The overall goal of the following experiment is to assay thermal nociception in drosophila larvae. This is achieved by harvesting drosophila larvae to subsequently expose them to noxious thermal stimuli as an optional second step Larvae can be UV irradiated, which causes tissue damage and thermal nociceptive sensitization. Next larva can be probed locally with a heat probe or globally using a heat plate in order to provoke nociceptive responses that can be quantitatively measured.
The heat probe shows that larvae have a ceiling to the nociceptive response to heat, and a surprising relationship between input temperature and the amplitude of the provoked response. The heat plate assay shows that larvae exhibit a stereotyped sequence of nociceptive behaviors that depend on the temperature of the surrounding water and the activity of the nociceptive sensory neurons. The methods described here can help answer key questions in the thermal nociception field, such as do animals respond differently to noxious thermal stimuli that are presented either locally or globally.
Generally individuals new to the heat probe assay will struggle because mastering the assay requires practice to minimize variation in the pressure, the location, and the conduct angle of the applied probe Visual demonstration of the heat plate assay is critical. As behaviors elicited with increasing temperature need to be seen to be best appreciated. To obtain sufficient progeny larvae of a defined genotype of interest, grow the desired stock directly or set up crosses in bottles containing fly food using 20 to 30 virgin females and 15 to 20 males five days after egg laying harvest and clean early third instar larvae by scooping out the mushy fly food and straining with gently running water through a 630 micron pore size mesh transfer the early third instar larvae to a small, moist pad of food to prevent starvation and desiccation.
After constructing an authorization chamber, use forceps or a paintbrush to gently place 10 early third instar larvae along the inside of the lid and close. Working in a fume hood, placed the authorization chamber inside a coupling jar containing a 10 milliliter beaker carrying a cotton ball soaked with 1.5 milliliters of dathyl ether. Screw on the cap of the coplan jar to expose the larvae to ether fumes for two to 2.5 minutes.
Following e authorization, remove the e authorization chamber from the Coplan jar and give it a few seconds in the hood for any residual ether fumes to dissipate. Use a water squirt bottle to gently rinse larvae from the authorization chamber into a small Petri dish. Blot the anesthetized larvae dry and then gently affix them dorsal side up on a strip of double-sided scotch tape fasten into a three inch by one inch glass microscope slide.
Place the slide on the bottom surface of a UV irradiation chamber and expose the larvae to UV light for six seconds at 20 millijoules per centimeter squared intensity. After irradiation, immerse the slide in water to gently float the larvae off of the double-sided scotch tape. Use forceps or a paintbrush to gently place the irradiated larvae in a 15 by 45 millimeter.
One dram glass culture vial containing one milliliter of fly food to recover for eight to 24 hours at 25 degrees Celsius or the desired culture temperature before rehosting them. For thermal nociception assays first preset the temperature of the thermal probe to the desired set point. Use a paintbrush or forceps to gently transfer an individual larvae onto a flat platform, ensuring that the larvae 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 larvae at segment A four, applying light pressure with the tip at about a 45 degree angle between the probe and the surface of the larvae. The pressure should cause a slight indentation on the surface of the larvae and will usually be sufficient to prevent locomotion.
Continue stimulating the larvae until a withdrawal response is exhibited or until the 22nd 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 larvae is a non-responder. Position the fiber optic light guides to ensure that there is adequate high contrast lighting. For viewing the larvae, place the heating block into the heat plate with flat surface up and then place the heat plate onto the microscope base.
Turn on the heat plate to the high setting and allow approximately 15 minutes for the temperature to stabilize at 95 degrees Celsius. Measure 80 microliters of distilled water and place the water drop in the middle of a 60 by 15 millimeter polystyrene Petri dish with a micro pipetter. Use forceps to gently place a clean mid third instar larvae into the middle of the water drop, introducing as little additional water as possible.
Gently place the Petri dish onto the solid heating block on the heat plate. Quickly adjust the location of the dish so the entire larvae and drop of water are in view. Start the timer.
At the moment, the Petri dish is placed onto the solid heating block of the heat plate and record the time of onset of each behavior.Five. Stereotyped locomotive behaviors are observed upon transfer of the larva immersed in water to the heat plate. Normal behavior in the absence of heat involves parasol locomotion, accompanied by searching head movements.
Head thrash behavior can occur next where the larva moves its head quickly in a forward or lateral motion. This motion is similar to their normal locomotion in water, but occurs more jerkily and persistently rolling. Behavior occurs when the larvae rolls laterally at least a full 360 degrees.
This can occur a variable number of times and sometimes involves incomplete rolls. For the purpose of scoring the behavior only count full 360 degree rolls. This behavior is the most similar to that scene with local application of the heat probe during whip behavior.
The larva exhibits rapid contraction release movements along the antio posterior axis that bring the head very briefly close to the tail Whipping is often observed in quick succession two or at the same time as rolling. Finally, the larva will exhibit seizure behavior often followed by paralysis. During seizure behavior, the larva stretches out along the interop posterior axis and exhibits a high frequency whole body.
Shaking behavior without bending paralysis occurs when the larvae ceases movement In some larvae, this is permanent, whereas others then exhibit a low frequency pulsation movement. In this plot of the percent responders belonging to each category, there is a ceiling to the larval thermal nociception response. In the heat probe assay, 100%of the larvae are fast responders up to a probe temperature of 52 degrees Celsius.
However, at 54 degrees Celsius and higher, 90%or more of the larvae fail to respond even after 20 seconds of contact. Although these larvae do continue moving following the 22nd cutoff, the number of rolls or time spent in aversion withdrawal behavior can also be measured and plotted. Surprisingly, there appears to be an inverse relationship between the probe input temperature and the robustness of the response as lower temperatures appear to provoke a higher number of rolls.
Five stereotyped locomotive behaviors are observed upon transfer of the larva immersed in water to the heat plate. The average latencies at which these behaviors are observed are shown here as are the average water drop temperatures measured for each latency under the optimal assay conditions presented here, the percentage of larvae showing each distinct behavior ranged from 77 to 100%although occasionally the rolling and whipping behaviors are omitted, overlap, or occur in reverse order, the percentage of larvae that's survived following initiation of paralysis behavior in the heat plate assay is shown here. Larvae were heated until paralysis and then removed to standard culture conditions to recover.
The mock treated larvae received equivalent treatment except for heat exposure and the formation of PPE in viable adults was quantitative on day seven to 13. Here, MD G four drives expression of UAS regulated transgenes in all four classes of multi dendritic peripheral sensory neurons. UAS or one delta C expresses a modified version of the drosophila open rectifier potassium channel required for synaptic transmission and UAS orrc one delta NC expresses a further modified version of the same channel that does not interfere with synaptic transmission.
Note that only larvae bearing both the MD GALF four driver and the UAS ORC one delta C transgene show increased latencies for four of the five observed behaviors. After watching this video, you should have a good understanding of how to measure both local and global responses of GEs offal larvae to noxious thermal stimuli. These procedures can be combined with other methods like genetic analysis to identify the genes required for thermal nociception While attempting the heat probe assay, it's important to remember to minimize variation and pressure, location and contact angle of the probe.
