The overall goal of this procedure is to determine the temperature preference of drosophila. This is accomplished by first preparing the flies and the apparatus, which is assembled around a temperature controlled aluminum base. Once temperatures are established in the apparatus, a cohort of flies is loaded in and after half an hour their temperature preference is scored.
Ultimately, the assay can be used with a great variety of experimental conditions. For example, circadian rhythms of temperature preference. This method can help to answer the key questions in the target rhythms field.
Since this target rhythms of temperature preference are controlled by different mechanisms than that of the locomotive activity, this behavior assay will lead to the identification of new molecular and the cellular mechanisms that were not previously identified using locomotive activity approaches. This method also can help answer key questions in the sensory neuroscience field, such as temperature and pain sensation. Because this temperature behavioral assays very robust, this assay will allow us to map the neural circuit from temperature detection to processing, which will allow us more insight into the sensory integration.
Raise the flies in humidified incubators with a 1212 light dark cycle. This protocol requires two such incubators set to opposite schedules. The night incubator should be dark during normal work hours.
When accessing an incubator during its night cycle, use red light to illuminate the incubator. Collect cohorts of 20 to 30 newly hatched flies of either sex and at any ratio, keep them in their incubator for two to three days before running them in the assay. At least five cohorts are required for each tested condition.
To build a temperature preference testing chamber, a plexiglass cover must be engineered to fit to an aluminum plate under the plate. Place four peltier devices. Each of the peltier devices should be connected to the temperature controllers that can generate cold or hot temperatures to prevent the pelters from overheating.
A computer cooling system is connected to water tubes, air cooling fans, and power supplies to monitor the temperatures on the aluminum plate. Temperature probes are embedded into the edge of the aluminum plate and are connected to temperature controllers. Set the cold and hot sides of the plate to 12 and 36 degrees Celsius respectively in the cover.
Attach six temperature probes at various positions within one of the lanes. Place the apparatus in an environmentally controlled room at 25 degrees Celsius and between 65 and 75%relative humidity. Be sure to monitor these conditions.
The room must have light control and good air circulation to prevent any circulating air from destabilizing the temperature. Gradient shield the testing area with a shower curtain. Uniform light must be used on the apparatus because luminosity can influence temperature preference.
Turn on the apparatus for at least 30 minutes. In order to establish a stable temperature gradient on the surface of the plate. Coat the cover of the behavior apparatus with a water repellent to prevent the flies from climbing the walls or ceiling of the cover.
Wipe the excess water repellent off and leave the cover to drive for 25 to 30 minutes if necessary, double stick tape may be used half an hour later. Wipe off any condensation from the aluminum plate. Then place the plexiglass cover on the aluminum plate and secure with six C clamps.
It is very important that the cover is sealed. Well monitor the air temperature between the plate and the cover. Make sure the probes do not touch either the aluminum plate or the plexiglass cover.
Let the cover equilibrate on the plate for at least 15 minutes. Eventually, the air temperature gradient between the aluminum plate and the cover will range from 18 to 32 degrees Celsius In the case of a day behavioral experiment, take the fly vials out of the day incubator and test the behavioral assay in the light immediately before a night behavioral experiment. Take the fly vials out of the night incubator.
Wrap them in foil and place them in a box in the dark room under a red lamp. For experiments during dark periods, the room lights must remain off at all times until the behavioral experiments are done. However, red lamps can be used for illumination using aspiration.
Load 20 to 25 flies into the space between the aluminum plate. Then cover the holes with cover slips to prevent the flies from escaping. Allow the flies to move about for 30 minutes.
When the experiment is completed, take at least two photos for the light experiments. A flash needs to be used for the dark experiments. Check the temperature of the apparatus.
Take note of the room temperature as well as the humidity to remove the flies connected tube of carbon dioxide near the hole on top of the apparatus to anesthetize the flies, discard the flies. They should not be retested. Next, wipe any condensation or moisture off the plate.
Replace the cover on the plate and tighten with the clamps in preparation For the next experiment, calculate the temperature gradient as follows. First, determine where the temperature probes are placed using a ruler based on the probe locations and their recorded temperatures. Draw straight lines representing each degree of temperature in the appropriate position on the photo of the fly's final position.
Next count the numbers of flies between each one degree Celsius interval. Exclude any flies on the walls or on the ceiling of the cover. Calculate the percentage of the flies in each temperature range of each lane, and then the mean preferred temperature for each condition.
Collect data from at least five trials. Then use the mean values of each trial to determine the average preferred temperature with errors. Normal flies raised at 25 degrees Celsius, prefer a lower temperature in the morning than in the evening.
A one to 1.5 degree increase in temperature preference was measured during the daytime and a 1.5 to two degrees Celsius. Decrease was seen at the transition from day to night. The assay can also be used to test mutant flies.
For example, it was found that the temperature preference of flies bearing a defective mutation in the trip, a one ion channel is higher than that of controls. After watching this video, you should have a good understanding of how to perform the temperature preference behavior assay. This method allows for the investigation of molecular mechanisms and neuro circuits, ofa and clocks, and how the brain integrate the different environmental cues and in the biological clocks.
