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Antinociceptive modulation by the adhesion GPCR CIRL promotes mechanosensory signal discrimination

  1. Sven Dannhäuser
  2. Thomas J Lux
  3. Chun Hu
  4. Mareike Selcho
  5. Jeremy T-C Chen
  6. Nadine Ehmann
  7. Divya Sachidanandan
  8. Sarah Stopp
  9. Dennis Pauls
  10. Matthias Pawlak
  11. Tobias Langenhan
  12. Peter Soba
  13. Heike L Rittner  Is a corresponding author
  14. Robert J Kittel  Is a corresponding author
  1. Department of Animal Physiology, Institute of Biology, Leipzig University, Germany
  2. Carl-Ludwig-Institute for Physiology, Leipzig University, Germany
  3. Center for Interdisciplinary Pain Medicine, Department of Anaesthesiology, University Hospital Würzburg, Germany
  4. Neuronal Patterning and Connectivity, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Germany
  5. Department of Neurophysiology, Institute of Physiology, University of Würzburg, Germany
  6. Rudolf Schönheimer Institute of Biochemistry, Division of General Biochemistry, Medical Faculty, Leipzig University, Germany
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Cite this article as: eLife 2020;9:e56738 doi: 10.7554/eLife.56738

Abstract

Adhesion-type GPCRs (aGPCRs) participate in a vast range of physiological processes. Their frequent association with mechanosensitive functions suggests that processing of mechanical stimuli may be a common feature of this receptor family. Previously, we reported that the Drosophila aGPCR CIRL sensitizes sensory responses to gentle touch and sound by amplifying signal transduction in low-threshold mechanoreceptors (Scholz et al., 2017). Here, we show that Cirl is also expressed in high-threshold mechanical nociceptors where it adjusts nocifensive behaviour under physiological and pathological conditions. Optogenetic in vivo experiments indicate that CIRL lowers cAMP levels in both mechanosensory submodalities. However, contrasting its role in touch-sensitive neurons, CIRL dampens the response of nociceptors to mechanical stimulation. Consistent with this finding, rat nociceptors display decreased Cirl1 expression during allodynia. Thus, cAMP-downregulation by CIRL exerts opposing effects on low-threshold mechanosensors and high-threshold nociceptors. This intriguing bipolar action facilitates the separation of mechanosensory signals carrying different physiological information.

Introduction

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 Gq/11 or Gs proteins, like the prostaglandin EP2 receptor, increase the excitability of nociceptors by activating phospholipase C and adenylyl cyclase pathways, respectively. In contrast, Gi/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 Gi/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 Gi/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.

Results

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 (dCirlpGAL4 >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.

Drosophila Cirl is expressed in proprioceptors and nociceptors.

(A) The Cirl promoter drives Tomato photoprotein expression (magenta; dCirlpGAL4 >UAS-CD4::tdTomato) in type one larval pentascolopidial ChO (lch5) neurons and type 2 C4da nociceptors, identified by a GFP-ppk promoter fusion (green; ppk-CD4::tdGFP). Magnified view of (B) C4da and (C) ChO neurons. Shown are immunohistochemical stainings against the fluorescent proteins. Scale bars (A) 20 µm, (B,C) 10 µm.

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 (dCirlKO) 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-dCirlRNAi) 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.

Table 1
Behaviour and imaging.
FigureGenotypeMeanSEMNP-valueTest
Figure 2Bcontrol53.341.4587p<0.0001unpaired t-test
dCirlKO75.372.67610
RNAi control54.233.2798p=0.0169one-way ANOVA, Tukey correction
ppk > dCirlRNAi68.082.05210
GAL4 control53.673.21114one-way ANOVA, Tukey correction
KO ppk > dCirl45.73.62710p=0.3345 (GAL4 control)
ppk > dCirl37.122.77810p=0.0023 (GAL4 control)
ppk > dCirl (29°C)28.673.12510p<0.0001 (GAL4 control)
Figure 3Ccontrol (Canton-S)443.71210p<0.0001Mann-Whitney
dunce1721.10610
control (f36a)50.51.16710p<0.0001unpaired t-test
dunceML79.580.96512
Figure 3Dcontrol (+vehicle)49.671.44510p<0.0001one-way ANOVA, Tukey correction
dCirlKO(+vehicle)65.671.72510
control (+SQ22536)38.331.51110p=0.9805p=0.0002 (control +vehicle)
dCirlKO(+SQ22536)401.40610p<0.0001 (dCirlKO +vehicle)
control (+DDA)35.672.33410p=0.6315p<0.0001 (control +vehicle)
dCirlKO(+DDA)39.331.38810p<0.0001 (dCirlKO +vehicle)
Figure 4Bcontrol1.580.279410p=0.0032unpaired t-test
ppk > dCirlRNAi2.940.286610
Figure 4Econtrol54.281.96110one-way ANOVA, Tukey correction
dCirlTT>A552.27410p=0.9660 (control)
dCirlHH>A73.781.84510p<0.0001 (control)
Figure 5AGAL4 control (+vehicle)53.963.83119p>0.9999 (ppk > dCirl +paclitaxel)Kruskal-Wallis test
GAL4 control (+paclitaxel)95.51.7410p=0.0002 (GAL4 control +vehicle)
ppk > dCirl (+vehicle)37.122.77810p>0.9999 (ppk > dCirl +paclitaxel)
ppk > dCirl (+paclitaxel)46.342.37410p<0.0001 (GAL4 control +Taxol)
dCirlKO (+vehicle)69.683.5420p<0.0001Mann-Whitney
dCirlKO (+paclitaxel)97.051.09710
Figure 5CGAL4 control (+vehicle)8.2560.198510p=0.0117 (GAL4 control +Taxol)Kruskal-Wallis test
GAL4 control (+paclitaxel)6.270.557410p=0.0017 (ppk > dCirl +paclitaxel)
ppk > dCirl (+vehicle)5.1080.160310p<0.0001 (GAL4 control +vehicle)
ppk > dCirl (+paclitaxel)3.8610.596110p=0.1848 (ppk > dCirl +vehicle)
GAL4 control (+vehicle)22.170.701510p=0.0014 (GAL4 control +paclitaxel)one-way ANOVA, Tukey correction
GAL4 control (+paclitaxel)15.421.17410p>0.9999 (ppk > dCirl +paclitaxel)
ppk > dCirl (+vehicle)17.260.609110p=0.0296 (GAL4 control +vehicle)
ppk > dCirl (+paclitaxel)11.221.71610p=0.2557 (ppk > dCirl +vehicle)
Figure 5Dcontrol4.9710.17478p=0.0025unpaired t-test
dCirlKO4.2190.125510
control18.110.50298p=0.2829unpaired t-test
dCirlKO17.370.438410
Figure 6Acontrol day 06.9720.80566p=0.4062unpaired t-test
CCI day 08.0030.87556
control day 78.1520.66476p<0.0001
CCI day 70.8610.09826
Figure 6BIB4 (control)28.65.1486p=0.0018unpaired t-test
IB4 (CCI)6.7910.40346
CGRP (control)27.895.5296p=0.3358
CGRP (CCI)21.493.0786
NF200 (control)26.762.7376p=0.45
NF200 (CCI)33.578.2166
Figure 6CIB4 (control)10.162.0656p=0.1293unpaired t-test
IB4 (CCI)6.6620.45426
CGRP (control)11.52.1036p=0.0895
CGRP (CCI)7.2120.88516
NF200 (control)14.251.9656p=0.3939Mann-Whitney
NF200 (CCI)134.1336
Cirl reduces nocifensive behaviour.

(A) Characteristic nocifensive ‘corkscrew’ roll of larvae upon mechanical stimulation with a von Frey filament (40 mN force). (B) Quantification of nocifensive behaviour in different genotypes. Increased nocifensive responses were observed in dCirlKO and upon nociceptor-specific expression of an RNAi construct (ppk-GAL4 >UAS-dCirlRNAi). Cirl re-expression rescued the null mutant (dCirlKO ppk-GAL4 >UAS-dCirl) and Cirl overexpression (ppk-GAL4 >UAS-dCirl) reduced nocifensive responses. Raising animals at a higher temperature (29°C vs. 25°C) increases UAS/GAL4-dependent transgene expression (Duffy, 2002). Data are presented as mean and individual values (lower bar plot) and as the difference between means with 95% confidence intervals (upper dot plot). Asterisks denote level of significance, *p≤0.05, **p≤0.01, ***p≤0.001.

Video 1
Nocifensive behaviour - the 'corkscrew' body roll.

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 dCirlKO) 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 dCirlKO larvae (dCirlKO 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).

Table 2
0: no response, 1: stimulated rolling, 2: spontaneous bending, 3: spontaneous rolling.
FigureGenotypeMean 0Mean 1Mean 2Mean 3N
Figure 3Bwild-type (dark)47.2952.710.000.00203
wild-type (light)48.0052.000.000.00200
ppk > bPAC (dark)31.2553.1315.630.0096
ppk > bPAC (light)5.1356.4128.2110.2639
KO ppk > bPAC (dark)0.0070.0026.673.3330
KO ppk > bPAC (light)0.0058.0612.9029.0331
Potentiation of nociceptor function by cAMP.

(A) Schematic illustration of cAMP production by bPAC. (B) Optogenetic assay. Stimulated and spontaneous nocifensive responses can be promoted and elicited, respectively, by bPAC activation in C4da neurons (blue labels, photostimulation). Larval behaviour was observed during 3 min illumination (~200 µW/mm2 at 475 nm) followed by mechanical stimulation (40 mN von Frey filament). (C) Nocifensive behaviour of PDE mutants with ~73% (dunce1) and ~35% (dunceML) residual cAMP hydrolysis rates (Davis and Kiger, 1981). (D) The adenylyl cyclase inhibitors SQ22536 and DDA (500 µM) reduce nocifensive responses to comparable levels in control and dCirlKO larvae. Data are presented as mean and individual values. Asterisks denote level of significance, ***p≤0.001.

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 dCirlKO 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 Gi/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, dCirlRNAi 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 ChR2XXM (Nagel et al., 2003; Scholz et al., 2017) triggered nocifensive behaviour, consistent with previous work (Hwang et al., 2007). Notably, dCirlKO larvae (dCirlKO ppk-GAL4 >UAS-chop2XXM) responded more strongly than controls (ppk-GAL4 >UAS-chop2XXM) 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).

Table 3
0: no photoinduced response, 1: photoinduced response.
FigureGenotypeMean 0Mean 1N
Figure 4Cwild-type (10−3 mW/mm2)100020
wild-type (10−2 mW/mm2)100020
wild-type (10−1 mW/mm2)100020
ppk > chop2XXM (10−3 mW/mm2)95520
ppk > chop2XXM (10−2 mW/mm2)257520
ppk > chop2XXM (10−1 mW/mm2)36.3663.6322
dCirlKOppk > chop2XXM (10−3 mW/mm2)57.1442.8621
dCirlKOppk > chop2XXM (10−2 mW/mm2)9.5290.4821
dCirlKOppk > chop2XXM (10−1 mW/mm2)010020
Figure 4 with 2 supplements see all
Cirl decreases the excitability of nociceptors.

(A) Calcium imaging of C4da axon terminals expressing GCaMP6m (ppk-GAL4 >UAS-GCaMP6m) in semi-intact larval preparations. Representative baseline (F0) and maximum calcium responses (Fmax) are shown for control and CirlRNAi animals upon von Frey filament stimulation (45 mN). Scale bar, 10 μm. (B) Average calcium traces (arrow indicates stimulation) and quantification of the signals (ΔFmax/F0). CirlRNAi significantly elevates mechano-nociceptive responses of C4da neurons. (C) Nocifensive responses (red) elicited via ChR2XXM-mediated photostimulation of C4da neurons in control (ppk-GAL4 >UAS-chop2XXM) and dCirlKO larvae (dCirlKO ppk-GAL4 >UAS-chop2XXM). (D) Structure of the GPS region in Drosophila CIRL (Scholz et al., 2017). The Stachel sequence (light blue) is part of the GAIN domain (blue) contained in the CTF. Conserved, mutated amino acids required for receptor autoproteolysis at the GPS are shown in red (−2: dCirlH>A, +1: dCirlT>A). (E) Quantification of nocifensive behaviour in dCirlT>A and dCirlH>A receptor mutants. Data are presented as mean and individual values. Asterisks denote level of significance, **p≤0.01, ***p≤0.001. See also Figure 4—figure supplements 1 and 2.

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 dCirlT>A allele carries a mutation at the +1 position of the GPS within the Stachel sequence, the −2 mutation in the dCirlH>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 CirlKO 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 dCirlKO 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.

Sensitization of nociceptors through chemotherapy-induced neuropathy.

(A) Increased nocifensive behaviour following paclitaxel treatment (10 µM) is counteracted by overexpressing Cirl in nociceptors. (B) Example images of C4da neuron morphology upon paclitaxel administration and Cirl overexpression. Scale bars, 100 µm. (C, D) Morphometric quantification of dendritic complexity of C4da neurons in the different genotypes. Data are presented as mean and individual values. Asterisks denote level of significance, *p≤0.05, **p≤0.01, ***p≤0.001.

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, dCirlKO 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.

Neuropathy-induced mechanical allodynia correlates with decreased Cirl1 expression in mammalian non-peptidergic nociceptors.

(A) Traumatic injury of the sciatic nerve (CCI, green) in Wistar rats results in mechanical allodynia after one week as measured by von Frey Hairs (paw withdrawal threshold) in comparison to the contralateral side (grey). (B, C) Quantification of Cirl1 (B) and Cirl3 (C) mRNA levels in subpopulations of rat DRG neurons via in situ hybridization (RNAscope). Shown are control conditions (naïve DRGs, grey) and one week after injury (green) following the emergence of allodynia. Data are presented as mean and individual values. Asterisks denote level of significance, **p≤0.01, ***p≤0.001. (D, E) Example images of the RNAscope assay in DRG neurons. Shown are projections of confocal stacks stained against IB4 and labelled with probes against Cirl1 and Cirl3 under control conditions (D) and CCI (E). Scale bar 20 µm.

Discussion

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., 2017Kaneko et al., 2017; Herman et al., 2018) and can be negatively regulated by Gi/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).

cAMP downregulation by Drosophila CIRL adjusts mechanosensory submodalities in opposite directions.

Scheme summarizing how processing of different levels of mechanical force is bidirectionally modulated by CIRL’s downregulation of cAMP production. Whereas low threshold mechanosensory neurons (ChOs; gentle touch) are less responsive in Cirl mutants, high threshold mechanical nociceptors (C4da neurons; harsh touch) become sensitized.

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, Gi/o-coupled receptors, such as opioid, somatostatin, and GABAB receptors, counteract cAMP-dependent nociceptor sensitization (Yudin and Rohacs, 2018). In addition to this second messenger pathway, Gβγ subunits of Gi/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 Gi/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.

Materials and methods

Drosophila experiments

Fly stocks

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Animals were raised at 25°C on standard cornmeal and molasses medium. The following fly strains were used in this study:

  • from Scholz et al., 2015; Scholz et al., 2017:

    • dCirlpGAL4 (LAT84): w1118; dCirlKO {w+mC = pTL464[dCirlp::GAL4]}attPdCirl loxP/CyOGFP w-

    • dCirlKO (LAT26): w1118; dCirlKO attPdCirl loxP

    • dCirlRescue (LAT163): w1118; dCirlKO {w+mC = pTL370[dCirl]}attPdCirl loxP

    • 20xUAS-dCirl (LAT85): w1118;; {w+mC = pTL471[20xUAS-IVS-dCirl::3xFlag]}attP2

    • dCirlKO 20xUAS-dCirl (LAT111): w1118; dCirlKO attPdCirl loxP; {w+mC = pTL471[20xUAS-IVS-dCirl::3xFlag]}attP2/TM6B, Tb

    • dCirlT>A (LAT174): w1118; dCirlKO {w+mC = pMN9[dCirlT>A]}attPdCirlloxP/CyoGFP w-

    • dCirlH>A (LAT280): w1118; dCirlKO {w+mC = pMN44[dCirlH>A]}attPdCirlloxP/CyoGFP w-

    • dCirlN-RFP (LAT159): w1118; dCirlKO {w+mC = pMN4[dCirlN-RFP]}attPdCirlloxP/CyoGFP w-

    • UAS-chop2XXM (RJK300): w1118; {w+mC = pTL537[chop2-D156H(XXM)::tdtomato]}attPVK00018/CyoGFP w-

    • dCirlKO UAS-chop2XXM (LAT193): w1118; dCirlKO {w+mC = pTL537[chop2-D156H(XXM)::tdtomato]}attPVK00018/CyoGFP w-

  • from the Bloomington Drosophila Stock Center:

    • BDSC#42748: w1118; UAS-GCaMP6m P{y+t7.7w+mC = 20xUAS-IVS-GCaMP6m}attP40

    • BDSC#35843: w1118;; P{w+mC = ppk-CD4::tdGFP}8/TM6B,Tb

    • BDSC#35841: y1 w*; P{w+mC = UAS-CD4::tdTomato}7 M1

    • BDSC#32078: w*; P{w+mC = ppk-GAL4.G}2

    • BDSC#32079: w*;; P{w+mC = ppk-GAL4.G}3

    • BDSC#6020: dnc1

    • BDSC#43: f36a

    • BDSC#9407: y1 w1 dncML f36a/FM7a

  • Figure 1:

    • dCirlpGAL4/UAS-CD4::tdTomato; ppk-CD4::tdGFP/+

  • Figure 2:

    • control: dCirlRescue

    • dCirlKO

    • RNAi control: UAS-dCirlRNAi/+

    • ppk >dCirlRNAi: ppk-GAL4/UAS-dCirlRNAi

    • GAL4 control: dCirlRescue/+; ppk-GAL4/+

    • dCirlKO ppk >dCirl: dCirlKO; ppk-GAL4/20xUAS-dCirl

    • ppk >dCirl: ppk-GAL4/20xUAS-dCirl

  • Figure 3:

    • (B) wild-type: w1118

    • ppk >bPAC: ppk-GAL4/UAS-bpac

    • dCirlKO ppk >bPAC: dCirlKO, UAS-bpac/dCirlKO; ppk-GAL4/+

    • (C) control for dunce1: Canton-S

    • control for dunceML: f36a

    • (D) control: dCirlRescue

    • dCirlKO

  • Figure 4:

    • (A,B) control: UAS-GCaMP6m/+; ppk-GAL4/+

    • ppk >dCirlRNAi: UAS-GCaMP6m/UAS-dCirlRNAi; ppk-GAL4/+

    • (C) wild-type: w1118

    • ppk >chop2XXM: UAS-chop2XXM/+; ppk-GAL4/+

    • dCirlKO ppk >chop2XXM: dCirlKO UAS-chop2XXM/dCirlKO; ppk-GAL4/+

    • (E) control: dCirlRescue

    • dCirlT>A

    • dCirlH>A

  • Figure 5:

    • (A) GAL4 control: ppk-GAL4/+ 

    • ppk >dCirl: ppk-GAL4/20xUAS-dCirl

    • dCirlKO

    • (B, C) GAL4 control: ppk-GAL4/+; ppk-CD4::tdGFP/+

    • ppk >dCirl: ppk-GAL4/+; ppk-CD4::tdGFP/20xUAS-dCirl

    • (D) Control: ppk-Gal4, UAS-CD4::tdTomato/+

    • dCirlKO: dCirlKO; ppk-Gal4, UAS-CD4::tdTomato/+

Immunohistochemistry

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Stainings of the dCirlpGAL4- and ppk-positive neurons (Figure 1) and CIRLN-RFP (Figure 4—figure supplement 2) were performed essentially as previously reported (Ehmann et al., 2014). Third instar larvae were dissected in cold PBS, fixed in 4% paraformaldehyde for 10 or 40 min at room temperature (RT), washed 3 x for 10 min in 0.3% PBT (PBS with 0.3% Triton X-100, Sigma-Aldrich) and blocked for 30 min in 0.3% PBT supplemented with 5% normal goat serum (NGS). The preparations were incubated with primary antibodies (diluted in 0.3% PBT with 5% or 3% NGS) at 4°C overnight. After washing 6 x for 10 min with 0.3% PBT, the samples were incubated with secondary antibodies (diluted in 0.3% PBT with 5% or 3% NGS) for 120 min at RT or at 4°C overnight. Following 6 × 10 min washing steps with 0.3% PBT, the preparations were immersed in Vectashield (Vector Laboratories) and stored for at least one night at 4°C. The following primary antibodies were used: mouse-α-GFP (1:200; Sigma-Aldrich, SAB4200681; RRID:AB_2827519), rabbit-α-RFP (1:200; antibodies-online, ABIN129578; RRID:AB_10781500). Secondary antibodies: Alexa Fluor-488-conjugated goat-α-mouse (1:250; Invitrogen, A-11001; RRID:AB_2534069), Cy3-conjugated goat-α-rabbit (1:250; Jackson ImmunoResearch, 111-165-003; RRID:AB_2338006), Alexa Fluor-647 goat- α-rabbit (1:250; Invitrogen, A-21246; RRID:AB_2535814), and α-HRP conjugated with Alexa488 (1:250; Jackson ImmunoResearch, 123-545-021; RRID:AB_2338965). Samples were mounted in Vectashield and confocal images (Figure 1, Figure 4—figure supplement 2) were acquired with a LSM 800 (Zeiss) and a Leica TCS SP5. Intensity and contrast were set using Fiji (Schindelin et al., 2012) and Photoshop CC 2018 (Adobe).

Nociceptor morphometry

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For analyses of C4da neuron morphology, staged third instar larvae (96 ± 3 hr after egg laying, AEL), raised in density-controlled vials, were mounted in halocarbon oil. Confocal images of photoprotein signals (ppk-GAL4 >UAS-CD4::tdGFP or UAS-CD4::tdTomato) were collected with a Zeiss LSM700 laser scanning microscope. Image stacks with a Z step size between 0.5 and 2 μm were acquired from abdominal segment A5 with a 20×/0.8 objective and quantified with Imaris (Bitplane) using the filament tracer tool.

Nocifensive behaviour

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For mechanical nociception assays, wandering third instar larvae were collected in a sylgard covered Petri dish and stimulated with a 40 mN von Frey filament (made from fishing line, 0.22 mm diameter, Caperlan; calibrated with a precision balance). A single noxious mechanical stimulus was rapidly delivered to midabdominal segments (~A4–A6) on the dorsal side of the larva. A positive response was scored if stimulation elicited at least one nocifensive corkscrew body roll. For all behavioural experiments each animal was tested only once. The adenylyl cyclase inhibitors SQ22536 and DDA were added to the food (giving final concentrations of 500 µM) one day prior to the experiments (Figure 3D). All data were collected from at least seven trials (N, Table 1) each sampling 6–53 larvae.

Calcium imaging

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Calcium imaging of C4da axon terminals was performed as previously described (Figure 4—figure supplement 1; Hu et al., 2017). Briefly, staged third instar larvae (96 ± 3 hr AEL) were pinned on a Sylgard (Dow Corning) plate and partially dissected in physiological saline to expose the ventral nerve cord (VNC). C4da neuron axon terminals expressing GCaMP6m were live-imaged by confocal microscopy with a 40 × water objective (Olympus FV1000MP) with 3x zoom to image at least four segments ensuring the calcium response could be detected. Activation of sensory neurons was achieved by providing a mechanonociceptive cue using a micromanipulator-mounted von Frey filament (45 mN) for stimulation of midabdominal segments (A3–A5). The most robust responses to local von Frey filament stimulation are restricted to a single VNC hemisegment corresponding to the stimulation site on the body wall although the adjacent segment(s) could also be slightly activated. The transient dip in fluorescence intensity (Figure 4B) is due to the larval brain moving out of focus briefly during and after mechanical stimulation. Baseline (F0) and relative maximum intensity change (ΔFmax) of GCaMP6m fluorescence were analysed.

Optogenetics

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bPAC
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Larvae were placed in a drop of water on a Sylgard-coated Petri dish and monitored with a stereomicroscope (Olympus SZX16). After applying blue light (~200 µW/mm2 at 475 nm) for 3 min, the animals were mechanically stimulated with a von Frey filament (see above). Each larva was scored according to the following criteria. no response: no nocifensive or irregular behaviour during light or upon mechanical stimulation; stimulated rolling: no nocifensive or irregular behaviour during light, corkscrew body roll upon mechanical stimulation; spontaneous bending: head-swinging or bending during light; spontaneous rolling: corkscrew body roll during light. For each set of experiments, three larvae were analysed simultaneously and each animal was tested only once. In Table 2, N refers to the number of individuals tested.

ChR2XXM
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Larvae were placed in a Petri dish and illuminated with blue light (either ~10−1 mW/mm2, ~10−2 mW/mm2, or ~10−3 mW/mm2 at 475 nm) for 30 s. Each larva was scored according to the following criteria. No photoinduced response: no nocifensive or irregular behaviour during light exposure; photoinduced response: nocifensive responses such as massive head-swinging, bending, or corkscrew body roll during light exposure. For each experiment, two or three larvae were analysed simultaneously and each animal was tested only once. In Table 3, N refers to the number of individuals tested. Larvae were raised on food supplemented with 100 µM all-trans-retinal (Sigma-Aldrich).

Drosophila neuropathy model

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DMSO (dimethyl sulfoxide, vehicle, Sigma Aldrich) or paclitaxel (S1150 Absource Diagnostics, dissolved in DMSO) were carefully mixed into the food vials once ~90% of first instar larvae had hatched. This way, paclitaxel treatment (10 µM) occurred after completion of neurogenesis and axonal pathfinding (Bhattacharya et al., 2012).

Rat experiments

Traumatic neuropathy (Chronic constriction injury, CCI)

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Animal care and protocols were performed in accordance with international guidelines for the care and use of laboratory animals (EU Directive 2010/63/EU for animal experiments) and were approved by the Government of Unterfranken (protocol numbers 2–733 and 2–264). Humane endpoints and criteria for discontinuation of the experiments with approved score sheets were defined, and the animals were treated accordingly. Animal studies were reported according to the ARRIVE guidelines (McGrath and Lilley, 2015). Male Wistar rats (200–250 g, Janvier labs, Le Genest-St-Isle, France) were housed in groups of six on dry litter (12 hr:12 hr light/dark cycle, 21–25°C, 45–55% humidity) with food and water ad libitum. All experiments were performed during the light phase and equal test groups (n = 6) were planned. Surgery of the Wistar rats was performed under deep isoflurane anaesthesia (1.8 Vol%, fiO2). After skin incision and exposure of the sciatic nerve by blunt preparation, four loose silk ligatures were made (Perma Silk 6.0, Ethicon Inc) with approximately 1 mm spacing in between (Sauer et al., 2017). After loosely tightening the ligatures, the skin was stitched (Prolene 5.0, Ethicon Inc). Animals were euthanized with CO2 at the end of the experiment.

Mechanical nociceptive thresholds

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A series of von Frey filaments (Aesthesio set, Ugo Basile) were used to record the withdrawal threshold of the hind paw to identify the mechanical allodynia response (Lux et al., 2019) and touch sensitivity in neuropathy. Filaments were applied to the plantar surface of the hind paw and held for 1–3 s, until they were bent to a 45° angle. Each paw received stimuli with different filament forces, with a 30 s recovery period between each application. The 50% paw withdrawal threshold for von Frey filament responses was determined using Dixon’s up and down method (Dixon, 1980).

In situ hybridization and immunohistochemistry

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After euthanizing the rats, DRGs were harvested, embedded in Tissue-Tek O.C.T. Compound (Sakura Finetek Europe B.V.), snap frozen in liquid nitrogen, and stored at −80°C. 10 µm thick cryosections were cut at −20°C (CM3050 S Research Cryostat, Leica Biosystems) and the slides were stored at −80°C until further use. For fixation, tissue sections were placed in precooled 4% PFA in DEPC (diethyl pyrocarbonate)-treated distilled water. Following washing steps with DEPC-treated reagents, probes for rat ADGRL1 and ADGRL3 (tagged with Cy3 and Cy5, respectively) were added for the RNAscope fluorescent multiplex assay (Advanced Cell Diagnostics, Inc), which was performed according to the manufacturer’s instructions (document 320293-USM). After 15 min of incubation at 4°C the samples were dehydrated in ethanol at RT (50%, 70%, 100%, 100%; 5 min each). Hydrophobic barriers measuring approximately the same area, were drawn around the tissue sections and allowed to dry completely. Afterwards, each section was incubated with two drops of RNAscope Protease IV reagent (15 min at RT). Following the RNAscope assay, samples were washed, blocked with 10% donkey serum in PBS (1 hr at RT), and counterstained with neuronal markers (diluted in 10% donkey serum in PBS and added for two nights at 4°C). Non peptidergic nociceptors: isolectin B4 (IB4)-FITC conjugate (1:200, Sigma-Aldrich, L2895); large myelinated mechanosensors and proprioceptors: rabbit anti-NF200 (1:200, Sigma-Aldrich, RRID:AB_477272); peptidergic nociceptors: mouse anti-CGRP (1:150, Abcam, RRID:AB_1658411). Following incubation, the sections were washed and secondary antibodies were added (diluted in PBS and incubated for 1 hr at RT). For CGRP: donkey anti-mouse AlexaFluor488 (1:1000, Life Technologies, RRID:AB_141607), for NF200: donkey anti-rabbit AlexaFluor488 (1:1000, Life Technologies, RRID:AB_141708). After washing, the slides were mounted in Vectashield, dried for 15 min at RT, and stored at 4°C until imaging (<24 hr). All images were acquired in one session by confocal microscopy (Olympus FV1000) with a 20×/0.75 objective (Olympus UPlan SAPO) using the same parameters for z-stacks (1 µm step size) of Cy3 and Cy5 channels.

Image evaluation

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Images were processed with Fiji (Schindelin et al., 2012) by scientists blinded to the test groups. The markers of neuronal subpopulations were used to identify complete and distinguishable cells as regions of interest (ROIs) for further analysis. Thresholds were applied to maximal projections of confocal z-stacks for Cy3 (grey value 1200) and Cy5 (grey value 1150) signals (this procedure was also used for the example images in Figure 6D,E). Elements between 3 and 22 pixels in size, defined as mRNA clusters, were quantified for each channel following background subtraction. An independent approach based on computational image evaluation gave comparable results (data not shown). Here, a convolutional neural network (DeepFLaSh) (Segebarth et al., 2020) was trained with six images for each neuronal marker (NF200, IB4, CGRP) and then used to identify Cy3 and Cy5 clusters.

Statistics

Data were analysed with Prism 8.2 (GraphPad). Group means were compared by an unpaired two- tailed t-test, unless the assumption of normal sample distribution was violated, in which case group means were compared by a nonparametric Mann–Whitney rank sum test. To compare more than two groups an ordinary one-way ANOVA with Tukey correction (normal distribution) or a Kruskal–Wallis test (not normally distributed) were used.

Data availability

The presented data are summarized in Tables 1-3.

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Decision letter

  1. Hugo J Bellen
    Reviewing Editor; Baylor College of Medicine, United States
  2. Richard M White
    Senior Editor; Memorial Sloan Kettering Cancer Center, United States
  3. Kartik Venkatachalm
    Reviewer; UT, United States
  4. Dion K Dickman
    Reviewer; University of Southern California, United States

In the interests of transparency, eLife publishes the most substantive revision requests and the accompanying author responses.

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.

dCirlKO 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 dCirlKO 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).

https://doi.org/10.7554/eLife.56738.sa1

Author response

[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 dCirlT>A allele carries a mutation at the +1 position of the GPS within the Stachel sequence, the -2 mutation in the dCirlH>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 Fmax plot (Figure 4B) are from the same data as the calcium traces, yet the Fmax 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).

dCirlKO 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 dCirlKO 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 dCirlKO 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 Gi/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 ChR2XXM triggered nocifensive behaviour, consistent with previous work (Hwang et al., 2007). Notably, dCirlKO larvae (dCirlKOppk-GAL4 > UAS-chop2XXM) responded more strongly than controls (ppk-GAL4 > UAS-chop2XXM) 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 “ChR2XXM”.

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.

https://doi.org/10.7554/eLife.56738.sa2

Article and author information

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

Funding

Deutsche Forschungsgemeinschaft (PA3241/2-1)

  • Mareike Selcho

Deutsche Forschungsgemeinschaft (RI817/13-1)

  • Heike L Rittner

Deutsche Forschungsgemeinschaft (FOR 2149/P03)

  • Robert J Kittel

Deutsche Forschungsgemeinschaft (TRR 166/B04)

  • Robert J Kittel

Deutsche Forschungsgemeinschaft (KI1460/4-1)

  • Robert J Kittel

Deutsche Forschungsgemeinschaft (KI1460/5-1)

  • Robert J Kittel

Deutsche Forschungsgemeinschaft (SPP 1926/SO1337/2-2)

  • Peter Soba

Deutsche Forschungsgemeinschaft (SO1337/4-1)

  • Peter Soba

Deutsche Forschungsgemeinschaft (FOR 2149/P01 and P03)

  • Tobias Langenhan

Deutsche Forschungsgemeinschaft (PA1979/2-1)

  • Dennis Pauls

Deutsche Forschungsgemeinschaft (PA1979/3-1)

  • Dennis Pauls

Deutsche Forschungsgemeinschaft (CRC 1423/B06)

  • Tobias Langenhan

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank PA Stevenson, I Maiellaro, and J Eilers for discussions, R Blum for help with the computational image analysis of DRGs, and N Naumann and B Goettgens for technical assistance. This work was supported by the Graduate School of Life Sciences, University of Würzburg and grants from the German Research Foundation (DFG) to MS (PA3241/2-1), HLR (RI817/13-1), RJK (FOR 2149/P03, TRR 166/B04, KI1460/4-1, KI1460/5-1), PS (SPP 1926/SO1337/2-2, SO1337/4-1), DP (PA1979/2-1, PA1979/3-1), and TL (FOR 2149/P01 and P03, CRC 1423, project number 421152132, subproject B06). Stocks obtained from the Bloomington Drosophila Stock Center (NIH P40OD018537) and the Vienna Drosophila Resource Center (Dietzl et al., 2007) were used in this study. The authors declare no competing financial interests.

Ethics

Animal experimentation: Animal care and protocols were performed in accordance with international guidelines for the care and use of laboratory animals (EU Directive 2010/63/EU for animal experiments) and were approved by the Government of Unterfranken (protocol numbers 2-733 and 2-264).

Senior Editor

  1. Richard M White, Memorial Sloan Kettering Cancer Center, United States

Reviewing Editor

  1. Hugo J Bellen, Baylor College of Medicine, United States

Reviewers

  1. Kartik Venkatachalm, UT, United States
  2. Dion K Dickman, University of Southern California, United States

Publication history

  1. Received: March 13, 2020
  2. Accepted: September 17, 2020
  3. Accepted Manuscript published: September 30, 2020 (version 1)
  4. Version of Record published: October 9, 2020 (version 2)

Copyright

© 2020, Dannhäuser et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Further reading

    1. Cancer Biology
    2. Neuroscience
    Susu Pan et al.
    Research Article Updated

    Emerging evidence suggests that the nervous system is involved in tumor development in the periphery, however, the role of the central nervous system remains largely unknown. Here, by combining genetic, chemogenetic, pharmacological, and electrophysiological approaches, we show that hypothalamic oxytocin (Oxt)-producing neurons modulate colitis-associated cancer (CAC) progression in mice. Depletion or activation of Oxt neurons could augment or suppress CAC progression. Importantly, brain treatment with celastrol, a pentacyclic triterpenoid, excites Oxt neurons and inhibits CAC progression, and this anti-tumor effect was significantly attenuated in Oxt neuron-lesioned mice. Furthermore, brain treatment with celastrol suppresses sympathetic neuronal activity in the celiac-superior mesenteric ganglion (CG-SMG), and activation of β2 adrenergic receptor abolishes the anti-tumor effect of Oxt neuron activation or centrally administered celastrol. Taken together, these findings demonstrate that hypothalamic Oxt neurons regulate CAC progression by modulating the neuronal activity in the CG-SMG. Stimulation of Oxt neurons using chemicals, for example, celastrol, might be a novel strategy for colorectal cancer treatment.

    1. Neuroscience
    Anirudh Wodeyar et al.
    Tools and Resources Updated

    Brain rhythms have been proposed to facilitate brain function, with an especially important role attributed to the phase of low-frequency rhythms. Understanding the role of phase in neural function requires interventions that perturb neural activity at a target phase, necessitating estimation of phase in real-time. Current methods for real-time phase estimation rely on bandpass filtering, which assumes narrowband signals and couples the signal and noise in the phase estimate, adding noise to the phase and impairing detections of relationships between phase and behavior. To address this, we propose a state space phase estimator for real-time tracking of phase. By tracking the analytic signal as a latent state, this framework avoids the requirement of bandpass filtering, separately models the signal and the noise, accounts for rhythmic confounds, and provides credible intervals for the phase estimate. We demonstrate in simulations that the state space phase estimator outperforms current state-of-the-art real-time methods in the contexts of common confounds such as broadband rhythms, phase resets, and co-occurring rhythms. Finally, we show applications of this approach to in vivo data. The method is available as a ready-to-use plug-in for the Open Ephys acquisition system, making it widely available for use in experiments.