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Sensory integration and neuromodulatory feedback facilitate Drosophila mechanonociceptive behavior

Nature Neuroscience volume 20, pages 1085–1095 (2017)Cite this article

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Abstract

Nociception is an evolutionarily conserved mechanism to encode and process harmful environmental stimuli. Like most animals, Drosophila melanogaster larvae respond to a variety of nociceptive stimuli, including noxious touch and temperature, with stereotyped escape responses through activation of multimodal nociceptors. How behavioral responses to these different modalities are processed and integrated by the downstream network remains poorly understood. By combining trans-synaptic labeling, ultrastructural analysis, calcium imaging, optogenetics and behavioral analyses, we uncovered a circuit specific for mechanonociception but not thermonociception. Notably, integration of mechanosensory input from innocuous and nociceptive sensory neurons is required for robust mechanonociceptive responses. We further show that neurons integrating mechanosensory input facilitate primary nociceptive output by releasing short neuropeptide F, the Drosophila neuropeptide Y homolog. Our findings unveil how integration of somatosensory input and neuropeptide-mediated modulation can produce robust modality-specific escape behavior.

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Figure 1: A08n neurons receive synaptic input from C4da neurons.
Figure 2: DP-ilp7 neurons are connected to multiple classes of sensory neurons.
Figure 3: A08n neurons are necessary and sufficient for nociceptive responses downstream of C4da neurons.
Figure 4: DP-ilp7 neurons integrate multi-mechanosensory input and facilitate nociceptive behavior.
Figure 5: sNPF signaling from DP-ilp7 to mechanosensory neurons is necessary for mechanonociceptive behavior.
Figure 6: Mechanonociceptive circuit elements and sNPF-R function are not required for C4da neuron-dependent thermonociception.
Figure 7: DP-ilp7 activity and sNPF signaling are required for C4da neuron presynaptic facilitation and A08n neuron responses.

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Acknowledgements

Stocks obtained from the Bloomington (NIH P40OD018537), VDRC and Kyoto Drosophila Stock Centers were used in this study. Antibodies were obtained from the Developmental Studies Hybridoma Bank (created by the NICHD/NIH, maintained at The University of Iowa). We thank S. Sigrist for UAS-Brp-short-mCherry and UAS-Dα7-GFP, M. Gallio for the UAS-spGFP1-10-Syb, G. Tavosanis for the LexOP-brp-short-mCherry and K. Yu for sNPF and sNPF-R lines; J. Veenstra for sNPF antibodies; D. Pauls for discussion and reagents; T. Oertner, S. Wiegert, D. Pauls, J. Parrish, S. Zhu, C. Han, K. Duncan and F. Calderon for critical reading of the manuscript; and W. Grueber, A. Cardona and M. Zlatic for communicating results before publication. This work was supported by a US National Institutes of Health (NIH) grant R01GM100027 (to A.G. and C.H.Y.), the Landesförschungsförderung LFF-FV27 (to. P.S.) and the Deutsche Forschungsgemeinschaft priority program SPP1926, project SO1337/2-1 (to P.S.).

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Author notes

  1. Chun Hu, Meike Petersen and Nina Hoyer: These authors contributed equally to this study.

Authors and Affiliations

  1. Center for Molecular Neurobiology (ZMNH), University Medical Center Hamburg-Eppendorf, University of Hamburg, Hamburg, Germany

    Chun Hu, Meike Petersen, Nina Hoyer, Bettina Spitzweck, Federico Tenedini, Denan Wang, Alisa Gruschka, Lara S Burchardt, Emanuela Szpotowicz, Michaela Schweizer & Peter Soba

  2. Department of Neurobiology, Duke University Medical Center, Durham, North Carolina, USA

    Ananya R Guntur & Chung-Hui Yang

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  1. Chun Hu
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Contributions

The study was designed by C.H., M.P., N.H. and P.S. and was directed and coordinated by P.S. Morphological analyses were performed by C.H., M.P., B.S., F.T. and A.G. The behavioral experiments were performed by C.H., N.H., M.P., B.S., F.T. and L.S.B. Calcium imaging was performed by C.H. EM analysis was performed by D.W., F.T., E.S. and M.S. Transgenic lines were generated by M.P., A.R.G. and C.-H.Y. P.S. prepared the manuscript with input from all authors.

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Integrated supplementary information

Supplementary Figure 1 Characterization of A08n neuron connectivity

(a) Co-labeling of C4da (ppk-CD4-tdTomato) and A08n neurons (6.14.3-Gal4, UAS-CD4-tdGFP). C4da axon terminals overlap with ladder-like projections of A08n neurons. A08n somata and additional neurons labeled by 6.14.3-Gal4 are indicated by arrows. Scale bar, 50 μm. (b) Flip-out labeling of 6.14.3-Gal4 expressing cells (UAS-CD4-tdTomato) shows that ladder-like projections are specific to A08n neurons. Scale bars, 50 μm. (c) A08n neurons were co-labeled by 6.14.3-Gal4 and 82E12-LexA expressing CD4-tdGFP and CD4-tdTomato, respectively. A08n somata are indicated by arrows. Scale bar: 50μm. (d) A08n split-Gal4 (82E12-Gal4AD, 6.14.3-Gal4DBD, UAS-CD4-tdGFP) expression pattern and co-labeling with C4da axon terminals (ppk-CD4-tdTomato). A08n somata and additionally weakly labeled A01b neurons are indicated by arrows. Scale bar, 50 μm. (e) Model of A08n and C4da neuron innervation A08n and synaptic overlap in VNC cross-sections. (f) A08n neurons expressing presynaptic Brp-short-mCherry and postsynaptic Dα7-GFP markers. Scale bar: 30 μm. (g) Syb-GRASP control. Expression of spGFP1-10-Syb in C4da neurons alone shows no reconstituted GFP signal. Scale bar, 25 μm.

Supplementary Figure 2 Characterization of DP-ilp7 neuron connectivity

(a) Co-labeling of C4da axon terminals (ppk-CD4-tdTomato) with ilp7 neurons (ilp7-Gal4, UAS-CD4-tdGFP) with arrows indicating DP-ilp7 somata. Scale bar, 50 μm. (b,b’) DP-ilp7 neuron morphology visualized by Flp-mediated CD4-tdTomato labeling together with Fas3-labelled sensory axons (C2da-C4da). Scale bar, 25 μm. (b’) Enlarged view of boxed area in (a) and resliced maximal projections along the YZ and XZ axis (indicated by dotted lines) show overlap of DP-ilp7 neurites with anti-Fas3 labeled sensory terminals. Scale bars, 5 μm. (c) Schematic projection patterns of sensory da neurons and DP-ilp7 neurons is shown. Boxed region is displays enlarged dorsal view (top panel) and cross-section view (bottom panel) indicating sites of sensory and DP-ilp7 neuron contact in ventromedial and -lateral regions of the VNC neuropil. (d) ilp7 neurons expressing presynaptic Brp-short-mCherry and postsynaptic Dα7-GFP markers together with anti-Fas3 labeling. Scale bar, 25 μm. (d’) Corresponding 3D-view of the boxed regions in (d) shows specific overlap of DP-ilp7 postsynaptic sites with both ventro-medial and -lateral Fas3 positive sensory terminals. Scale bar, 5 μm. (e) Syb-GRASP control. Expression of spGFP1-10-Syb in sensory da neurons (2-17-Gal4) alone shows no reconstituted GFP signal. Scale bar: 25 μm. (f) Pan-da neuron expression of CD4-tdGFP (21-7-Gal4) shows sensory terminal overlap with DP-ilp7 neurons (ilp7-LexA, LexOP-CD4-tdTomato). XY maximum projections and XZ projections of indicated regions (dotted lines) are shown. Scale bars, 10 μm.

Supplementary Figure 3 Analysis of nociceptive circuit elements in larval locomotion and innocuous-touch response

(a) Larval locomotion analysis of freely moving larvae expressing Kir2.1 in C4da, A08n or ilp7 neurons and controls. Animals were video-captured for 1 min and average locomotion speed was analyzed. A mild increase in locomotion speed was observed for C4da neuron silencing with Kir2.1 compared to Gal4 control, but not for any other tested neuronal subsets (n as indicated, p<0.001, p>0.05, Mann-Whitney U test). (b) Cumulative gentle touch scores for C2/3da, C4da, A08n and ilp7 driver lines expressing Kir2.1. C2/3da neuron inactivation decreased gentle touch behavior (n as indicated, p<0.0001, Mann-Whitney U test), while silencing of other subsets showed no defect in innocuous touch behavior (n as indicated, p>0.05, Mann-Whitney U test). (b’) Touch response scores according to Kernan et al.79.

Supplementary Figure 4 Functional analysis of A08n and ilp7 neuron connectivity and behavior

(a-b) Expression pattern of CsChrimson-GFP using (a) ppk-Gal4 or (b) 27H06-LexA in the (a,b) periphery and (a’,b’) CNS. High level expression of CsChrimson with ppk-Gal4 resulted in partial dendrite and axon degeneration of C4da neurons. Scale bars, 50 μm. (c-f) A08n neurons are sufficient to elicit nociceptive responses. Flipase-induced mosaic expression of CsChrimson-GFP in only one or few neurons. 3rd instar larvae were tested for light induced nociceptive behavior and subdivided into rollers/non-rollers. Larval brains of each group were dissected and examined for their expression pattern. (c) Schematic larval brain with expression pattern of 82E12-Gal4 showing the nomenclature used for labeled cell clusters. (d) Representative images of larval brains dissected after behavioral analysis. CsChrimson-GFP expressing neurons of larvae displaying no reaction (“non-rollers”) or nocifensive rolling (“rollers”) after activation were identified and assigned according to (c). Scale bars, 50 μm. (e) Percentage of animals displaying non- vs. rolling with particular cells expressing CsChrimson. Labeling of A08n neurons was significantly enriched in rolling vs. non-rolling larvae (p<0.001, two-tailed unpaired Student’s t-test). All other neurons except s2 and l2 showed no enrichment in labeling in rolling larvae (p>0.05, two-tailed unpaired Student’s t-test). (f) To exclude a role for s2 and l2 cells, the number of larvae showing light induced rolling (“rollers”) labeling A08n plus the indicated cell types showed that s2 and l2 cells are not correlated with nocifensive responses, but were co-expressing CsChrimson together with A08n. (g) Kir2.1 mediated inactivation of C4da neurons did not impair A08n induced optogenetic rolling responses (n as indicated, p>0.05, χ 2 test). (h) Rolling induced by optogenetic activation of A08n neurons was not affected by co-activation or silencing of ilp7 neurons (n as indicated, p>0.05, χ2 test). (i) Syb-GRASP between ilp7 presynaptic sites and A08n neurons shows no reconstituted GFP signal. Scale bar, 25 μm. (j) Syb-GRASP between A08n presynaptic sites and ilp7 neurons shows reconstituted GFP signal along the medial axon projections of A08n neurons. Scale bar, 25 μm. (j’) Enlarged view from (j’). Scale bar, 10 μm. (k) Syb-GRASP control. Expression of spGFP1-10-Syb in A08n neurons alone shows no reconstituted GFP signal. Scale bar, 25 μm. (l) Optogenetic activation of A08n or ilp7 neurons does not result in calcium responses of DP-ilp7 or A08n neurons respectively (n as indicated, p>0.05, Mann-Whitney U test).

Supplementary Figure 5 Behavioral analyses of DP-ilp7 and innocuous-touch neuron function

(a) CsChrimson-GFP expression pattern using DP-ilp7-LexA shows specific signal in DP-ilp7 neurons only. Scale bar: 50 μm. (b) Characterization of C2da neuron specific expression of R78A01-Gal4 using UAS-CD4-tdGFP together with ppk-CD4-tdTomato co-labeling C4da neurons. C2da neuron specific axon terminals are labeled in abdominal segments. Thoracic segments show partial labeling of C1da neuron projections. Arrows indicate medial terminal projections, arrowheads lateral, C2da neuron specific terminal projections. (b’) R78A01-Gal4 expresses in a subset of C2da neurons located on the dorsal (ddaB) and ventral (ldaA) body wall. However, the two ventral C2da neurons are not labeled. Scale bars, 30 μm. (c) Mechanonociceptive behavior of 3rd instar larvae at 96h AEL after the first (I) and second (II) mechanical stimulation with a 45 mN von Frey filament. Kir2.1 mediated silencing of C3da or C2/3da neurons inhibits mechano-nociceptive responses (n as indicated, p<0.001, χ2 test). (d) Tetanus toxin light chain (TnT) mediated silencing of C2/3da or A08n neurons strongly inhibits mechano-nociceptive responses (n as indicated, p<0.001, χ 2 test). (e) Tetanus toxin light chain (TnT) mediated silencing of C2/3da but not C2da neurons alone impairs innocuous touch behavior (n as indicated, p<0.001, Mann-Whitney U test). (f) Animals lacking ilp7 peptide (ilp7ko) show significantly increased mechano-nociceptive responses after the 1st stimulus (p<0.001, chi2 test) but not after the 2nd stimulus (n as indicated, p>0.05, χ 2 test).

Supplementary Figure 6 Ilp7 neuron-derived sNPF is required for mechanonociception

(a) sNPF is expressed in DP-ilp7 but not in other ilp7 neurons. The sNPF expression pattern was visualized with a MIMIC enhancer trap expressing GFP under control of the endogenous sNPF promoter (sNPFMI01807). Co-labeling of ilp7 neurons (ilp7-LexA,LexAop-CD4-tdTomato) showed overlapping expression in DP-ilp7 (arrowheads) but not posterior ilp7 neurons (arrows). Scale bar, 50 μm. (b) Anti-sNPF and anti-Fas3 staining of 3rd instar larval brain preparations of w1118 control, sNPFc00448 or sNPFMi01807 homozygous animals showed strongly reduced sNPF levels in both mutant alleles. Scale bar, 50 μm. (c) sNPF-RNAi in ilp7 but not C4da neurons impairs mechano-nociceptive responses (n as indicated, p<0.05, χ2 test). (d) Heterozygous sNPF mutant animals (sNPFc00448) show impaired mechano-nociceptive responses which are enhanced by ilp7 neuron specific sNPF knockdown by RNAi. Expression of sNPF1/2 in ilp7 neurons partially rescues rolling behavior compared to sNPF-RNAi (n as indicated, p<0.05, χ2 test). (e) Inhibition of sNPF-R function in C2/3da or C4da neurons expressing a dominant negative variant did not impair innocuous touch responses (n as indicated, p>0.05, Mann-Whitney U test). (f) Inhibition of sNPF-R function in basin neurons using RNAi mediated knockdown or a dominant negative variant did not impair mechano-nociceptive responses (n as indicated, p>0.05, χ2 test).

Supplementary Figure 7 Modality-specific requirement of DP-ilp7 neuron activity and sNPF function in mechanonociception

(a) Latency of nociceptive rolling responses was measured in 3rd instar larvae at 96h AEL after local simulation with a probe heated to 46 °C. Kir2.1 mediated silencing of ilp7 neurons does not induce thermo-nociceptive defects (n as indicated, p>0.05, Kruskal-Wallis ANOVA). (b) Inhibition of sNPF-R function in C4da neurons expressing a dominant negative variant did not significantly impair thermo-nociceptive responses (n as indicated, p>0.05, Kruskal-Wallis ANOVA). (c) Schematic of testing activity and sNPF function in C4da presynaptic and A08n neuron calcium responses. (c’) shows larval preparation used for calcium imaging with optical access to the VNC and von Frey filament stimulation of an abdominal segment (a4) on the intact posterior body wall. (d) Mechano-nociceptive calcium responses of A08n neurons (82E12-Gal4, UAS-GCaMP6m) after mechanical stimulation of abdominal segment 4-6 (45 mN) with and without ilp7 neuron inhibition by Kir2.1. (d’) Maximum responses (ΔFmax/F0) were plotted and compared (n=11/genotype, p<0.05, Mann-Whitney test). (e) Modality specific nociceptive circuit function requires distinct circuit and neuromodulatory components. Harsh mechanical touch activates C2-C4da neurons, which results in activation of DP-ilp7 neurons (indicated by color coded arrows). sNPF derived from DP-ilp7 neurons is required for paracrine feedback signaling via sNPF-R in C2/3/4da neurons (light blue arrows). sNPF-R signaling in turn results in C4da neuron presynaptic facilitation and successive increase in A08n response to trigger nociceptive escape behavior. Thermo-nociceptive behavior is likely encoded by additional circuit components independent of the identified network elements. (f) Synaptic model of sNPF and sNPF-R action. Activity results in local release of sNPF from DP-ilp7 neurons. Released sNPF can then activate sNPF-Rs expressed in sensory C2/C3/C4da neurons in a paracrine fashion.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 and Supplementary Table 1. (PDF 2042 kb)

Supplementary Methods Checklist (PDF 231 kb)

A08n response to C4da neuron activation

A08n Calcium response after light induced C4da neuron activation (6.14.3-Gal4, UAS-GCaMP6m;27H06-LexA, LexAop-CsChrimson). Note that local interneurons labeled by 6.14.3-Gal4 are not activated by the 625nm light stimulus. (AVI 3278 kb)

Optogenetic activation of C4da neurons

Retinal-fed 3rd instar larvae expressing CsChrimson in C4da neurons (27H06-LexA, LexAop-CsChrimson) were exposed to 625nm light for 5s which induced larval rolling, writhing and locomotion speedup. (AVI 14234 kb)

Optogenetic activation of A08n neurons

Retinal-fed 3rd instar larvae expressing CsChrimson in A08n neurons (6.14.3-Gal4, UAS-CsChrimson) were exposed to 625nm light for 5s which recapitulates the C4da induced nocifensive response. (AVI 14041 kb)

DP-ilp7response to C1da-C4da neuron activation

DP-ilp7 Calcium response after light induced C1-C4da neuron activation (21-7-Gal4>CsChrimson;ilp7-LexA>GCaMP6m). (AVI 374 kb)

Optogenetic activation of C2da neurons

Retinal-fed 3rd instar larvae expressing CsChrimson in C2da neurons (ppk-Gal80; R78A01-Gal4,UAS-CsChrimson) were exposed to 625nm light for 5s which induced C-shaped bending and slow larval rolling without writhing and locomotion speedup. (AVI 8280 kb)

Optogenetic activation of C3da neurons

Retinal-fed 3rd instar larvae expressing CsChrimson in C3da neurons (tsh-Gal80;19-12-Gal4,UAS-CsChrimson) were exposed to 625nm light for 5s which induced contraction (“hunching”) of all body segments. (AVI 4740 kb)

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Hu, C., Petersen, M., Hoyer, N. et al. Sensory integration and neuromodulatory feedback facilitate Drosophila mechanonociceptive behavior. Nat Neurosci 20, 1085–1095 (2017). https://doi.org/10.1038/nn.4580

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