An Improved Assay and Tools for Measuring Mechanical Nociception in Drosophila Larvae

This protocol is significant because it allows the user to build a tool that can be used to precisely measure mechanical nociception responses in genetically tractable Drosophila larvae. The main advantage of this technique is that it uses simple materials to build custom tools that can be used to measure mechanical nociception from the subthreshold to fully-responsive range. These tools and methods can be used to measure baseline mechanical nociception and nociceptive sensitization following injury or disease.

Probing larvae with custom filaments takes practice. Users should be able to generate dose-response curves with control larvae before attempting an experiment. To construct a mechanical probe, use a small wire cutter to cut each Nitinol filament to the specified length perpendicular to its long axis.

The filaments come in three different preset diameters. Examine the tip of the filaments under a stereo microscope to ensure that no sharp or irregular edges remain that could cause tissue damage to the larva skin and interfere with the calibration. Then use a sharpening stone to manually smooth the edges of the probe until no sharp irregularities persist.

Next, use a hypodermic needle to poke a hole near the end of a wooden Popsicle stick. Apply wood glue to a single Nitinol filament and insert the glue-coated filament into the needle slot. When the glue has dried, press the probe against the scale until the probe bends to determine the maximum force that can be recorded in grams.

Use the formula to convert the recorded mass to force in millinewtons. Then divide the measured force by the surface area of the filament tip to convert the calculated force to kilopascals of pressure. Preparing multiple probes using filaments of different diameters and lengths will generate a full set, spanning the responsive range for Drosophila larvae.

To prepare Drosophila larvae for an experiment, raise the larval progeny on standard food in a 25 degree Celsius incubator for about 96 hours. When the larvae reach the third instar stage, prepare a plug of fly food in a small Petri dish for larvae to be tested and use forceps to gently sort the medium sized mid-third instar larvae from the smaller second and early third instar and the larger late or wandering third instar larvae. Then use the forceps to transfer 20 to 30 mid-third instar larvae into a small Petri dish containing a small plug of fly food moistened with water at room temperature.

To perform a mechanical nociception assay, use forceps to place a mid-third instar larva onto a dark thin vinyl pad under a Brightfield stereo microscope and arrange optical fiber lights between the microscope objective lenses and the pad. Use a paper towel to wipe away any excess water surrounding the larva and move the pad to orient the head and mouth of the larva toward the non-dominant hand of the researcher. Select a mechanical probe and apply the probe to the posterior dorsal side of the larva at approximately abdominal segment A8 for one to two seconds, carefully compressing the larva into the underlying pad at the point of probe contact until the probe bends and elicits the previously measured amount of pressure.

A positive nociceptive response is indicated if the larva shows a complete corkscrew roll of 360 degrees along the axis of its body within three seconds. Record the behavioral response for each larva. Then discard the tested larva and prepare the next larva for the assay.

These customized mechanical probes with Nitinol filaments can be used to elicit mechanically evoked behaviors and to generate a full behavioral dose-response curve using both innocuous and noxious mechanical probes of varying intensity. As these behavioral assay results demonstrate, probes that exert pressure of 200 kilopascals or less do not provoke an aversive rolling response in Drosophila larvae. As expected, these subthreshold or non-noxious mechanical probes do not elicit visible neuronal tissue damage.

Conversely, super threshold or noxious probes elicit an augmented behavioral response as well as tissue damage to the peripheral sensory neurons themselves in a dose-dependent manner. These probes can also be used to measure mechanical hypersensitivity after injury. Approximately 20%of larvae respond with aversive rolling as early as two hours after UV treatment, while 50%responded four hours compared to mock UV irradiated animals.

Because the probe used for this analysis was a normally subthreshold 200 kilopascal, this response was considered to be mechanical allodynia. At later time points, the behavioral response of the UV-treated larvae is slightly increased but not statistically different from that of the mock irradiated control group. Larvae probed with a 462 kilopascal probe at four, eight, and 16 hours following UV treatment exhibit a significant increase in the aversive rolling response with four hours being the peak of the behavioral hypersensitivity as the stimulus was initially noxious.

The response was considered to be mechanical hyperalgesia. Probe preparation and larva selection and compression are critical to the success of the process. Using these probes brought the baseline nociception and mechanical nociceptive hypersensitivity can be measured following injury.

Using these tools and assay, the molecular and genetic bases of mechanical nociception and nociceptive hypersensitivity can be measured in the genetically tractable Drosophila model.