Nerve injury drives a heightened state of vigilance and neuropathic sensitization in Drosophila

Abstract

Injury can lead to devastating and often untreatable chronic pain. While acute pain perception (nociception) evolved more than 500 million years ago, virtually nothing is known about the molecular origin of chronic pain. Here we provide the first evidence that nerve injury leads to chronic neuropathic sensitization in insects. Mechanistically, peripheral nerve injury triggers a loss of central inhibition that drives escape circuit plasticity and neuropathic allodynia. At the molecular level, excitotoxic signaling within GABAergic (γ-aminobutyric acid) neurons required the acetylcholine receptor nAChRα1 and led to caspase-dependent death of GABAergic neurons. Conversely, disruption of GABA signaling was sufficient to trigger allodynia without injury. Last, we identified the conserved transcription factor twist as a critical downstream regulator driving GABAergic cell death and neuropathic allodynia. Together, we define how injury leads to allodynia in insects, and describe a primordial precursor to neuropathic pain may have been advantageous, protecting animals after serious injury.

Fig. 1. Drosophila exhibit thermal allodynia after injury.

(A) Adult thermal nociception assay developed to measure nociceptive sensitization over time in the fly. (B) Uninjured wild-type animals exhibit escape behavior in response to temperatures of ≥42°C; this response is dependent on painless and TrpA1 (n = 9 replicates, 10 animals per replicate). (C) Amputation injury used in this study. (D) Example tracking data from adult thermal pain assay, showing allodynia in the escape response (38°C) following injury. (E) Time course of allodynia response (38°C) following injury. (F) Dose response to temperature 14 days after injury (n = 9 replicates, 10 animals per replicate). (G) Average speed of movement for uninjured intact control or animals 7 days after injury in Canton S (n = 7 replicates, 10 animals per replicate). Data are represented as means ± SEM. ***P < 0.001; ns, not significant, two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for (B), (E), and (F) and Student’s t test for (G).

Fig. 2. TrpA1 is required in ppk+ sensory neurons for allodynia after injury.

(A) ppk+ sensory neuron projections in the fly leg, labeled with CD8-GFP, n ≥ 7. (B) ppk+ cell bodies in the leg, labeled with Lamin-GFP, n ≥ 7. (C) ppk+ sensory neuron projections from the dissected leg to the VNC and brain. (D) Expression of active tetanus toxin (TNT) but not inactive tetanus toxin (iTNT) in ppk+ sensory neurons blocked all adult thermal nocifensive behavior (n = 9 animals, 10 animals per replicate). (E) TrpA1 and painless mutants are resistant to thermal allodynia (38°C) (n = 9 replicates, 10 animals per replicate). (F) TrpA1 is specifically required in ppk+ sensory neurons for allodynia after injury (n = 9 replicates, 10 animals per replicate). (G) Reintroduction of TrpA1 specifically in ppk+ sensory neurons rescue allodynia response (n = 9 replicates, 10 animals per replicate). Data are represented as means ± SEM. ***P < 0.001; two-way ANOVA followed by Tukey’s post hoc test.

Fig. 3. Peripheral injury leads to sensory neuropathy, central sensitization, and augmentation of the nociceptive escape circuit.

(A) ppk+ sensory neuropathy is observed after leg amputation. (B) Quantification of sensory neuropathy (ppk1+ projection length) in the amputated leg over time (n ≥ 7). (C) Adult nociception electrophysiology preparation after injury. (D to F) Leg amputation results in contralateral sensitization of the escape response circuit with (D and E) increase in escape circuit velocity (velocity difference highlighted in purple) and (D and F) an increase in the duration of the escape response (injured duration highlighted in green) (n ≥ 9). Data are represented as means ± SEM. **P < 0.01; ***P < 0.001, two-way ANOVA followed by Tukey’s post hoc test (B) and Mann-Whitney-Wilcoxon test (E and F).

Fig. 4. GABA gates peripheral activity; peripheral nerve injury reduces GABAergic function.

(A) ppk+ sensory neuron projections to the VNC. ppk-Gal4>UAS-CD8-GFP is shown in yellow, anti-GABA is shown in green, nc82 contrast stain is shown in purple, showing ventral top, close-up view of anti-GABA and ppk-Gal4>UAS-CD8-GFP colocalization, and tangential side, close-up view of anti-GABA and ppk-Gal4>UAS-CD8-GFP colocalization, n ≥ 7. (B) Reduction in GABA immunoreactivity after injury of VNC stained for GABA and nc82 from uninjured and injured animals (7 days after leg amputation), showing ventral top, close-up view. (C) Quantification of (B), n ≥ 9. (D) Imaging of VNC GABAergic interneurons expressing ppk-Gal4>TNT, stained for GABA and nc82. (E) Quantification of (D), n ≥ 9. (F) Imaging of VNC with nuclear-labeled Lamin-GFP (Gad1-Gal4>UAS-Lamin-GFP) and an active caspase antibody. (G) Quantification of GABAergic cells in (F), n ≥ 9. (H) Quantification of active caspase/Gad1+ cells in (F), n ≥ 9. Data are represented as means ± SEM. ***P < 0.001, two-way ANOVA followed by Tukey’s post hoc test.

Fig. 5. Preventing GABAergic cell death blocks changes in the nociception circuit and suppresses neuropathic allodynia.

(A) Blocking GABAergic cell death after leg injury by Gad1-Gal4–driven expression of UAS-p35 prevents GABA loss. (B) Quantification of (A), n ≥ 9. (C and D) GABAergic-specific expression of p35 (Gad1-Gal4>UAS-p35) rescues contralateral sensitization of the escape response circuit measured by (C) escape circuit velocity, n ≥ 9; (D) escape response duration, n ≥ 9; and (E) prevents neuropathic allodynia behavior (n = 9 replicates, 10 animals per replicate). (F) Nociceptive sensory neuron–specific (ppk-Gal4) knockdown of GABA receptor D-GABA-B-R2 is sufficient to cause thermal allodynia (38°C) in uninjured flies (n ≥ 9 replicates, 10 animals per replicate). (G) Knockdown of nAChRα1 in GABAergic neurons (Gad1-Gal4>nAChRα1-IR) prevents GABA loss after injury. (H) Quantification of active caspase in GABAergic neurons (active caspase/Gad1+ cells) in control intact and injured flies expressing Lamin-GFP and nAChRα1-IR (Gad1-Gal4>UAS-Lamin-GFP; UAS-nAChRα1-IR). (I) Knockdown of nAChRα1 in GABAergic neurons prevents neuropathic allodynia behavior (n ≥ 9 replicates, 10 animals per replicate). Data are represented as means ± SEM. **P < 0.01; ***P < 0.001, two-way ANOVA followed by Tukey’s post hoc test.

Fig. 6. Twist mediates GABAergic excitotoxicity and central sensitization after neuropathic injury.

(A) GABAergic (Gad1-Gal4) knockdown of twist blocks GABAergic cell death after peripheral nerve injury, n ≥ 9. (B) Quantification of (A). (C) twist is required in GABAergic neurons for neuropathic allodynia to develop after nerve injury (n ≥ 9 replicates, 10 animals per replicate). (D) Leg amputation results in the induction of Twist protein in GABAergic neurons of the VNC. (E) GABAergic (Gad1-Gal4) knockdown of nAChR prevents the induction of Twist protein caused by leg injury. (F) Quantification of (D), n ≥ 9. (G) Quantification of (E), n ≥ 9. (H) GABAergic (Gad1-Gal4) knockdown of twist prevents increased active caspase/Gad1+ cells, n ≥ 9. (I) Model for how injury leads to thermal allodynia in the fly. Data are represented as means ± SEM. ***P < 0.001; **P < 0.01; two-way ANOVA followed by Tukey’s post hoc test.