In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors 1,2 while the second involves a wholesale (global) activation of most or all such neurons 3 . Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species 1,2 . The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology [4][5][6] In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus 7 . Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus 8 . In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli 9 in addition to their recently discovered role as photoreceptors 10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells 11,12 . The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system 11 where they may connect to second-order neurons that project to the brain.Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates. Video LinkThe video component of this article can be found at https://www.jove.com/video/3837/ ProtocolA typical outline of the experimental steps for preparing normal or UV-sensitized larvae for the two thermal nociception assays is shown in Figure 1.

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