Drosophila Insulin receptor regulates the persistence of injury-induced nociceptive sensitization

Abstract

Diabetes-associated nociceptive hypersensitivity affects diabetic patients with hard-to-treat chronic pain. Because multiple tissues are affected by systemic alterations in insulin signaling, the functional locus of insulin signaling in diabetes-associated hypersensitivity remains obscure. Here, we used Drosophila nociception/nociceptive sensitization assays to investigate the role of Insulin receptor (Insulin-like receptor, InR) in nociceptive hypersensitivity. InR mutant larvae exhibited mostly normal baseline thermal nociception (absence of injury) and normal acute thermal hypersensitivity following UV-induced injury. However, their acute thermal hypersensitivity persists and fails to return to baseline, unlike in controls. Remarkably, injury-induced persistent hypersensitivity is also observed in larvae that exhibit either type 1 or type 2 diabetes. Cell type-specific genetic analysis indicates that InR function is required in multidendritic sensory neurons including nociceptive class IV neurons. In these same nociceptive sensory neurons, only modest changes in dendritic morphology were observed in the InR^RNAi -expressing and diabetic larvae. At the cellular level, InR-deficient nociceptive sensory neurons show elevated calcium responses after injury. Sensory neuron-specific expression of InR rescues the persistent thermal hypersensitivity of InR mutants and constitutive activation of InR in sensory neurons ameliorates the hypersensitivity observed with a type 2-like diabetic state. Our results suggest that a sensory neuron-specific function of InR regulates the persistence of injury-associated hypersensitivity. It is likely that this new system will be an informative genetically tractable model of diabetes-associated hypersensitivity.

Keywords: Diabetes; Drosophila; Hyperalgesia; Insulin receptor; Nociceptive sensitization; Sensory neurons.

Fig. 1.

InR mutant larvae exhibit persistent thermal hyperalgesia. (A,B) Schematics of the nociception (A) and persistent nociceptive sensitization (B) assays. (C-E) Quantitation of nociceptive behavioral responses to thermal stimulation at 43°C of InR mutant larvae. w¹¹¹⁸ control larvae, two heterozygous hypomorphic alleles and a transheterzygous allelic combination of InR were tested: InR^e19/+, InR^93Dj4/+, InR^e19/93Dj4. Baseline responses without UV tissue damage (n=60 for w¹¹¹⁸, n=90 for others) (C), thermal sensitivity at 8 h post-UV (n=90 for w¹¹¹⁸, n=80 for InR^e19/+, n=88 for InR^93Dj4/+, n=76 for InR^e19/93Dj4) (D), thermal sensitivity at 24 h post-UV (n=88 for InR^e19/93Dj4, n=90 for others) (E). Statistical significance was determined by the Log-rank test. ***P<0.001, ****P<0.0001.

Fig. 2.

A type 1 diabetes-like state induces persistent thermal hyperalgesia in Drosophila larvae. (A) Schematic of the genetic manipulation that induces a type 1 diabetes-like state in Drosophila larvae by silencing IPCs. (B) Representative in vivo confocal images of class IV md neuron dendritic morphology in controls and in larvae exhibiting a type 1 diabetes-like state±UV irradiation. In all panels, dendritic morphology was visualized using a ppk-CD4::tdTomato transgene. Controls: dilp2-Gal4 alone and UAS-Kir2.1 alone. Type 1 Diabetes: dilp2-Gal4>UAS-Kir2.1. (C,D) Quantitative dendritic morphology analysis measuring number of branches (C) and total dendritic length (D) presented as mean±s.e.m. n=8 neurons. Statistical significance was determined by one-way ANOVA with Bonferroni multiple comparison post hoc test. (E-G) Quantitation of nociceptive behavioral responses to thermal stimulation (43°C) in control larvae and when IPCs were silenced genetically. In all behavioral analyses, accumulated total responses were plotted as a function of latency to aversive withdrawal. Baseline behavioral responses in the absence of UV irradiation (E), thermal sensitivity at 8 h post-UV (F), thermal sensitivity at 24 h post-UV (G). n=90 larvae tested for each condition. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig. 3.

A type 2 diabetes-like state induces persistent thermal hyperalgesia in Drosophila larvae. (A) Schematic of the diet condition (high sugar) that induces a type 2 diabetes-like state in exposed larvae. (B) Representative in vivo confocal images of class IV md neuron dendritic morphology in controls (normal diet) and in larvae exhibiting a type 2 diabetes-like state (high-sugar diet)±UV irradiation. Dendritic morphology was visualized using a ppk-CD4::tdTomato transgene. (C,D) Quantitative dendritic morphology analysis measuring number of branches (C) and total dendritic length (D) presented as mean±s.e.m. n=8 neurons. Statistical significance was determined by one-way ANOVA with Bonferroni multiple comparison post hoc test. (E-H) Quantitation of nociceptive behavioral responses to thermal stimulation (43°C) in control larvae and larvae fed a high-sugar diet. Baseline responses in the absence of UV irradiation (n=90 for each condition) (E), thermal sensitivity at 8 h post-UV (n=88 for control, n=90 for high sugar) (F), thermal sensitivity at 16 h post-UV (n=53 for control, n=90 for high sugar) (G), thermal sensitivity at 24 h post-UV (n=90 for control, n=97 for high sugar) (H). **P<0.01, ****P<0.0001.

Fig. 4.

Sensory neuron-specific interference with InR function causes persistent thermal hyperalgesia. (A-D) Quantitation of thermal nociceptive behavioral responses (43°C) when UAS-InR^RNAi is expressed in md neurons. n=90 for each condition. Baseline responses in the absence of UV irradiation (A), thermal sensitivity at 8 h post-UV (B), thermal sensitivity at 24 h post-UV (C), thermal sensitivity at 42 h post-UV (D). (E) Representative in vivo confocal images of class IV md neuron dendritic morphology labeled with ppk1.9-GAL4,UAS-mCD8::GFP. Dendritic morphology was compared between control larvae expressing UAS-Luc^RNAi and larvae expressing UAS-InR^RNAi±UV irradiation. (F,G) Quantitative dendritic morphology analysis measuring number of branches (F) and total dendritic length (G) presented as mean±s.e.m. n=8 neurons. Statistical significance was determined by one-way ANOVA with Bonferroni multiple comparison post hoc test. **P<0.01.

Fig. 5.

CaMPARI analysis reveals increased cellular calcium in sensory neurons. (A) Schematic of CaMPARI experimental outline. (B) Quantitative analysis of CaMPARI responses in class IV md neurons of larvae expressing UAS-CaMPARI via md-Gal4±UAS-InR^RNAi. The CaMPARI response is calculated as the F_Red/F_Green ratio presented as mean±s.e.m. and is represented graphically, where each measured neuron is represented by a single data point, and also as a heatmap depicting the averaged CaMPARI response. On the heatmap, magenta indicates a higher F_Red/F_Green ratio and green indicates a lower ratio. n=24-45 neurons. Statistical significance was determined by one-way ANOVA with Bonferroni multiple comparison post hoc test. A key to relevant experimental variables (PC light, thermal stimulation, UV and genotype) is provided and applies to the quantitative data in B and the micrographs in C. (C) Representative in vivo confocal images of class IV md neuronal cell bodies. For each condition the F_Green, F_Red and F_RedLUT (a heatmap representation of photoconverted CaMPARI intensity) are shown. **P<0.01, ****P<0.0001.

Fig. 6.

Constitutive activation of InR causes hyposensitivity during the acute phase. (A-C) Quantitation of thermal nociceptive behavioral responses (43°C) when UAS-InR^CA is expressed in md neurons versus Gal4 and UAS alone controls. Baseline responses in the absence of UV irradiation (A), thermal sensitivity at 8 h post-UV (B), thermal sensitivity at 24 h post-UV (C). n=90 for each condition/genotype. ****P<0.0001.

Fig. 7.

Expression of InR in md neurons rescues the persistent thermal hyperalgesia of InR mutants and type 2 diabetic larvae. (A-C) Quantitation of nociceptive behavioral responses (43°C) when UAS-InR is expressed in md sensory neurons in the heterozygous InR mutant background. Baseline responses without UV irradiation (A), UV-induced acute hyperalgesia (8 h post-UV) (B), UV-induced persistent hyperalgesia (24 h post-UV) (C) (n=90 for each condition/genotype). (D-F) Quantitation of nociceptive behavior responses (43°C) when UAS-InR^CA is expressed in md sensory neurons in the high-sugar fed larvae. Baseline responses without UV irradiation (n=90) (D), UV-induced acute hyperalgesia (8 h post-UV) (n=55 for Gal4 alone, n=87 for UAS alone, n=80 for md>InR^CA) (E), UV-induced persistent hyperalgesia (24 h post-UV) (n=90) (F). ****P<0.0001.

Fig. 8.

Graphical representation of pain levels versus time postinjury, annotated across control, persistently hypersensitive genotypes and diabetic conditions, and upon constitutive activation of InR. Control, blue solid line; persistently hypersensitive genotypes and diabetic conditions, red dashed line; constitutive activation of InR, green dashed line. Landmark time points (gray vertical bars) and the likely window of ILS activity in nociceptive sensory neurons (orange arrow/text) are indicated.