Novel Assay for Cold Nociception in <em>Drosophila</em> Larvae

Abstract

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (≤10 °C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated. Here we describe and characterize the first noxious cold (≤10 °C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Identification of a neural basis for cold acclimation in Drosophila larvae

“…Cold-evoked behaviors were assessed using the previously developed cold-plate and cold-probe assays (Patel and Cox, 2017;Turner et al, 2016Turner et al, , 2017. For cold-plate, wandering 3 rd instar larvae were acclimated to a room-temperature aluminum arena.…”

Drosophila as a Model to Study the Mechanism of Nociception

“…Flies cannot be exposed to low temperatures for a long time, and their behavior prefers warmer temperatures, but the mechanism by which they perceive and avoid cold stimuli has not been studied until recently. The method of measuring the cold nociception of Drosophila larvae is to place the cold probe at a 45° angle to the back of the larva, and apply enough pressure downward to make the surface of the larva slightly concave while allowing it to move forward or backward ( Turner et al, 2017 ). Keep the cold probe still for 10 s until the cold-induced response of the Drosophila larvae is observed, which is the response latency period ( Figure 1C ).…”

Protocols for measuring cold-evoked neural activity and cold tolerance in Drosophila larvae following fictive cold acclimation

“… After a length of time determined by your experimental design, remove each Petri dish and continue at either step 8 ( larval dissection and single unit electrophysiological recordings ) or step 13 ( cold-tolerance assay by cold plate ). Note: Following fictive cold acclimation, one might also perform behavioral analyses by either the cold-plate ( Himmel et al., 2020 ; Patel and Cox, 2017 ) or cold-probe assays ( Turner et al., 2017 ), as previously described. …”

“…Note: Following fictive cold acclimation, one might also perform behavioral analyses by either the cold-plate ( Himmel et al., 2020 ; Patel and Cox, 2017 ) or cold-probe assays ( Turner et al., 2017 ), as previously described.…”