Antinociceptive modulation by the adhesion GPCR CIRL promotes mechanosensory signal discrimination
- Department of Animal Physiology, Institute of Biology, Leipzig University, Germany
- Carl-Ludwig-Institute for Physiology, Leipzig University, Germany
- Center for Interdisciplinary Pain Medicine, Department of Anaesthesiology, University Hospital Würzburg, Germany
- Neuronal Patterning and Connectivity, Center for Molecular Neurobiology, University Medical Center Hamburg-Eppendorf, Germany
- Department of Neurophysiology, Institute of Physiology, University of Würzburg, Germany
- Rudolf Schönheimer Institute of Biochemistry, Division of General Biochemistry, Medical Faculty, Leipzig University, Germany
Figures
Figure 1
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 …
Figure 2
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. …
Figure 3
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 …
Figure 4 with 2 supplements
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 …
Figure 4—figure supplement 1
Larval preparation for calcium imaging.
Figure 4—figure supplement 2
CIRL protein expression in mechanical nociceptors.
(A,B) The genomic transgene dCirlN-RFP (Scholz et al., 2017) reports low protein expression levels in C4da neurons (arrows). (B) Shown are confocal images of immunohistochemical stainings against …
Figure 5
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 …
Figure 6
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 …
Figure 7
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;…
Videos
Video 1
Nocifensive behaviour - the 'corkscrew' body roll.
Tables
Table 1
Behaviour and imaging.
| Figure | Genotype | Mean | SEM | N | P-value | Test | |
|---|---|---|---|---|---|---|---|
| Figure 2B | control | 53.34 | 1.458 | 7 | p<0.0001 | unpaired t-test | |
| dCirlKO | 75.37 | 2.676 | 10 | ||||
| RNAi control | 54.23 | 3.279 | 8 | p=0.0169 | one-way ANOVA, Tukey correction | ||
| ppk > dCirlRNAi | 68.08 | 2.052 | 10 | ||||
| GAL4 control | 53.67 | 3.211 | 14 | one-way ANOVA, Tukey correction | |||
| KO ppk > dCirl | 45.7 | 3.627 | 10 | p=0.3345 (GAL4 control) | |||
| ppk > dCirl | 37.12 | 2.778 | 10 | p=0.0023 (GAL4 control) | |||
| ppk > dCirl (29°C) | 28.67 | 3.125 | 10 | p<0.0001 (GAL4 control) | |||
| Figure 3C | control (Canton-S) | 44 | 3.712 | 10 | p<0.0001 | Mann-Whitney | |
| dunce1 | 72 | 1.106 | 10 | ||||
| control (f36a) | 50.5 | 1.167 | 10 | p<0.0001 | unpaired t-test | ||
| dunceML | 79.58 | 0.965 | 12 | ||||
| Figure 3D | control (+vehicle) | 49.67 | 1.445 | 10 | p<0.0001 | one-way ANOVA, Tukey correction | |
| dCirlKO(+vehicle) | 65.67 | 1.725 | 10 | ||||
| control (+SQ22536) | 38.33 | 1.511 | 10 | p=0.9805 | p=0.0002 (control +vehicle) | ||
| dCirlKO(+SQ22536) | 40 | 1.406 | 10 | p<0.0001 (dCirlKO +vehicle) | |||
| control (+DDA) | 35.67 | 2.334 | 10 | p=0.6315 | p<0.0001 (control +vehicle) | ||
| dCirlKO(+DDA) | 39.33 | 1.388 | 10 | p<0.0001 (dCirlKO +vehicle) | |||
| Figure 4B | control | 1.58 | 0.2794 | 10 | p=0.0032 | unpaired t-test | |
| ppk > dCirlRNAi | 2.94 | 0.2866 | 10 | ||||
| Figure 4E | control | 54.28 | 1.961 | 10 | one-way ANOVA, Tukey correction | ||
| dCirlTT>A | 55 | 2.274 | 10 | p=0.9660 (control) | |||
| dCirlHH>A | 73.78 | 1.845 | 10 | p<0.0001 (control) | |||
| Figure 5A | GAL4 control (+vehicle) | 53.96 | 3.831 | 19 | p>0.9999 (ppk > dCirl +paclitaxel) | Kruskal-Wallis test | |
| GAL4 control (+paclitaxel) | 95.5 | 1.74 | 10 | p=0.0002 (GAL4 control +vehicle) | |||
| ppk > dCirl (+vehicle) | 37.12 | 2.778 | 10 | p>0.9999 (ppk > dCirl +paclitaxel) | |||
| ppk > dCirl (+paclitaxel) | 46.34 | 2.374 | 10 | p<0.0001 (GAL4 control +Taxol) | |||
| dCirlKO (+vehicle) | 69.68 | 3.54 | 20 | p<0.0001 | Mann-Whitney | ||
| dCirlKO (+paclitaxel) | 97.05 | 1.097 | 10 | ||||
| Figure 5C | GAL4 control (+vehicle) | 8.256 | 0.1985 | 10 | p=0.0117 (GAL4 control +Taxol) | Kruskal-Wallis test | |
| GAL4 control (+paclitaxel) | 6.27 | 0.5574 | 10 | p=0.0017 (ppk > dCirl +paclitaxel) | |||
| ppk > dCirl (+vehicle) | 5.108 | 0.1603 | 10 | p<0.0001 (GAL4 control +vehicle) | |||
| ppk > dCirl (+paclitaxel) | 3.861 | 0.5961 | 10 | p=0.1848 (ppk > dCirl +vehicle) | |||
| GAL4 control (+vehicle) | 22.17 | 0.7015 | 10 | p=0.0014 (GAL4 control +paclitaxel) | one-way ANOVA, Tukey correction | ||
| GAL4 control (+paclitaxel) | 15.42 | 1.174 | 10 | p>0.9999 (ppk > dCirl +paclitaxel) | |||
| ppk > dCirl (+vehicle) | 17.26 | 0.6091 | 10 | p=0.0296 (GAL4 control +vehicle) | |||
| ppk > dCirl (+paclitaxel) | 11.22 | 1.716 | 10 | p=0.2557 (ppk > dCirl +vehicle) | |||
| Figure 5D | control | 4.971 | 0.1747 | 8 | p=0.0025 | unpaired t-test | |
| dCirlKO | 4.219 | 0.1255 | 10 | ||||
| control | 18.11 | 0.5029 | 8 | p=0.2829 | unpaired t-test | ||
| dCirlKO | 17.37 | 0.4384 | 10 | ||||
| Figure 6A | control day 0 | 6.972 | 0.8056 | 6 | p=0.4062 | unpaired t-test | |
| CCI day 0 | 8.003 | 0.8755 | 6 | ||||
| control day 7 | 8.152 | 0.6647 | 6 | p<0.0001 | |||
| CCI day 7 | 0.861 | 0.0982 | 6 | ||||
| Figure 6B | IB4 (control) | 28.6 | 5.148 | 6 | p=0.0018 | unpaired t-test | |
| IB4 (CCI) | 6.791 | 0.4034 | 6 | ||||
| CGRP (control) | 27.89 | 5.529 | 6 | p=0.3358 | |||
| CGRP (CCI) | 21.49 | 3.078 | 6 | ||||
| NF200 (control) | 26.76 | 2.737 | 6 | p=0.45 | |||
| NF200 (CCI) | 33.57 | 8.216 | 6 | ||||
| Figure 6C | IB4 (control) | 10.16 | 2.065 | 6 | p=0.1293 | unpaired t-test | |
| IB4 (CCI) | 6.662 | 0.4542 | 6 | ||||
| CGRP (control) | 11.5 | 2.103 | 6 | p=0.0895 | |||
| CGRP (CCI) | 7.212 | 0.8851 | 6 | ||||
| NF200 (control) | 14.25 | 1.965 | 6 | p=0.3939 | Mann-Whitney | ||
| NF200 (CCI) | 13 | 4.133 | 6 | ||||
Table 2
0: no response, 1: stimulated rolling, 2: spontaneous bending, 3: spontaneous rolling.
| Figure | Genotype | Mean 0 | Mean 1 | Mean 2 | Mean 3 | N |
|---|---|---|---|---|---|---|
| Figure 3B | wild-type (dark) | 47.29 | 52.71 | 0.00 | 0.00 | 203 |
| wild-type (light) | 48.00 | 52.00 | 0.00 | 0.00 | 200 | |
| ppk > bPAC (dark) | 31.25 | 53.13 | 15.63 | 0.00 | 96 | |
| ppk > bPAC (light) | 5.13 | 56.41 | 28.21 | 10.26 | 39 | |
| KO ppk > bPAC (dark) | 0.00 | 70.00 | 26.67 | 3.33 | 30 | |
| KO ppk > bPAC (light) | 0.00 | 58.06 | 12.90 | 29.03 | 31 |
Table 3
0: no photoinduced response, 1: photoinduced response.
| Figure | Genotype | Mean 0 | Mean 1 | N |
|---|---|---|---|---|
| Figure 4C | wild-type (10−3 mW/mm2) | 100 | 0 | 20 |
| wild-type (10−2 mW/mm2) | 100 | 0 | 20 | |
| wild-type (10−1 mW/mm2) | 100 | 0 | 20 | |
| ppk > chop2XXM (10−3 mW/mm2) | 95 | 5 | 20 | |
| ppk > chop2XXM (10−2 mW/mm2) | 25 | 75 | 20 | |
| ppk > chop2XXM (10−1 mW/mm2) | 36.36 | 63.63 | 22 | |
| dCirlKOppk > chop2XXM (10−3 mW/mm2) | 57.14 | 42.86 | 21 | |
| dCirlKOppk > chop2XXM (10−2 mW/mm2) | 9.52 | 90.48 | 21 | |
| dCirlKOppk > chop2XXM (10−1 mW/mm2) | 0 | 100 | 20 |
