The overall goal of this noxious cold assay is to be able to quantitate and assess changes in innocuous to noxious cold detection. This method can help answer key questions in the nociception field, including facilitating identification of novel, conserved, cold nociception genes and modulators of pain responses in disease and or injury. The main advantage of this technique is it allows for precise manipulation of the noxious cold stimulus and quantification of cold evoked responses over a variety of environmental and genetics contexts.
Generally individuals new to this method will need to practice consistent application of the probe before beginning an experiment. We first had the idea for this method when we found the that drosophila larvae respond to noxious heat and we became curious about whether they do the same to noxious cold. Raise stocks or genetic crosses of drosophila in a 25 degrees Celsius incubator.
Note, if culturing a cross, use 20 to 25 virgin females and 15 to 20 males per vial containing regular corn meal fly media and culture for approximately 48 hours before transferring flies to a new vial of food. Five days after the culture is started, collected third instar larvae of the desired genotype by gently squirting a stream of water into the mushy food and larvae. Then pour out the contents into a medium sized clean Petri Dish.
Next, gently sort larvae by size, using forceps to preclude larger, wandering third instar, or small, sickly larvae from being tested. Discard larvae containing any undesired genetic markers or balancers. Have another lab member label containers where sorted larvae will be placed to keep the experimenter blind to the experimental condition or genotype of the larvae.
Use a scoopula to transfer a small amount of fresh food into a clean labeled Petri Dish filled halfway with room temperature water. Finally, use forceps to gently move the sorted larvae onto the food to prevent starvation or dessication, if testing is anticipated to take more than 20 minutes. Begin by turning on the cold probe unit and allow it a few minutes to cool to the desired temperature.
Wipe away any excess moisture on the probe with a laboratory wipe. When not in immediate use, place an insulating cap on the probe to both insulate and keep the tip clean and prevent damage. Next, place a middle third instar larva onto a thin piece of dark movable vinyl to help with contrast visualization and alignment of the probe.
Then, place this piece of vinyl with the larva under a bright field microscope. Adjust the microscope and light unit to medium brightness to provide contrast and prevent the larva from drying out too quickly. Using a damp paintbrush, ensure that the larva is moist from the Petri Dish water so that it will not stick to the vinyl and remove any excess water surrounding it using a KimWipe.
Advanced the probe by hand towards the larva at a 90 degree angle to the anteroposterior body axis, using the vinyl to move the larva into correct position. The orient the tip of the probe to gently lay across the mid-dorsal surface of the larva with the probe at approximately a 45 degree angle to the microscope stage. Upon probe contact, start a timer.
Apply enough downward pressure to slightly indent the surface of the larval cuticle, while still allowing forward or backward movement. Next, hold the probe in place for up to 10 seconds or until a cold evoked behavioral response is observed, whichever occurs first. Then, remove the probe.
Record the observed behavioral response and latency of the response. Take note of any non-responders or larvae that do not respond within 10 seconds. Finally, discard the larva and prepare the next.
Repeat the procedure until the desired number of test larvae is reached. After contact with the cold probe, results from this experiment indicate that the larvae respond with the following cold-evoked behaviors, crunching their anterior and posterior segments towards the center of their body in a contraction, CT, raising of their anterior and posterior segments into the air to make a U-Shape, US, raising only the posterior segments into the air, PR.Furthermore, different behaviors peak over varying cold ranges such that PR and US peak at three to eight degrees Celsius, while CT peaks around nine to 14 degrees Celsius. Lastly, when comparing latencies of the different cold behaviors, these data show that CT responses were significantly different from PR and US at three degrees Celsius and 10 degrees Celsius and from US at 20 degrees Celsius.
Once mastered, this technique can be completed in approximately 30 to 40 minutes for one set of 30 larvae. While attempting this procedure, it's important to be aware of abnormal locomotion, the moisture level of the larva, and the pressure and placement of the cold probe onto the larva. Visual demonstration of this method is critical as improper application of the cold probe could result in different dose response curves or a lack of the cold response behavior.
Following this procedure, other methods can be used, such as fluorescent light imaging, gene expression analysis and behavioral assays. All will determine how cold exposure effects cellular activity, morphology, gene expression, or other behaviors. This technique paves the way for researchers in the field of cold nociception to study the genetic, environmental, and disease effects in drosophila.
After watching this video, you should have a good understanding of how to use the cold probe tool and assay to analyze cold-evoked responses and understand the cellular and genetic mechanisms of cold nociception.
