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Assaying Thermo-nociceptive Behavior in Drosophila Larvae   

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Original research article

A brief version of this protocol appeared in:
Nature Neuroscience
Aug 2017


Thermo-nociception, the detection and behavioral response to noxious temperatures, is a highly conserved action to avoid injury and ensure survival. Basic molecular mechanisms of thermal responses have been elucidated in several model organisms and are of clinical relevance as thermal hypersensitivity (thermos-allodynia) is common in neuropathic pain syndromes. Drosophila larvae show stereotyped escape behavior upon noxious heat stimulation, which can be easily quantified and coupled with molecular genetic approaches. It has been successfully used to elucidate key molecular components and circuits involved in thermo-nociceptive responses. We provide a detailed and updated protocol of this previously described method (Tracey et al., 2003) to apply a defined local heat stimulus to larvae using a fast-regulating hot probe.

Keywords: Drosophila, Nociception, Temperature, Thermo-nociception, Sensory neurons, Larvae, Hot probe assay


Drosophila larvae respond to thermal stimuli above 40 °C with an escape response (Tracey et al., 2003), likely to prevent cell damage and injury. The activation of nociceptive sensory neurons, class IV dendritic arborization (C4da) neurons, is necessary and sufficient for this response (Hwang et al., 2007). Applying a local thermo-nociceptive stimulus using a heated probe (> 40 °C, Figures 1, 5, 6) typically triggers a stereotyped behavior consisting of a 360° rolling motion along the larval body axis and increased speed of locomotion. Previous studies have shown that the transient receptor potential (Trp) channel painless (Tracey et al., 2003) and TrpA1 (Neely et al., 2011; Zhong et al., 2012) are the sensory channels responding to noxious heat in this system, as their expression and function in C4da neurons is required for nociceptive escape response.

Although mechano- and thermos-nociceptive stimulation of larvae result in very similar rolling escape responses (Hwang et al., 2007; Tracey et al., 2003; Zhong et al., 2012), the involved neuronal networks seem to differ. Despite the need to touch the animal with the heated probe, gentle touch-sensitive neurons (C2da and C3da) do not play a role in the behavioral response to noxious temperatures, but are essential for mechano-nociceptive responses (Hu et al., 2017). Activation of C4da neurons with noxious heat elicits in neuronal burst firing (Terada et al., 2016), which might be sufficient to elicit strong downstream network responses to induce escape behavior. Moreover, thermo-sensitive TrpA1 expressing neurons in the CNS respond to temperature gradients and are sufficient to induce rolling behavior (Luo et al., 2017). Thus thermo- and mechano-nociception might employ distinct subsets of the nociceptive network.

Using thermo-nociceptive assays together with genetic approaches in this system has led to the identification of several molecular components (Neely et al., 2010; Honjo et al., 2016), including pathways regulating thermal sensitivity (Babcock et al., 2011; Im et al., 2015). This makes the larval nociceptive system an attractive model to identify key components and circuit mechanisms regulating thermo-nociception.

Here, we provide a detailed protocol to conduct hot probe thermo-nociception assays as used in our recent work (Hu et al., 2017), which is based on the previously developed and described method by Tracey et al. (2003). We built a custom hot probe (Figure 1) with fast control of temperature required for consistent behavioral responses. Similar setups and protocols have also been employed in various other studies with comparable results (Neely et al., 2011; Chattopadhyay et al., 2012; Zhong et al., 2012). Our protocol and setup allow assaying behavioral responses to a locally applied hot stimulus depending on C4da and downstream neuron function (Figure 6).

Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Petersen, M., Tenedini, F. M., Hoyer, N., Kutschera, F. and Soba, P. (2018). Assaying Thermo-nociceptive Behavior in Drosophila Larvae. Bio-protocol 8(4): e2737. DOI: 10.21769/BioProtoc.2737.

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