Volkow, 2016). Opioids are powerful analgesics but have severe side effects and lead to addiction mainly through activation of receptors in the central nervous system. There is thus a strong incentive to develop novel peripherally acting pain therapeutics.

The specificity theory, put forward more than 100 years ago (Sherrington, 1906), defines nociceptors as a functionally distinct subtype of nerve endings, which are specifically tuned to detect harmful, high-intensity stimuli. The results reported in the present study are consistent with this validated concept and identify a physiological mechanism, which contributes to the functional specialization. On the one hand, CIRL helps set the high activation threshold of mechanical nociceptors, while on the other hand, CIRL lowers the activation threshold of touch sensitive neurons (Figure 7). This bidirectional adjustment, attributable to cell-specific effects of cAMP, moves both submodalities further apart and sharpens the contrast of mechanosensory signals carrying different information.

The presented data are summarized in Tables 1-3.

1. 1. Abraira VE
   2. Ginty DD

   (2013) The sensory neurons of touch

   Neuron 79:618–639.

   https://doi.org/10.1016/j.neuron.2013.07.051

   • PubMed
   • Google Scholar
2. 1. Basbaum AI
   2. Bautista DM
   3. Scherrer G
   4. Julius D

   (2009) Cellular and molecular mechanisms of pain introduction

   Acute Versus Persistent Pain 14:1–22.

   https://doi.org/10.1016/j.cell.2009.09.028

   • Google Scholar
3. 1. Bhattacharya MR
   2. Gerdts J
   3. Naylor SA
   4. Royse EX
   5. Ebstein SY
   6. Sasaki Y
   7. Milbrandt J
   8. DiAntonio A

   (2012) A model of toxic neuropathy in Drosophila reveals a role for MORN4 in promoting axonal degeneration

   Journal of Neuroscience 32:5054–5061.

   https://doi.org/10.1523/JNEUROSCI.4951-11.2012

   • PubMed
   • Google Scholar
4. 1. Bodmer R
   2. Jan YN

   (1987) Morphological differentiation of the embryonic peripheral neurons in Drosophila

   Roux's Archives of Developmental Biology 196:69–77.

   https://doi.org/10.1007/BF00402027

   • Google Scholar
5. 1. Boiko N
   2. Medrano G
   3. Montano E
   4. Jiang N
   5. Williams CR
   6. Madungwe NB
   7. Bopassa JC
   8. Kim CC
   9. Parrish JZ
   10. Hargreaves KM
   11. Stockand JD
   12. Eaton BA

   (2017) TrpA1 activation in peripheral sensory neurons underlies the ionic basis of pain hypersensitivity in response to Vinca alkaloids

   PLOS ONE 12:e0186888.

   https://doi.org/10.1371/journal.pone.0186888

   • PubMed
   • Google Scholar
6. 1. Bourinet E
   2. Altier C
   3. Hildebrand ME
   4. Trang T
   5. Salter MW
   6. Zamponi GW

   (2014) Calcium-permeable ion channels in pain signaling

   Physiological Reviews 94:81–140.

   https://doi.org/10.1152/physrev.00023.2013

   • PubMed
   • Google Scholar
7. 1. Brand AH
   2. Perrimon N

   (1993)

   Targeted gene expression as a means of altering cell fates and generating dominant phenotypes

   Development 118:401–415.

   • PubMed
   • Google Scholar
8. 1. Chatzigeorgiou M
   2. Yoo S
   3. Watson JD
   4. Lee WH
   5. Spencer WC
   6. Kindt KS
   7. Hwang SW
   8. Miller DM
   9. Treinin M
   10. Driscoll M
   11. Schafer WR

   (2010) Specific roles for DEG/ENaC and TRP channels in touch and thermosensation in C. elegans nociceptors

   Nature Neuroscience 13:861–868.

   https://doi.org/10.1038/nn.2581

   • PubMed
   • Google Scholar
9. 1. Chen TW
   2. Wardill TJ
   3. Sun Y
   4. Pulver SR
   5. Renninger SL
   6. Baohan A
   7. Schreiter ER
   8. Kerr RA
   9. Orger MB
   10. Jayaraman V
   11. Looger LL
   12. Svoboda K
   13. Kim DS

   (2013) Ultrasensitive fluorescent proteins for imaging neuronal activity

   Nature 499:295–300.

   https://doi.org/10.1038/nature12354

   • PubMed
   • Google Scholar
10. Preprint

    1. Christianson MG
    2. Mauthner SE
    3. Tracey D

    (2016) A gunshot approach to identify mechanical nociception genes with a high throughput behavioral assay

    bioRxiv.

    https://doi.org/10.1101/077644

    • Google Scholar
11. 1. Davis RL
    2. Kiger JA

    (1981) Dunce mutants of Drosophila melanogaster: mutants defective in the cyclic AMP phosphodiesterase enzyme system

    The Journal of Cell Biology 90:101–107.

    https://doi.org/10.1083/jcb.90.1.101

    • PubMed
    • Google Scholar
12. 1. Delmas P
    2. Hao J
    3. Rodat-Despoix L

    (2011) Molecular mechanisms of mechanotransduction in mammalian sensory neurons

    Nature Reviews Neuroscience 12:139–153.

    https://doi.org/10.1038/nrn2993

    • PubMed
    • Google Scholar
13. 1. Dietzl G
    2. Chen D
    3. Schnorrer F
    4. Su KC
    5. Barinova Y
    6. Fellner M
    7. Gasser B
    8. Kinsey K
    9. Oppel S
    10. Scheiblauer S
    11. Couto A
    12. Marra V
    13. Keleman K
    14. Dickson BJ

    (2007) A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila

    Nature 448:151–156.

    https://doi.org/10.1038/nature05954

    • PubMed
    • Google Scholar
14. 1. Dixon WJ

    (1980) Efficient analysis of experimental observations

    Annual Review of Pharmacology and Toxicology 20:441–462.

    https://doi.org/10.1146/annurev.pa.20.040180.002301

    • PubMed
    • Google Scholar
15. 1. Dubin AE
    2. Schmidt M
    3. Mathur J
    4. Petrus MJ
    5. Xiao B
    6. Coste B
    7. Patapoutian A

    (2012) Inflammatory signals enhance piezo2-mediated mechanosensitive currents

    Cell Reports 2:511–517.

    https://doi.org/10.1016/j.celrep.2012.07.014

    • PubMed
    • Google Scholar
16. 1. Duffy JB

    (2002) GAL4 system in Drosophila: a fly geneticist's Swiss army knife

    Genesis 34:1–15.

    https://doi.org/10.1002/gene.10150

    • PubMed
    • Google Scholar
17. 1. Effertz T
    2. Nadrowski B
    3. Piepenbrock D
    4. Albert JT
    5. Göpfert MC

    (2012) Direct gating and mechanical integrity of Drosophila auditory transducers require TRPN1

    Nature Neuroscience 15:1198–1200.

    https://doi.org/10.1038/nn.3175

    • PubMed
    • Google Scholar
18. 1. Ehmann N
    2. van de Linde S
    3. Alon A
    4. Ljaschenko D
    5. Keung XZ
    6. Holm T
    7. Rings A
    8. DiAntonio A
    9. Hallermann S
    10. Ashery U
    11. Heckmann M
    12. Sauer M
    13. Kittel RJ

    (2014) Quantitative super-resolution imaging of bruchpilot distinguishes active zone states

    Nature Communications 5:4650.

    https://doi.org/10.1038/ncomms5650

    • PubMed
    • Google Scholar
19. 1. Emery EC
    2. Young GT
    3. Berrocoso EM
    4. Chen L
    5. McNaughton PA

    (2011) HCN2 ion channels play a central role in inflammatory and neuropathic pain

    Science 333:1462–1466.

    https://doi.org/10.1126/science.1206243

    • PubMed
    • Google Scholar
20. 1. Fang X
    2. Djouhri L
    3. McMullan S
    4. Berry C
    5. Waxman SG
    6. Okuse K
    7. Lawson SN

    (2006) Intense isolectin-B4 binding in rat dorsal root ganglion neurons distinguishes C-fiber nociceptors with broad action potentials and high Nav1.9 expression

    Journal of Neuroscience 26:7281–7292.

    https://doi.org/10.1523/JNEUROSCI.1072-06.2006

    • PubMed
    • Google Scholar
21. 1. Ghysen A
    2. Dambly-Chaudière C
    3. Aceves E
    4. Jan L-
    5. Jan Y-

    (1986) Sensory neurons and peripheral pathways in Drosophila embryos

    Roux's Archives of Developmental Biology 195:281–289.

    https://doi.org/10.1007/BF00376060

    • PubMed
    • Google Scholar
22. 1. Gorczyca DA
    2. Younger S
    3. Meltzer S
    4. Kim SE
    5. Cheng L
    6. Song W
    7. Lee HY
    8. Jan LY
    9. Jan YN

    (2014) Identification of Ppk26, a DEG/ENaC channel functioning with Ppk1 in a mutually dependent manner to guide locomotion behavior in Drosophila

    Cell Reports 9:1446–1458.

    https://doi.org/10.1016/j.celrep.2014.10.034

    • PubMed
    • Google Scholar
23. 1. Griffith LC
    2. Budnik V

    (2006) Plasticity and second messengers during synapse development

    International Review of Neurobiology 75:237–265.

    https://doi.org/10.1016/S0074-7742(06)75011-5

    • PubMed
    • Google Scholar
24. 1. Grueber WB
    2. Jan LY
    3. Jan YN

    (2002)

    Tiling of the Drosophila epidermis by multidendritic sensory neurons

    Development 129:2867–2878.

    • PubMed
    • Google Scholar
25. 1. Grueber WB
    2. Ye B
    3. Moore AW
    4. Jan LY
    5. Jan YN

    (2003) Dendrites of distinct classes of Drosophila sensory neurons show different capacities for homotypic repulsion

    Current Biology 13:618–626.

    https://doi.org/10.1016/S0960-9822(03)00207-0

    • PubMed
    • Google Scholar
26. 1. Guo Y
    2. Wang Y
    3. Wang Q
    4. Wang Z

    (2014) The role of PPK26 in Drosophila larval mechanical nociception

    Cell Reports 9:1183–1190.

    https://doi.org/10.1016/j.celrep.2014.10.020

    • PubMed
    • Google Scholar
27. 1. Hamann J
    2. Aust G
    3. Araç D
    4. Engel FB
    5. Formstone C
    6. Fredriksson R
    7. Hall RA
    8. Harty BL
    9. Kirchhoff C
    10. Knapp B
    11. Krishnan A
    12. Liebscher I
    13. Lin HH
    14. Martinelli DC
    15. Monk KR
    16. Peeters MC
    17. Piao X
    18. Prömel S
    19. Schöneberg T
    20. Schwartz TW
    21. Singer K
    22. Stacey M
    23. Ushkaryov YA
    24. Vallon M
    25. Wolfrum U
    26. Wright MW
    27. Xu L
    28. Langenhan T
    29. Schiöth HB

    (2015) International union of basic and clinical pharmacology. XCIV. adhesion G protein-coupled receptors

    Pharmacological Reviews 67:338–367.

    https://doi.org/10.1124/pr.114.009647

    • PubMed
    • Google Scholar
28. 1. Han C
    2. Jan LY
    3. Jan YN

    (2011) Enhancer-driven membrane markers for analysis of nonautonomous mechanisms reveal neuron-glia interactions in Drosophila

    PNAS 108:9673–9678.

    https://doi.org/10.1073/pnas.1106386108

    • PubMed
    • Google Scholar
29. 1. Herman JA
    2. Willits AB
    3. Bellemer A

    (2018) Gαq and phospholipase cβ signaling regulate nociceptor sensitivity in Drosophila melanogaster larvae

    PeerJ 6:e5632.

    https://doi.org/10.7717/peerj.5632

    • PubMed
    • Google Scholar
30. 1. Herradon G
    2. Ezquerra L
    3. Nguyen T
    4. Wang C
    5. Siso A
    6. Franklin B
    7. Dilorenzo L
    8. Rossenfeld J
    9. Silos-Santiago I
    10. Alguacil LF

    (2008) Noradrenergic and opioidergic alterations in neuropathy in different rat strains

    Neuroscience Letters 438:186–189.

    https://doi.org/10.1016/j.neulet.2008.03.095

    • PubMed
    • Google Scholar
31. 1. Hervera A
    2. Negrete R
    3. Leánez S
    4. Martín-Campos JM
    5. Pol O

    (2011) Peripheral effects of morphine and expression of μ-opioid receptors in the dorsal root ganglia during neuropathic pain: nitric oxide signaling

    Molecular Pain 7:8–10.

    https://doi.org/10.1186/1744-8069-7-25

    • PubMed
    • Google Scholar
32. 1. Hoffman BD
    2. Grashoff C
    3. Schwartz MA

    (2011) Dynamic molecular processes mediate cellular mechanotransduction

    Nature 475:316–323.

    https://doi.org/10.1038/nature10316

    • PubMed
    • Google Scholar
33. 1. Honjo K
    2. Mauthner SE
    3. Wang Y
    4. Skene JHP
    5. Tracey WD

    (2016) Nociceptor-Enriched genes required for normal thermal nociception

    Cell Reports 16:295–303.

    https://doi.org/10.1016/j.celrep.2016.06.003

    • PubMed
    • Google Scholar
34. 1. Hu C
    2. Petersen M
    3. Hoyer N
    4. Spitzweck B
    5. Tenedini F
    6. Wang D
    7. Gruschka A
    8. Burchardt LS
    9. Szpotowicz E
    10. Schweizer M
    11. Guntur AR
    12. Yang CH
    13. Soba P

    (2017) Sensory integration and neuromodulatory feedback facilitate Drosophila mechanonociceptive behavior

    Nature Neuroscience 20:1085–1095.

    https://doi.org/10.1038/nn.4580

    • PubMed
    • Google Scholar
35. 1. Hwang RY
    2. Zhong L
    3. Xu Y
    4. Johnson T
    5. Zhang F
    6. Deisseroth K
    7. Tracey WD

    (2007) Nociceptive neurons protect Drosophila larvae from parasitoid wasps

    Current Biology 17:2105–2116.

    https://doi.org/10.1016/j.cub.2007.11.029

    • PubMed
    • Google Scholar
36. 1. Im SH
    2. Galko MJ

    (2012) Pokes, sunburn, and hot sauce:Drosophila as an emerging model for the biology of nociception

    Developmental Dynamics 241:16–26.

    https://doi.org/10.1002/dvdy.22737

    • PubMed
    • Google Scholar
37. 1. Julius D

    (2013) TRP channels and pain

    Annual Review of Cell and Developmental Biology 29:355–384.

    https://doi.org/10.1146/annurev-cellbio-101011-155833

    • PubMed
    • Google Scholar
38. 1. Kaneko T
    2. Macara AM
    3. Li R
    4. Hu Y
    5. Iwasaki K
    6. Dunnings Z
    7. Firestone E
    8. Horvatic S
    9. Guntur A
    10. Shafer OT
    11. Yang CH
    12. Zhou J
    13. Ye B

    (2017) Serotonergic modulation enables Pathway-Specific plasticity in a developing sensory circuit in Drosophila

    Neuron 95:623–638.

    https://doi.org/10.1016/j.neuron.2017.06.034

    • PubMed
    • Google Scholar
39. 1. Kernan MJ

    (2007) Mechanotransduction and auditory transduction in Drosophila

    Pflügers Archiv - European Journal of Physiology 454:703–720.

    https://doi.org/10.1007/s00424-007-0263-x

    • Google Scholar
40. 1. Kim SE
    2. Coste B
    3. Chadha A
    4. Cook B
    5. Patapoutian A

    (2012) The role of Drosophila piezo in mechanical nociception

    Nature 483:209–212.

    https://doi.org/10.1038/nature10801

    • PubMed
    • Google Scholar
41. 1. Kim M-J
    2. Johnson WA

    (2014) ROS-mediated activation of Drosophila larval nociceptor neurons by UVC irradiation

    BMC Neuroscience 15:14.

    https://doi.org/10.1186/1471-2202-15-14

    • Google Scholar
42. 1. Kishore A
    2. Purcell RH
    3. Nassiri-Toosi Z
    4. Hall RA

    (2016) Stalk-dependent and Stalk-independent signaling by the adhesion G Protein-coupled receptors GPR56 (ADGRG1) and BAI1 (ADGRB1)

    Journal of Biological Chemistry 291:3385–3394.

    https://doi.org/10.1074/jbc.M115.689349

    • PubMed
    • Google Scholar
43. 1. Langenhan T
    2. Piao X
    3. Monk KR

    (2016) Adhesion G protein-coupled receptors in nervous system development and disease

    Nature Reviews Neuroscience 17:550–561.

    https://doi.org/10.1038/nrn.2016.86

    • PubMed
    • Google Scholar
44. 1. Lehnert BP
    2. Baker AE
    3. Gaudry Q
    4. Chiang AS
    5. Wilson RI

    (2013) Distinct roles of TRP channels in auditory transduction and amplification in Drosophila

    Neuron 77:115–128.

    https://doi.org/10.1016/j.neuron.2012.11.030

    • PubMed
    • Google Scholar
45. 1. Liebscher I
    2. Schön J
    3. Petersen SC
    4. Fischer L
    5. Auerbach N
    6. Demberg LM
    7. Mogha A
    8. Cöster M
    9. Simon KU
    10. Rothemund S
    11. Monk KR
    12. Schöneberg T

    (2014) A tethered agonist within the ectodomain activates the adhesion G protein-coupled receptors GPR126 and GPR133

    Cell Reports 9:2018–2026.

    https://doi.org/10.1016/j.celrep.2014.11.036

    • PubMed
    • Google Scholar
46. 1. Logothetis DE
    2. Kurachi Y
    3. Galper J
    4. Neer EJ
    5. Clapham DE

    (1987) The βγ subunits of GTP-binding proteins activate the muscarinic K+ channel in heart

    Nature 325:321–326.

    https://doi.org/10.1038/325321a0

    • Google Scholar
47. 1. Lopes DM
    2. Malek N
    3. Edye M
    4. Jager SB
    5. McMurray S
    6. McMahon SB
    7. Denk F

    (2017) Sex differences in peripheral not central immune responses to pain-inducing injury

    Scientific Reports 7:1–8.

    https://doi.org/10.1038/s41598-017-16664-z

    • Google Scholar
48. 1. Lux TJ
    2. Hu X
    3. Ben-Kraiem A
    4. Blum R
    5. Chen JT-C
    6. Rittner HL

    (2019) Regional differences in tight junction protein expression in the Blood–DRG Barrier and Their Alterations after Nerve Traumatic Injury in Rats

    International Journal of Molecular Sciences 21:270.

    https://doi.org/10.3390/ijms21010270

    • Google Scholar
49. 1. Marker CL
    2. Luján R
    3. Loh HH
    4. Wickman K

    (2005) Spinal G-protein-gated potassium channels contribute in a dose-dependent manner to the analgesic effect of mu- and Delta- but not kappa-opioids

    Journal of Neuroscience 25:3551–3559.

    https://doi.org/10.1523/JNEUROSCI.4899-04.2005

    • PubMed
    • Google Scholar
50. 1. Mauthner SE
    2. Hwang RY
    3. Lewis AH
    4. Xiao Q
    5. Tsubouchi A
    6. Wang Y
    7. Honjo K
    8. Skene JH
    9. Grandl J
    10. Tracey WD

    (2014) Balboa binds to pickpocket in vivo and is required for mechanical nociception in Drosophila larvae

    Current Biology 24:2920–2925.

    https://doi.org/10.1016/j.cub.2014.10.038

    • PubMed
    • Google Scholar
51. 1. McGlone F
    2. Wessberg J
    3. Olausson H

    (2014) Discriminative and affective touch: sensing and feeling

    Neuron 82:737–755.

    https://doi.org/10.1016/j.neuron.2014.05.001

    • PubMed
    • Google Scholar
52. 1. McGrath JC
    2. Lilley E

    (2015) Implementing guidelines on reporting research using animals (ARRIVE etc.): new requirements for publication in BJP

    British Journal of Pharmacology 172:3189–3193.

    https://doi.org/10.1111/bph.12955

    • PubMed
    • Google Scholar
53. 1. Mederos y Schnitzler M
    2. Storch U
    3. Meibers S
    4. Nurwakagari P
    5. Breit A
    6. Essin K
    7. Gollasch M
    8. Gudermann T

    (2008) Gq-coupled receptors as mechanosensors mediating myogenic vasoconstriction

    The EMBO Journal 27:3092–3103.

    https://doi.org/10.1038/emboj.2008.233

    • PubMed
    • Google Scholar
54. 1. Monk KR
    2. Hamann J
    3. Langenhan T
    4. Nijmeijer S
    5. Schöneberg T
    6. Liebscher I

    (2015) Adhesion G Protein-Coupled receptors: from in vitro pharmacology to in vivo mechanisms

    Molecular Pharmacology 88:617–623.

    https://doi.org/10.1124/mol.115.098749

    • PubMed
    • Google Scholar
55. 1. Murthy SE
    2. Dubin AE
    3. Patapoutian A

    (2017) Piezos thrive under pressure: mechanically activated ion channels in health and disease

    Nature Reviews Molecular Cell Biology 18:771–783.

    https://doi.org/10.1038/nrm.2017.92

    • PubMed
    • Google Scholar
56. 1. Nagel G
    2. Szellas T
    3. Huhn W
    4. Kateriya S
    5. Adeishvili N
    6. Berthold P
    7. Ollig D
    8. Hegemann P
    9. Bamberg E

    (2003) Channelrhodopsin-2, a directly light-gated cation-selective membrane channel

    PNAS 100:13940–13945.

    https://doi.org/10.1073/pnas.1936192100

    • PubMed
    • Google Scholar
57. 1. Pinto LG
    2. Souza GR
    3. Kusuda R
    4. Lopes AH
    5. Sant'Anna MB
    6. Cunha FQ
    7. Ferreira SH
    8. Cunha TM

    (2019) Non-Peptidergic nociceptive neurons are essential for mechanical inflammatory hypersensitivity in mice

    Molecular Neurobiology 56:5715–5728.

    https://doi.org/10.1007/s12035-019-1494-5

    • PubMed
    • Google Scholar
58. 1. Price MP
    2. McIlwrath SL
    3. Xie J
    4. Cheng C
    5. Qiao J
    6. Tarr DE
    7. Sluka KA
    8. Brennan TJ
    9. Lewin GR
    10. Welsh MJ

    (2001) The DRASIC cation channel contributes to the detection of cutaneous touch and acid stimuli in mice

    Neuron 32:1071–1083.

    https://doi.org/10.1016/S0896-6273(01)00547-5

    • PubMed
    • Google Scholar
59. 1. Reinhold A-K
    2. Yang S
    3. Chen JT-C
    4. Hu L
    5. Sauer R-S
    6. Krug SM
    7. Mambretti EM
    8. Fromm M
    9. Brack A
    10. Rittner HL

    (2019) Tissue plasminogen activator and neuropathy open the blood-nerve barrier with upregulation of microRNA-155-5p in male rats

    Biochimica Et Biophysica Acta (BBA) - Molecular Basis of Disease 1865:1160–1169.

    https://doi.org/10.1016/j.bbadis.2019.01.008

    • Google Scholar
60. 1. Salzman GS
    2. Zhang S
    3. Gupta A
    4. Koide A
    5. Koide S
    6. Araç D

    (2017) Stachel-independent modulation of GPR56/ADGRG1 signaling by synthetic ligands directed to its extracellular region

    PNAS 114:10095–10100.

    https://doi.org/10.1073/pnas.1708810114

    • PubMed
    • Google Scholar
61. 1. Sando R
    2. Jiang X
    3. Südhof TC

    (2019) Latrophilin GPCRs direct synapse specificity by coincident binding of FLRTs and teneurins

    Science 363:eaav7969.

    https://doi.org/10.1126/science.aav7969

    • PubMed
    • Google Scholar
62. 1. Sauer RS
    2. Kirchner J
    3. Yang S
    4. Hu L
    5. Leinders M
    6. Sommer C
    7. Brack A
    8. Rittner HL

    (2017) Blood-spinal cord barrier breakdown and pericyte deficiency in peripheral neuropathy

    Annals of the New York Academy of Sciences 1405:71–88.

    https://doi.org/10.1111/nyas.13436

    • PubMed
    • Google Scholar
63. 1. Schindelin J
    2. Arganda-Carreras I
    3. Frise E
    4. Kaynig V
    5. Longair M
    6. Pietzsch T
    7. Preibisch S
    8. Rueden C
    9. Saalfeld S
    10. Schmid B
    11. Tinevez JY
    12. White DJ
    13. Hartenstein V
    14. Eliceiri K
    15. Tomancak P
    16. Cardona A

    (2012) Fiji: an open-source platform for biological-image analysis

    Nature Methods 9:676–682.

    https://doi.org/10.1038/nmeth.2019

    • PubMed
    • Google Scholar
64. 1. Scholz N
    2. Gehring J
    3. Guan C
    4. Ljaschenko D
    5. Fischer R
    6. Lakshmanan V
    7. Kittel RJ
    8. Langenhan T

    (2015) The Adhesion GPCR Latrophilin/CIRL Shapes Mechanosensation

    Cell Reports 11:866–874.

    https://doi.org/10.1016/j.celrep.2015.04.008

    • Google Scholar
65. Book

    1. Scholz N
    2. Monk KR
    3. Kittel RJ
    4. Langenhan T

    (2016) Adhesion GPCRs as a Putative Class of Metabotropic Mechanosensors

    In: Langenhan T, Schöneberg T, editors. Adhesion G Protein-Coupled Receptors: Molecular, Physiological and Pharmacological Principles in Health and Disease. Cham: Springer International Publishing. pp. 221–247.

    https://doi.org/10.1007/978-3-319-41523-9

    • Google Scholar
66. 1. Scholz N
    2. Guan C
    3. Nieberler M
    4. Grotemeyer A
    5. Maiellaro I
    6. Gao S
    7. Beck S
    8. Pawlak M
    9. Sauer M
    10. Asan E
    11. Rothemund S
    12. Winkler J
    13. Prömel S
    14. Nagel G
    15. Langenhan T
    16. Kittel RJ

    (2017) Mechano-dependent signaling by latrophilin/CIRL quenches cAMP in proprioceptive neurons

    eLife 6:e28360.

    https://doi.org/10.7554/eLife.28360

    • PubMed
    • Google Scholar
67. Preprint

    1. Segebarth D
    2. Griebel M
    3. Duerr A
    4. von CCR
    5. Martin C
    6. Fiedler D
    7. Comeras LB
    8. Sah A
    9. Stein N
    10. Gupta R
    11. Sasi M
    12. Lange MD
    13. Tasan RO
    14. Singewald N
    15. Pape H-C
    16. Sendtner M
    17. Flath CM
    18. Blum R

    (2020) On the objectivity, reliability, and validity of deep learning enabled bioimage analyses

    bioRxiv.

    https://doi.org/10.1101/473199

    • Google Scholar
68. Book

    1. Sherrington CS

    (1906)

    The Integrative Action of the Nervous System

    New Haven: Yale University Press.

    • Google Scholar
69. 1. Sisignano M
    2. Angioni C
    3. Park CK
    4. Meyer Dos Santos S
    5. Jordan H
    6. Kuzikov M
    7. Liu D
    8. Zinn S
    9. Hohman SW
    10. Schreiber Y
    11. Zimmer B
    12. Schmidt M
    13. Lu R
    14. Suo J
    15. Zhang DD
    16. Schäfer SM
    17. Hofmann M
    18. Yekkirala AS
    19. de Bruin N
    20. Parnham MJ
    21. Woolf CJ
    22. Ji RR
    23. Scholich K
    24. Geisslinger G

    (2016) Targeting CYP2J to reduce paclitaxel-induced peripheral neuropathic pain

    PNAS 113:12544–12549.

    https://doi.org/10.1073/pnas.1613246113

    • PubMed
    • Google Scholar
70. 1. Skolnick P
    2. Volkow ND

    (2016) Re-energizing the development of pain therapeutics in light of the opioid epidemic

    Neuron 92:294–297.

    https://doi.org/10.1016/j.neuron.2016.09.051

    • PubMed
    • Google Scholar
71. 1. Stierl M
    2. Stumpf P
    3. Udwari D
    4. Gueta R
    5. Hagedorn R
    6. Losi A
    7. Gärtner W
    8. Petereit L
    9. Efetova M
    10. Schwarzel M
    11. Oertner TG
    12. Nagel G
    13. Hegemann P

    (2011) Light modulation of cellular cAMP by a small bacterial photoactivated adenylyl cyclase, bPAC, of the soil bacterium Beggiatoa

    Journal of Biological Chemistry 286:1181–1188.

    https://doi.org/10.1074/jbc.M110.185496

    • PubMed
    • Google Scholar
72. 1. Stoveken HM
    2. Hajduczok AG
    3. Xu L
    4. Tall GG

    (2015) Adhesion G protein-coupled receptors are activated by exposure of a cryptic tethered agonist

    PNAS 112:6194–6199.

    https://doi.org/10.1073/pnas.1421785112

    • PubMed
    • Google Scholar
73. 1. Szczot M
    2. Pogorzala LA
    3. Solinski HJ
    4. Young L
    5. Yee P
    6. Le Pichon CE
    7. Chesler AT
    8. Hoon MA

    (2017) Cell-Type-Specific splicing of Piezo2 regulates mechanotransduction

    Cell Reports 21:2760–2771.

    https://doi.org/10.1016/j.celrep.2017.11.035

    • PubMed
    • Google Scholar
74. 1. Thakur M
    2. Crow M
    3. Richards N
    4. Davey GI
    5. Levine E
    6. Kelleher JH
    7. Agley CC
    8. Denk F
    9. Harridge SD
    10. McMahon SB

    (2014) Defining the nociceptor transcriptome

    Frontiers in Molecular Neuroscience 7:87.

    https://doi.org/10.3389/fnmol.2014.00087

    • PubMed
    • Google Scholar
75. 1. Tracey WD
    2. Wilson RI
    3. Laurent G
    4. Benzer S

    (2003) Painless, a Drosophila gene essential for nociception

    Cell 113:261–273.

    https://doi.org/10.1016/S0092-8674(03)00272-1

    • PubMed
    • Google Scholar
76. 1. Xiang Y
    2. Yuan Q
    3. Vogt N
    4. Looger LL
    5. Jan LY
    6. Jan YN

    (2010) Light-avoidance-mediating photoreceptors tile the Drosophila larval body wall

    Nature 468:921–926.

    https://doi.org/10.1038/nature09576

    • PubMed
    • Google Scholar
77. 1. Yudin Y
    2. Rohacs T

    (2018) Inhibitory G _i/O -coupled receptors in somatosensory neurons: Potential therapeutic targets for novel analgesics

    Molecular Pain 14:1–16.

    https://doi.org/10.1177/1744806918763646

    • Google Scholar
78. 1. Zhang W
    2. Yan Z
    3. Jan LY
    4. Jan YN

    (2013) Sound response mediated by the TRP channels NOMPC, NANCHUNG, and INACTIVE in chordotonal organs of Drosophila larvae

    PNAS 110:13612–13617.

    https://doi.org/10.1073/pnas.1312477110

    • PubMed
    • Google Scholar
79. 1. Zhang M
    2. Wang Y
    3. Geng J
    4. Zhou S
    5. Xiao B

    (2019) Mechanically activated piezo channels mediate touch and suppress acute mechanical pain response in mice

    Cell Reports 26:1419–1431.

    https://doi.org/10.1016/j.celrep.2019.01.056

    • Google Scholar
80. 1. Zhong L
    2. Hwang RY
    3. Tracey WD

    (2010) Pickpocket is a DEG/ENaC protein required for mechanical nociception in Drosophila larvae

    Current Biology 20:429–434.

    https://doi.org/10.1016/j.cub.2009.12.057

    • PubMed
    • Google Scholar
81. 1. Zhong L
    2. Bellemer A
    3. Yan H
    4. Ken H
    5. Jessica R
    6. Hwang RY
    7. Pitt GS
    8. Tracey WD

    (2012) Thermosensory and nonthermosensory isoforms of Drosophila melanogaster TRPA1 reveal heat-sensor domains of a thermoTRP channel

    Cell Reports 1:43–55.

    https://doi.org/10.1016/j.celrep.2011.11.002

    • PubMed
    • Google Scholar

Acceptance summary:

You provide compelling evidence that aGPCR is expressed in high-threshold mechanosensory neurons where it regulates nocifensive (pain-like) behaviors. The systemic ablation of cirl, or its specific knockdown in nociceptive neurons, leads to an enhanced nocifensive response, whereas overexpression of the gene elicits an attenuated response. You also provide evidence that vertebrate chronic pain models are associated with diminished expression of the cirl homologs, indicating a potential role in the onset of chronic and pathological pain in vertebrates. Based on these observations the three reviewers agree that your manuscript is appropriate for eLife.

Decision letter after peer review:

[Editors’ note: the authors submitted for reconsideration following the decision after peer review. What follows is the decision letter after the first round of review.]

Thank you for choosing to send your work, "Antinociceptive modulation by the adhesion-GPCR CIRL promotes mechanosensory signal discrimination", for consideration at eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. Although the work is of interest, we regret to inform you that the findings at this stage are too preliminary for consideration at eLife at this time.

The reviewers felt that the data are interesting but have raised many concerns that will require additional experiments. We realize that at this time, during COVID, most labs are not open and you will not be able to do these experiments. For these reasons, we will give you the option of either going to a different journal entirely, or coming back to eLife with a substantially revised version at some point in the future. If you come back to eLife, we would consider that a new submission but would do everything we can to engage the same reviewers for this process.

We do not intend any criticism of the quality of the data or the rigor of the science. We wish you good luck with your work and we hope you will consider eLife for future submissions.

Reviewer #1:

In a study that is a follow-up of the original characterization of CIRL in Drosophila mechanotransduction, Dannhäuser et al. demonstrate that the aGPCR is also expressed in high-threshold mechanosensory neurons where it regulates nocifensive (pain-like) behaviors. The authors demonstrate that owing to its coupling with Gi, CIRL lowers cAMP levels in a cell autonomous manner. Either the systemic ablation of cirl, or its specific knockdown in nociceptive neurons, led to an enhanced nocifensive response, whereas overexpression of the gene in those same neurons elicited an attenuated response. The authors claim CIRL influences the overall activity of the nociceptive neurons, an argument based on Ca²⁺ imaging of nociceptive neurons. Finally, the authors provide experimental evidence that vertebrate chronic pain models are associated with diminished expression of the cirl homologs, which suggests a potential role in in the onset of chronic and pathological pain in vertebrates.

On the whole the manuscript is well written and makes good use of Drosophila as a model to examine the neurogenetics of nociception. There are, however, some concerns that diminish enthusiasm and need to be addressed before publication in ELife.

1) The authors make no reference to the ligand that is needed for the activation of CIRL in the context of nocifensive behavior. They write that CIRL is activated by a tethered protein ligand encoded by Strachel. Have they not examined whether deletion or knockdown of Strachel leads to similar nocifensive phenotypes? Given that ligand-induced activation of GPCRs is central to our understanding of biological function of those receptors, identification of ligand is critical to the proposed model. This reviewer appreciates the difficulties associated with de novo identification of GPCR ligands, and is not asking for a full examination. However, the role for Strachel in nocifensive behavior can and should be examined.

2) In the Ca²⁺ imaging data shown in Figure 4 it appears that the authors are imaging regions of the larval brain that harbor the axon termini of nociceptive neurons. They argue that the response amplitudes are larger in following cirl knockdown. These data are not compelling for several reasons. First, the observed Ca²⁺ responses are probably a function of the pressure applied to the periphery of the animals. Given the involvement of second messengers that typically amplify signals, minute variations in the pressure of the initial 'poke' could elicit Ca²⁺ responses of wildly different amplitudes. How can the authors know that the location and pressure of the stimulus is comparable between different data points? Second, control animals show increased GCaMP signals in segments A3 and A4, whereas the mutant animals show responses in A4 and A5. Which segments were stimulated in these recordings, and did the responses correlate with the segments that were stimulated? Third, the data should be presented as Ca²⁺ traces (amplitudes vs. time) so that readers can evaluate the temporal relationships between stimulation and Ca²⁺ elevation. Fourth, the authors should indicate the frequency of spontaneous activity at the axon termini.

3) The authors demonstrate that in mice with sciatic ligatures, expression of the homologs of cirl decreases. This is a very interesting finding that could be of broad interest to the field of chronic pain. Can the authors provide any evidence that the knockdown of cirl homologs in DRG neurons is associated with an increase in cAMP levels or cAMP-dependent signalling? Is the knockdown of cirl homologs in DRG neurons sufficient to trigger hyperexcitability of elevated Ca²⁺ responses?

4) A minor textual issue relates to the authors' statement that CIRL modulates the processing of innocuous and noxious stimuli a bidirectional manner. In the opinion of this reviewer, this statement is not fully accurate. In both populations of neurons, CIRL couples to Gi such that engagement of the agonist leads to a decrease in cAMP levels. The molecular function of CIRL – lowering cAMP levels – is the same in both populations. The difference in behavior is a function of the neuron type under consideration. In one population, lowering cAMP leads to increased activity whereas the same signal correlates with decreased activity in the other population. If anything, it is cAMP that modulates the processing of innocuous and noxious stimuli a bidirectional manner. Any receptor that mimics CIRL in the regulation of cAMP in those neurons would elicit the similar behaviors. If the authors agree with this notion, they should consider rewording the relevant portions of the manuscript.

5) Another minor comment relates to the use of the term, "quenches." Gi activation decreases the activity of AC leading to a decrease in the synthesis of cAMP. This is not equivalent to a "quench", a term that implies the breakdown of cAMP molecules already present in the cell. It is recommended that the authors change the text suitably.

Reviewer #2:

This study by Dannhauser et al. investigates the function of the adhesion GPCR Cirl in modulating nociceptive signalling in pain-sensing neurons in the Drosophila periphery. Building from a previous study published in eLife in which the authors demonstrated that Cirl worked to sensitize sensory responses in low-threshold mechanosensory neurons, this group now characterizes the function of Cirl in nociceptive neurons. As such, this study is well suited as a Research Advance. Using a combination of genetic manipulations, behavioral assays, and functional imaging, the authors show that Cirl essentially functions similarly in nociceptive neurons as it does in mechanosensory neurons, working to reduce cAMP levels. However, the downstream signal transduction machinery in nociceptive neurons apparently interprets the Cirl-dependent reduction in cAMP levels differently, ultimately diminishing the response of nociceptors to mechanical stimulation. Rather than Cirl itself functioning differently between touch and pain neurons, it appears that it is the downstream cAMP signal transduction machinery that operates in different ways.

The demonstration that the same signal transduction molecules can be interpreted differently by distinct cell types is not particularly surprising, as this has been well understood for decades in diverse fields (developmental patterning, axon guidance, etc). Importantly, Cirl levels are capable of bi-directionally tuning the sensitivity of nociceptive neurons, and the optogenetic experiments manipulating cAMP levels along with the calcium imaging serve to link Cirl-dependent signalling to neuronal output. Overall, this is an interesting study, the manuscript is well written and nicely sets up the framework and significance of the findings. What is missing is an understanding of to what extent CIRL is a target for physiological modulation of nociception in Drosophila – i.e., in what context and how would Cirl-dependent signalling adaptively (or maladaptively) calibrate the sensitivity of mechanosensation in touch and/or nociception? Similar to the previous study, it is also not clear how cAMP levels are transformed to specific changes in mechanosensitivity. Given the understandable efforts to avoid imposing additional experiments during this time of covid-19 precautions, I would suggest the following to improve the manuscript.

1) Physiological signals that regulate CIRL-mediated modulation of nociception: The rat data shown in Figure 6 is used by the authors to argue that Cirl1 may function in mammals similarly to Drosophila: "these correlative results are consistent with the Drosophila data linking low Cirl expression levels to nociceptor sensitization": This language is problematic, as the authors have not shown that downregulation of Cirl expression is an actual mechanism used in vivo in Drosophila to sensitize nociception. Rather, the authors have deliberately manipulated Cirl expression and observed sensitization. This holds true for the experiments in Figure 5 using the paclitaxel assay as well. However, in the rat experiment, the authors seem to imply an endogenous signalling system that ultimately served to reduce Cirl expression in response to chronic activation of neuropathic pain. The authors should make clear these distinctions and the lack of evidence from their Drosophila studies that Cirl activity is directly modulated to calibrate pain sensitivity.

The authors should also show the primary in situ hybridization images used for quantification in Figure 6 so readers can assess the data.

2) Re-framing the understanding of Cirl and cAMP signalling: Throughout this study, the authors really demonstrate that Cirl functions similarly in nociceptors and low-threshold mechanosensitive neurons – in both neuronal subtypes, Cirl functions to reduce cAMP signalling. Ultimately, is it simply the signal transduction downstream of cAMP that is unique to the two cell types, not Cirl itself. The authors do a good job of suggesting that the distinctions may be in the separate mechanosensitive proteins expressed in the neuronal subtypes in the Discussion. But at minimum the authors should make it clear that 1) Cirl functions similarly in both neurons to reduce cAMP signalling and 2) there is no evidence yet that Cirl-related signalling actually changes to adapt or tune mechanosensitivity in Drosophila.

Reviewer #3:

The authors examine the role of an adhesion GPCR dCirl in the function of the nociceptive neurons of the Drosophila larva. They show 1) that a reporter for dCirl is expressed in the cIVda nociceptors, 2) mutants for dCirl show hypersensitive mechanical nociception behaviors and the reverse is seen with dCirl over expression. 3) There is elevated Ca in the axon terminals is observed upon mechanical stimulation. 4) increasing cAMP in the neurons via an optogenetic sensitizes nociception responses 5) manipulating dCirl levels affects nociceptor branching 6) dCirl over expression reduces paclitaxel induced hyperalgesia 7) mammalian Cirl genes are down regulated in rats with sciatic nerve ligation injury. Overall, the data appear to be quite robust and the authors do make a convincing case that dCirl negatively regulates the sensitivity of the nociceptive neurons. Nevertheless, the paper is largely descriptive and provides little mechanistic understanding of how the phenotype actually occurs. Neither the mechanisms of how dCirl may be activated in the nociceptors nor the downstream effectors of its action have been determined. The rat experiments seem gratuitously added on and the paclitaxel experiments also provide little insight.

The authors find that dCirl negatively regulates nociceptor excitability and their prior studies found that dCirl has the opposite effect in chordotonal organs. Based on this they claim that they report "a new molecular principle underlying the processing of mechanical input." This is a vastly overstated claim as it is extremely well understood that same GPCR can have opposing effects in different contexts depending on the downstream effectors. Most GPCRs can be coupled to various G α subunits and therefore can have variable effects in different cell-types. As well, signalling can occur through β-arrestin, GRKs, Srcs etc… the present study does little to address the mechanism of the opposing effects of dCirl in Cho's vs. cIVda neurons.

dCirl^KO enhances the effects of increasing cAMP in the neurons may indicate dCirl loss is increasing cAMP but it could also indicate a parallel pathway. Epistasis experiments of the dCirl^KO are needed to resolve this issue.The authors really should investigate the effects of dCirl on thermal nociception and optogenetic triggered to determine if the effect is specific to mechanical nociception or to excitability of the cells in general.

Prior literature in the field that has implicated GPCR signalling in the Drosophila nociception pathways needs to be cited and incorporated into the Discussion:

Kaneko et al., 2017; Herman et al., 2018; Honjo et al., 2016 (found G α o as a negative regulator of thermal nociception); Christianson, Mauthner and Tracey, 2016 (found G α o as a negative regulator of mechanical nociception).

[Editors’ note: The authors appealed the original decision. What follows is the authors’ response to the first round of review.]

Reviewer #1:

[…] On the whole the manuscript is well written and makes good use of Drosophila as a model to examine the neurogenetics of nociception. There are, however, some concerns that diminish enthusiasm and need to be addressed before publication in eLife.

1) The authors make no reference to the ligand that is needed for the activation of CIRL in the context of nocifensive behavior. They write that CIRL is activated by a tethered protein ligand encoded by Strachel. Have they not examined whether deletion or knockdown of Strachel leads to similar nocifensive phenotypes? Given that ligand-induced activation of GPCRs is central to our understanding of biological function of those receptors, identification of ligand is critical to the proposed model. This reviewer appreciates the difficulties associated with de novo identification of GPCR ligands, and is not asking for a full examination. However, the role for Strachel in nocifensive behavior can and should be examined.

We would like to thank the reviewer for pointing out that the role of Cirl’s tethered agonist should be examined in more detail. The additional experiments have added an important new aspect to the manuscript.

Several studies have shown that aGPCR activation can occur by means of an intramolecular tethered agonist, termed Stachel (stalk; Liebscher et al., 2014; Monk et al., 2015; Stoveken et al., 2015). This linker sequence of approximately 20 amino acids begins at the extracellular GPCR proteolysis site (GPS) and connects the GAIN domain to the 7-transmembrane unit. In the revised manuscript, we now illustrate this layout in Figure 4D.

In line with tethered agonism, our previous study in eLife, which this Research Advance builds upon, showed that CIRL function requires an intact Stachel in touch-sensitive ChO neurons (Scholz et al., 2017). Notably, though, mutating the Stachel sequence of CIRL3 does not disrupt receptor function in the mouse hippocampus (Sando et al., 2019). Moreover, work in cell culture has put forward a model in which Stachel-dependent and -independent aGPCR activation may co-exist and trigger different downstream signalling pathways (Kishore et al., 2016; Salzman et al., 2017).

In order to test for a role of the Stachel sequence in activating CIRL in mechanical nociceptors, we now used two established mutants. Whereas the dCirl^T>A allele carries a mutation at the +1 position of the GPS within the Stachel sequence, the -2 mutation in the dCirl^H>A allele leaves the Stachel intact but reduces protein expression (Scholz et al., 2017). Analysing nocifensive behaviour in these mutants shows normal responses for the T>A allele and a Cirl null-mutant phenocopy for the H>A allele (Figure 4E). Thus, the nociceptors appear to be sensitive to reduced CIRL expression, which is consistent with very low protein levels in wild-type (close to the detection threshold; see new Figure 4—figure supplement 2). An intact tethered agonist, in turn, is dispensable for CIRL function in nociceptive neurons, contrasting the situation in touch-sensitive ChO neurons, where mutating the Stachel sequence disrupts receptor function.

This highly interesting finding argues that the mechanism of CIRL activation differs in these two types of mechanosensory neurons and provides evidence in support of a dual activation model of an aGPCR in a physiological setting. Moreover, the new results raise the possibility that this functional differentiation may be connected to specific downstream effects (see also reviewer #3 response 4).

The new results are described and discussed in detail in the revised manuscript (Introduction, last paragraph; Results, sixth paragraph; Discussion, third paragraph).

2) In the Ca²⁺ imaging data shown in Figure 4 it appears that the authors are imaging regions of the larval brain that harbor the axon termini of nociceptive neurons. They argue that the response amplitudes are larger in following cirl knockdown. These data are not compelling for several reasons. First, the observed Ca²⁺ responses are probably a function of the pressure applied to the periphery of the animals. Given the involvement of second messengers that typically amplify signals, minute variations in the pressure of the initial 'poke' could elicit Ca²⁺ responses of wildly different amplitudes. How can the authors know that the location and pressure of the stimulus is comparable between different data points? Second, control animals show increased GCaMP signals in segments A3 and A4, whereas the mutant animals show responses in A4 and A5. Which segments were stimulated in these recordings, and did the responses correlate with the segments that were stimulated? Third, the data should be presented as Ca²⁺ traces (amplitudes vs. time) so that readers can evaluate the temporal relationships between stimulation and Ca²⁺ elevation. Fourth, the authors should indicate the frequency of spontaneous activity at the axon termini.

We thank the reviewer for this comment. For these experiments, we use semi-intact preparations (opening segment A1-A2 to expose the ventral nerve cord while keeping A2/A3-A9 segments intact), leaving the posterior larval body wall intact but exposing the larval brain (see new Figure 4—figure supplement 1). The stimulus is then applied to an abdominal segment (segment A3-A5 as spelled out in the Materials and methods section) using a calibrated von Frey filament (45mN) mounted on a micromanipulator. Typically, the filament will only elicit responses in 1 segment (always targeting A3-A5), which means only the poked segment showed the axonal calcium response. Stimulation force is determined mainly by the filament strength, which bends upon reaching the maximum force (45 mN). In addition, micromanipulator movement speed is kept constant for stimulation. However, the exact placement of the filament on a body wall segment can only be reconciled by correlating it with the corresponding calcium responses in the nociceptor axon terminals, as imaging (brain) and stimulation (posterior body wall) occur in different regions of the animal. This established and published method (Hu et al., 2017) faithfully reports calcium responses in axon terminals of nociceptors, and has been successfully used to report differences due to genetic perturbations as shown here as well. Responses in the targeted segments (A3-A5) are indistinguishable due to the presence of the same number and type of nociceptors in all segments, and repeated stimulation gives rise to highly comparable responses. Equally, stimulation applied to A3-A5 segments of intact larvae gives rise to identical behavioral escape responses with comparable frequency.

The examples originally shown for the two conditions differ in terms of the targeted segment, yet they are still representative for the entire dataset and fully comparable to previous data obtained under the same conditions (Hu et al., 2017). Nonetheless, we have now chosen two examples showing maximum stimulation in the same segment (new Figure 4A). In all cases, we quantified the signal in the segment showing the highest response, as only one body wall segment is strongly stimulated by the von Frey filament. We however agree with the reviewer that calcium traces allow a more objective evaluation of the data, which we now provide in Figure 4B. Please note that due to the mechanical stimulation and corresponding body wall movement, the larval brain is briefly out of focus during and after stimulation (recognizable by the characteristic dip in fluorescence intensity). Of note, the extracted maxima used for the F_max plot (Figure 4B) are from the same data as the calcium traces, yet the F_max values appear larger as the maximum fluorescence change in each sample is not occurring at precisely the same time, thus reducing the mean fluorescence change over time.

Lastly, the sensitivity of the GCaMP6m reporter under these conditions does not allow us to visualize spontaneous activity in the axon termini. Baseline fluorescence signals show very little fluctuation and detectable calcium responses as shown here occur only after stimulation (<0.5 s), i.e. they are tightly linked to the mechanical stimulus and never observed without stimulation.

3) The authors demonstrate that in mice with sciatic ligatures, expression of the homologs of cirl decreases. This is a very interesting finding that could be of broad interest to the field of chronic pain. Can the authors provide any evidence that the knockdown of cirl homologs in DRG neurons is associated with an increase in cAMP levels or cAMP-dependent signalling? Is the knockdown of cirl homologs in DRG neurons sufficient to trigger hyperexcitability of elevated Ca²⁺ responses?

Answering these questions will require RNAi targeting of Cirl1 and Cirl3 in rat DRG neurons and/or knockout mice. For the mutant studies, we will likely need to use nociceptor-specific knockouts, which in turn will necessitate extended crossing and breeding. Both approaches will require new IRB approvals. We fully agree that the suggested experiments are of interest and should be carried out in detail in future studies. As a Research Advance, however, we feel that this work is beyond the scope of the present manuscript.

4) A minor textual issue relates to the authors' statement that CIRL modulates the processing of innocuous and noxious stimuli a bidirectional manner. In the opinion of this reviewer, this statement is not fully accurate. In both populations of neurons, CIRL couples to Gi such that engagement of the agonist leads to a decrease in cAMP levels. The molecular function of CIRL – lowering cAMP levels – is the same in both populations. The difference in behavior is a function of the neuron type under consideration. In one population, lowering cAMP leads to increased activity whereas the same signal correlates with decreased activity in the other population. If anything, it is cAMP that modulates the processing of innocuous and noxious stimuli a bidirectional manner. Any receptor that mimics CIRL in the regulation of cAMP in those neurons would elicit the similar behaviors. If the authors agree with this notion, they should consider rewording the relevant portions of the manuscript.

We have now taken care to explain more clearly how CIRL exerts opposing/bidirectional effects on the activity of neurons that process innocuous vs. noxious stimuli. That is, while CIRL lowers cAMP in both neuron types, the effect of cAMP on cellular activity differs in ChO and C4da neurons. In addition, to rewording the text (Abstract; Introduction, last paragraph; Discussion, first and last paragraphs; Figure 7 legend) we have modified Figure 7 to illustrate this principle.

5) Another minor comment relates to the use of the term, "quenches." Gi activation decreases the activity of AC leading to a decrease in the synthesis of cAMP. This is not equivalent to a "quench", a term that implies the breakdown of cAMP molecules already present in the cell. It is recommended that the authors change the text suitably.

“quench” has now been replaced throughout the text.

Reviewer #2:

[…] Overall, this is an interesting study, the manuscript is well written and nicely sets up the framework and significance of the findings. What is missing is an understanding of to what extent CIRL is a target for physiological modulation of nociception in Drosophila – i.e., in what context and how would Cirl-dependent signalling adaptively (or maladaptively) calibrate the sensitivity of mechanosensation in touch and/or nociception? Similar to the previous study, it is also not clear how cAMP levels are transformed to specific changes in mechanosensitivity. Given the understandable efforts to avoid imposing additional experiments during this time of covid-19 precautions, I would suggest the following to improve the manuscript.

1) Physiological signals that regulate CIRL-mediated modulation of nociception: The rat data shown in Figure 6 is used by the authors to argue that Cirl1 may function in mammals similarly to Drosophila: "these correlative results are consistent with the Drosophila data linking low Cirl expression levels to nociceptor sensitization": This language is problematic, as the authors have not shown that downregulation of Cirl expression is an actual mechanism used in vivo in Drosophila to sensitize nociception. Rather, the authors have deliberately manipulated Cirl expression and observed sensitization. This holds true for the experiments in Figure 5 using the paclitaxel assay as well. However, in the rat experiment, the authors seem to imply an endogenous signalling system that ultimately served to reduce Cirl expression in response to chronic activation of neuropathic pain. The authors should make clear these distinctions and the lack of evidence from their Drosophila studies that Cirl activity is directly modulated to calibrate pain sensitivity.

We thank the reviewer for the thoughtful comments and for pointing out this important consideration. In the revised manuscript, we have clarified that “It remains to be determined whether expression of DrosophilaCirl can be regulated physiologically to adapt or tune nociceptor sensitivity and whether activation of mammalian CIRL, in turn, can provide analgesia.”

The authors should also show the primary in situ hybridization images used for quantification in Figure 6 so readers can assess the data.

Example images are now shown in Figure 6D and E.

2) Re-framing the understanding of Cirl and cAMP signalling: Throughout this study, the authors really demonstrate that Cirl functions similarly in nociceptors and low-threshold mechanosensitive neurons – in both neuronal subtypes, Cirl functions to reduce cAMP signalling. Ultimately, is it simply the signal transduction downstream of cAMP that is unique to the two cell types, not Cirl itself. The authors do a good job of suggesting that the distinctions may be in the separate mechanosensitive proteins expressed in the neuronal subtypes in the Discussion. But at minimum the authors should make it clear that 1) Cirl functions similarly in both neurons to reduce cAMP signalling and 2) there is no evidence yet that Cirl-related signalling actually changes to adapt or tune mechanosensitivity in Drosophila.

1) We agree that this signalling principle should be made quite clear. Indeed, this point was also raised by reviewer #1 (see response 4). We have now made appropriate changes to the text (Abstract; Introduction, last paragraph; Discussion, first and last paragraphs; Figure 7 legend) and have included additional illustrations in Figure 7.

2) This clarification has now been added to the text (Results, last paragraph).

Reviewer #3:

The authors examine the role of an adhesion GPCR dCirl in the function of the nociceptive neurons of the Drosophila larva. They show 1) that a reporter for dCirl is expressed in the cIVda nociceptors, 2) mutants for dCirl show hypersensitive mechanical nociception behaviors and the reverse is seen with dCirl over expression. 3) There is elevated Ca in the axon terminals is observed upon mechanical stimulation. 4) increasing cAMP in the neurons via an optogenetic sensitizes nociception responses 5) manipulating dCirl levels affects nociceptor branching 6) dCirl over expression reduces paclitaxel induced hyperalgesia 7) mammalian Cirl genes are down regulated in rats with sciatic nerve ligation injury. Overall, the data appear to be quite robust and the authors do make a convincing case that dCirl negatively regulates the sensitivity of the nociceptive neurons. Nevertheless, the paper is largely descriptive and provides little mechanistic understanding of how the phenotype actually occurs. Neither the mechanisms of how dCirl may be activated in the nociceptors nor the downstream effectors of its action have been determined. The rat experiments seem gratuitously added on and the paclitaxel experiments also provide little insight.

The authors find that dCirl negatively regulates nociceptor excitability and their prior studies found that dCirl has the opposite effect in chordotonal organs. Based on this they claim that they report "a new molecular principle underlying the processing of mechanical input." This is a vastly overstated claim as it is extremely well understood that same GPCR can have opposing effects in different contexts depending on the downstream effectors. Most GPCRs can be coupled to various G α subunits and therefore can have variable effects in different cell-types. As well, signalling can occur through β-arrestin, GRKs, Srcs etc… the present study does little to address the mechanism of the opposing effects of dCirl in Cho's vs. cIVda neurons.

The cited statement has been removed. The new results on CIRL activation, i.e. Stachel-dependent vs. -independent (see reviewer #1 response 1, Introduction, last paragraph; Results, sixth paragraph, Figure 4D, E), and downstream signalling, i.e. enhancing mechanotransduction vs. reducing cellular excitability (see below), now provide further details on the opposing effects of CIRL in ChOs vs. C4da neurons (Figure 7, Discussion, third paragraph).

dCirl^KO enhances the effects of increasing cAMP in the neurons may indicate dCirl loss is increasing cAMP but it could also indicate a parallel pathway. Epistasis experiments of the dCirl^KO are needed to resolve this issue.

We thank the reviewer for pointing out this critical experiment. To substantiate that CIRL influences nociception by acting on cAMP-dependent signalling, we inhibited the endogenous adenylyl cyclase activity by pharmacological means. Consistent with nociceptor sensitization by cAMP, dietary supplementation with the adenylyl cyclase inhibitors SQ22536 or DDA significantly decreased nocifensive behaviour of Drosophila larvae. Importantly, this treatment produced indistinguishable responses of control and dCirl^KO animals to von Frey filament stimulation. This result shows that CIRL acts in the same pathway as cAMP production by the adenylyl cyclase and supports the notion that the aGPCR exerts its antinociceptive effect by lowering cAMP levels through G_i/o -mediated inhibition of adenylyl cyclase activity. These new data are presented in Figure 3D and are included in the fourth paragraph of the Results, in the third paragraph of the Discussion and in the Materials and methods subsection “Nocifensive behaviour”.

The authors really should investigate the effects of dCirl on thermal nociception and optogenetic triggered to determine if the effect is specific to mechanical nociception or to excitability of the cells in general.

We fully agree, this is a valid point. In principle, CIRL could influence the activity of C4da neurons by modulating cellular excitability or by modulating the mechanotransduction process, as is the case in touch-sensitive ChO neurons (Scholz et al., 2017). To differentiate between these possibilities, we chose the optogenetic strategy and circumvented mechanotransduction by stimulating the nociceptors with light. Photostimulation of C4da neurons via the optimized Channelrhodpsin-2 (ChR2) variant ChR2^XXM triggered nocifensive behaviour, consistent with previous work (Hwang et al., 2007). Notably, dCirl^KO larvae (dCirl^KOppk-GAL4 > UAS-chop2^XXM) responded more strongly than controls (ppk-GAL4 > UAS-chop2^XXM) over a range of different light intensities. This demonstrates that CIRL decreases the excitability of mechanical nociceptors, contrasting its role in ChO neurons where the aGPCR specifically enhances mechanotransduction. These results are shown in Figure 4C (now titled “Cirl decreases the excitability of nociceptors”) and are included in the fifth paragraph of the Results, the third paragraph of the Discussion and in the Materials and methods subsection “ChR2^XXM”.

Prior literature in the field that has implicated GPCR signalling in the Drosophila nociception pathways needs to be cited and incorporated into the Discussion:

Kaneko et al., 2017; Herman et al., 2018; Honjo et al., 2016 (found G α o as a negative regulator of thermal nociception); Christianson, Mauthner and Tracey, 2016 (found G α o as a negative regulator of mechanical nociception).

The recommended literature has now been cited in the first paragraph of the Discussion.

Author details

1. Sven Dannhäuser

   1. Department of Animal Physiology, Institute of Biology, Leipzig University, Leipzig, Germany
   2. Carl-Ludwig-Institute for Physiology, Leipzig University, Leipzig, Germany

   Contribution

   Formal analysis, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

2. Thomas J Lux

   Center for Interdisciplinary Pain Medicine, Department of Anaesthesiology, University Hospital Würzburg, Würzburg, Germany

   Contribution

   Formal analysis, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1049-9872

3. Chun Hu

   Neuronal Patterning and Connectivity, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

   Contribution

   Formal analysis, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

4. Mareike Selcho

   1. Department of Animal Physiology, Institute of Biology, Leipzig University, Leipzig, Germany
   2. Carl-Ludwig-Institute for Physiology, Leipzig University, Leipzig, Germany

   Contribution

   Formal analysis, Supervision, Funding acquisition, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

5. Jeremy T-C Chen

   Center for Interdisciplinary Pain Medicine, Department of Anaesthesiology, University Hospital Würzburg, Würzburg, Germany

   Contribution

   Formal analysis, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

6. Nadine Ehmann

   1. Department of Animal Physiology, Institute of Biology, Leipzig University, Leipzig, Germany
   2. Carl-Ludwig-Institute for Physiology, Leipzig University, Leipzig, Germany

   Contribution

   Formal analysis, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

7. Divya Sachidanandan

   1. Department of Animal Physiology, Institute of Biology, Leipzig University, Leipzig, Germany
   2. Carl-Ludwig-Institute for Physiology, Leipzig University, Leipzig, Germany

   Contribution

   Formal analysis, Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8219-8177

8. Sarah Stopp

   1. Department of Animal Physiology, Institute of Biology, Leipzig University, Leipzig, Germany
   2. Carl-Ludwig-Institute for Physiology, Leipzig University, Leipzig, Germany

   Contribution

   Investigation, Writing - review and editing

   Competing interests

   No competing interests declared

9. Dennis Pauls

   1. Department of Animal Physiology, Institute of Biology, Leipzig University, Leipzig, Germany
   2. Carl-Ludwig-Institute for Physiology, Leipzig University, Leipzig, Germany

   Contribution

   Investigation, Writing - review and editing, Funding acquisition

   Competing interests

   No competing interests declared

10. Matthias Pawlak

    Department of Neurophysiology, Institute of Physiology, University of Würzburg, Würzburg, Germany

    Contribution

    Conceptualization, Formal analysis, Writing - review and editing

    Competing interests

    No competing interests declared

11. Tobias Langenhan

    Rudolf Schönheimer Institute of Biochemistry, Division of General Biochemistry, Medical Faculty, Leipzig University, Leipzig, Germany

    Contribution

    Conceptualization, Supervision, Funding acquisition, Writing - review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-9061-3809

12. Peter Soba

    Neuronal Patterning and Connectivity, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany

    Contribution

    Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing - review and editing

    Competing interests

    No competing interests declared

13. Heike L Rittner

    Center for Interdisciplinary Pain Medicine, Department of Anaesthesiology, University Hospital Würzburg, Würzburg, Germany

    Contribution

    Conceptualization, Formal analysis, Supervision, Funding acquisition, Writing - review and editing

    For correspondence

    rittner_h@ukw.de

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-4867-0188

14. Robert J Kittel

    1. Department of Animal Physiology, Institute of Biology, Leipzig University, Leipzig, Germany
    2. Carl-Ludwig-Institute for Physiology, Leipzig University, Leipzig, Germany

    Contribution

    Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Writing - original draft, Project administration, Writing - review and editing

    For correspondence

    rjkittel@me.com

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-9199-4826

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