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

- To prepare Drosophila larvae for a heat probe assay, collect animals of the desired developmental stage, and transfer single larvae onto a test plate with a thin film of water. The water allows the larvae to move freely without sticking to the plate surface.

Drosophila larvae detect noxious thermal stimuli through multidendritic sensory neurons in their epidermis known as nociceptors. These don't fire until a certain sensory threshold is reached, such as potentially harmful high temperatures.

Set the heat probe to a temperature that will overcome this sensory threshold, and cause the nociceptors to fire. Gently press the tip of the probe against the larva to apply heat. The larva will respond to the noxious temperature with thermo-nociceptive behaviors, withdrawing from the probe. Stop when the larva exhibits a 360-degree role away from the heat probe, or when a defined time has elapsed. Record the response time.

In the example protocol, we will see a demonstration of a localized heat probe assay on early third instar Drosophila larvae.

- First, preset the temperature of the thermal probe to the desired set point. Use a paintbrush or forceps to gently transfer an individual larva onto a flat platform, ensuring that the lava is covered by a thin film of water.

- In order to minimize the variation in pressure, as well as to ensure the correct location and angle of the heat probe, practice makes perfect.

- Gently press the probe tip against the larva at segment A4, applying light pressure with the tip at about a 45-degree angle between the probe and the surface of the larva. The pressure should cause a slight indentation on the surface of larva, and will usually be sufficient to prevent locomotion. Continue stimulating the larva until a withdrawal response is exhibited or until the 20-second cutoff is reached.

Responding larvae typically first show a preliminary behavior of lifting the head and tail, followed by the withdrawal behavior of rolling at least 360 degrees. Once the withdrawal behavior is initiated, release contact with the probe, and record the latency, or time taken to withdrawal. If no withdrawal behavior is observed within 20 seconds, then the larva is a non-responder.