Chloride-dependent mechanisms of multimodal sensory discrimination and neuropathic sensitization in Drosophila

Abstract

Individual sensory neurons can be tuned to many stimuli, each driving unique, stimulus-relevant behaviors, and the ability of multimodal nociceptor neurons to discriminate between potentially harmful and innocuous stimuli is broadly important for organismal survival. Moreover, disruptions in the capacity to differentiate between noxious and innocuous stimuli can result in neuropathic pain. Drosophila larval Class III (CIII) neurons are peripheral noxious cold nociceptors and gentle touch mechanosensors; high levels of activation drive cold-evoked contraction (CT) behavior, while low levels of activation result in a suite of touch-associated behaviors. However, it is unknown what molecular factors underlie CIII multimodality. Here, we show that the TMEM16/anoctamins subdued and white walker (wwk; CG15270) are required for cold-evoked CT, but not for touch-associated behavior, indicating a conserved role for anoctamins in nociception. We also evidence that CIII neurons make use of atypical depolarizing chloride currents to encode cold, and that overexpression of ncc69—a fly homologue of NKCC1—results in phenotypes consistent with neuropathic sensitization, including behavioral hypersensitization and spontaneous nociceptor activity, making Drosophila CIII neurons a candidate system for future studies of the basic mechanisms underlying neuropathic pain.

Modality specific roles for metabotropic GABAergic signaling and calcium induced calcium release mechanisms in regulating cold nociception

“…To assess innocuous mechanical sensitivity of Drosophila larvae, we performed gentle touch assays using a fine brush bristle on animals with CIII md neuron specific gene knockdowns for GABA B -R2, IP 3 R or RyR (Kernan et al, 1994). Responses of Drosophila larvae to innocuous mechanical stimulation include pause, head withdrawal, turn and/or reverse locomotion, where an animal receives a score for performing each behavior, max score of 4 for each trail ( Figure 3H ) (Himmel, Sakurai, et al, 2021; Kernan et al, 1994; Turner et al, 2016). Gentle touch-evoked responses of GABA B -R2 and IP 3 R knockdown in CIII md neurons were not significantly different from controls ( Figure 3I-J ).…”

“…Additionally, increasing intracellular Cl − concentration in CIII md neurons via ncc69 overexpression, orthologous to human NKCC1, resulted in increased bursting activity. Cl − ion homeostasis is not required for innocuous mechanical sensation but it is required for cold nociception, which suggests similar functional roles in sensory discrimination for GABA B -R2, IP 3 R and stimulus evoked Cl − current in Drosophila multimodal CIII neurons (Himmel, Sakurai, et al, 2021). However, it remains unclear whether Drosophila Anoctamins function in conjunction with GPCRs, IP 3 R or RyR for stimulus evoked activation of cold sensitive pathways.…”

“…We tested age matched third instar Drosophila larvae for behavioral responses to innocuous mechanical stimulation. (Himmel, Sakurai, et al, 2021; Kernan, 1994; Turner et al, 2016) We placed third instar larvae on lightly misted black metal plate, same as cold plate assay, and allowed the animals to acclimate to the new arena. Using a single brush bristle, we gently touch the anterior segments.…”

“…Specifically, Pkd2, Trpm and nompC are required in cold sensitive CIII md neurons for noxious cold thermosensation and innocuous mechanosensation (Turner et al, 2016). Recent work has revealed chloride ion homeostasis and calcium-activated chloride channel function via the TMEM16/anoctamins, subdued and white walker ( wwk, CG15270 ), as required for cold nociception but dispensable for detection of innocuous mechanical sensory information (Himmel, Sakurai, et al, 2021) providing initial clues to molecular bases of multimodal sensory discrimination in these neurons. Here, we sought to identify molecular bases of multimodal sensory discrimination that arise from differential Ca 2+ dynamics in CIII md neurons.…”

Steroid hormone signaling activates thermal nociception during Drosophila peripheral nervous system development

“…This mechanism of Subdued activity would be dependent on the intracellular Cl − concentration created by symporters and cotransporters. The importance of Cl − regulation has recently been highlighted in the Class III da neurons, as both Subdued and depolarizing Cl − currents have recently been found to promote cold-evoked nociception ( Himmel et al, 2021 ). Therefore, identifying regulators of Cl − concentration during the development of C4da neurons and their impact on nociception will be of interest in future studies.…”