Background

Nociception, the innate ability to detect and avoid noxious stimuli, is highly conserved across the animal kingdom. Drosophila melanogaster larvae are capable to detect and avoid a variety of noxious stimuli including noxious touch, heat and light (Tracey et al., 2003; Hwang et al., 2007; Xiang et al., 2010). Mechanical stimulation above a certain threshold (> 30 mN) elicits a stereotyped rolling escape response at all larval stages (Almeida-Carvalho et al., 2017), which is thought to have evolved to avoid ovipositor injection by parasitic wasps such as L. boulardi (Hwang et al., 2007). This escape response is mediated by activation of nociceptive C4da neurons, which possess sensory dendrites covering the entire body wall allowing the animal to detect noxious cues. C4da neurons express several mechanosensory channels belonging to the DEG/ENaC family (pickpocket [ppk], ppk26/balboa) (Zhong et al., 2010; Gorczyca et al., 2014; Guo et al., 2014; Mauthner et al., 2014), a mechanosensitive TrpA1 isoform (Zhong et al., 2012), piezo (Kim et al., 2012) and the Trp channel painless (Tracey et al., 2003), all of which are required for normal mechanonociceptive responses.

The escape response can be assayed by using a von Frey filament exerting a force between 30-120 mN, which activates mechanosensory channels in C4da neurons (Hwang et al., 2007; Kim et al., 2012). Recent work has also shed light on circuit mechanisms required for mechanonociceptive responses. Mechanically induced escape responses require co-activation of class II da (C2da) and class III da (C3da) sensory neurons, as silencing of either subset impaired rolling behavior (Hu et al., 2017). Moreover, this sensory integration is specific for mechanonociception and in addition requires neuropeptide-mediated feedback. We provide a detailed protocol from our recent work (Hu et al., 2017), which employed a mechanonociception assay based on previously described methods (Hwang et al., 2007; Caldwell and Tracey, 2010; Zhong et al., 2010). We typically use a mechanical force of 45-50 mN, which elicits weak responses after the first stimulus, but enhanced responses after a second subsequent stimulus. This approach allows assaying changes in sensitivity and sensitization of mechanonociceptive responses, which can be coupled with genetic approaches to identify molecular and network components required for normal escape behavior.