Local Heat Probe Assay: A Method to Assess Nociception of High Temperatures in Drosophila Larvae

Overview

Drosophila larvae have dedicated sensory neurons–nociceptors–whose response to potentially harmful thermal stimuli triggers aversive–or so-called "nocifensive"–behaviors in the animal. This video describes a method to assess thermal nociception by recording larval behavior in response to local application of a heat probe onto the cuticle. This method is, therefore, called the local heat probe assay.

Protocol

This protocol is an excerpt from Chattopadhyay et al., Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae, J. Vis. Exp. (2012).

1. Local Heat Probe Assay

We deliver a noxious thermal stimulus to an individual larval body segment using a custom-built thermal probe manufactured by Pro-Dev Engineering (see Table of materials). Although this probe has optimal design features (a small metal tip of ~ .07 mm² area and the ability to precisely maintain a set point temperature from 23 °C to 65 °C) in principle any tool with a small tip that can be heated to a defined temperature for a period of up to 20 seconds should suffice. The probe tip is used to stimulate early 3^rd instar larvae precisely on the dorsal midline at abdominal segment A4 (see Figure 1). In response to this thermal stimulus, larvae will generally exhibit an aversive withdrawal behavior of rolling laterally by 360 degrees or more. This behavior is distinct from their light touch response to a non-noxious room temperature metal probe which generally involves a brief pause in their locomotory activity.

Protocol for the heat probe assay:

1. Pre-set temperature of the thermal probe to desired set point.
2. Use a paintbrush or forceps to gently transfer an individual larva onto a flat platform (we usually use a small piece of vinyl cut from a binder) on which the larvae will be subsequently stimulated. The larva should be covered by a thin film of water before contact with the thermal probe. The film of water covering the larva should be as little as possible and completely cover the larva, while ensuring that the larva is not dry when touching the vinyl.
3. Gently press the probe tip against the larva at segment A4 applying light pressure with the tip at about a 45° angle between the probe and the surface of the larva (see Figure 1). The pressure should cause a slight indentation on the surface of the larva and will usually be sufficient to prevent locomotion. If the larva continues to move, apply slightly more pressure and it will usually stop. Do not record data from larvae that move beyond the field of view or for which constant probe contact cannot be achieved until response or the 20 s cutoff.
4. Continue stimulating the larva until a withdrawal response is exhibited or until the 20 second cutoff is reached, whichever occurs first. Responding larvae typically first show a preliminary behavior of lifting the head and tail. This is usually followed by the withdrawal behavior of rolling at least 360 degrees. Only a complete roll of 360 degrees is scored as nocifensive behavior (the preliminary head or tail raise is not).
5. Once the withdrawal behavior is initiated release contact with the probe and record the latency or time to withdrawal. If no withdrawal behavior is observed within 20 seconds, then the larva is a non-responder. The responders can be further divided into 2 categories. If the withdrawal behavior is shown within 5 seconds, then the larva is a fast-responder. If the withdrawal behavior is shown between 5-20 seconds, then the larva is a slow-responder (see Figure 1 in Babcock et al., Curr. Biol. (2009)).

Representative Results

Figure 1: Experimental set up for local heat probe assay. The heat probe is controlled by a thermal control unit which is used to set and maintain the temperature of the probe. The probe is held perpendicular to the anteroposterior axis and used to stimulate the larva at an angle of 45° to the horizontal. Probe contact is made specifically at abdominal segment A4 as shown. The user must maintain this contact with gentle pressure up until the 20 second cutoff or until the rolling behavior commences. If the temperature is perceived as noxious, the larva will show an aversive withdrawal behavior characterized by at least one 360° roll. The number of rolls can be single or multiple (See Figure 2B). Please click here to view a larger version of this figure.

Figure 2: Quantification of behavioral response using the heat probe assay. (A) Plot of the percent of responders belonging to each category (fast-, slow-, and non-responders) versus temperature. (B) Plot of the latency to aversive withdrawal behavior versus the number of rolls each larva exhibited at four different test temperatures of increasing noxiousness (42 °C, 44 °C, 46 °C, 48 °C, 50 °C, and 52 °C). Please click here to view a larger version of this figure.

Materials

Name	Company	Catalog Number	Comments
Thermal Probe	Pro-Dev Engineering	Custom-built on demand	Contact information can be provided on request
Leica DFC290 12v/400mA Color camera	Leica Microsystems	12730080	Any equivalent camera will do.
Leica MZ6 microscope	Leica Microsystems	Part number for MZ6 zoom body (optics carrier) is 10445614
Schott Ace Modulamp Unit	Schott AG	A20500
Schott Dual Gooseneck 23 inch Fiber Optic Light Guide	Schott AG	Schott A08575
Thermal Control Unit	TSCI corp.	Custom Built	Details can be provided on request
Zeiss Stemi 2000 microscope	Carl Zeiss, Inc.	NT55-605	Any equivalent microscope will do.
Forceps	Fine Science Tools	FS-1670
Paintbrush	Blick Art Materials	06762-1002
35 X 10 mm Polystyrene Petri Dish	Falcon BD	351008	We have not tested alternative dishes.
Piece of vinyl	Office Depot	480009