We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here we describe the design and analysis of TPR in Drosophila.
The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.
Temperature is a ubiquitous environmental cue. Animals exhibit a variety of behaviors in order to avoid harmful temperatures and seek comfortable ones. Drosophila exhibit a robust temperature preference behavior6,7. When flies are released into a temperature gradient from 18-32 °C, the flies avoid both warm and cold temperatures and finally choose a preferred temperature of 25 °C in the morning3. The warm temperature sensors are a set of thermosensory neurons, AC neurons, which express Drosophila transient receptor potential (TPR) channel, TRPA16,9. The cold temperature sensors are located in the 3rd antennal segments, since ablating the 3rd antennal segments causes the lack of cold temperature avoidance6. Recently, the TRPP protein Brivido (Brv) was identified10. Since Brv is expressed in the 3rd antennal segments and mediates cold detection, Brv is a possible cold sensing molecule, which is critical for the temperature preference behavior. In sum, the flies use these two temperature sensors to avoid the warm and cold temperatures and find a preferred temperature.
While mammals generate heat to regulate their body temperature, ectotherms generally adapt their body temperatures to the ambient temperature11. Some ectotherms are known to exhibit a daily TPR behavior which is believed to be a strategy for the ectotherms to regulate their BTR12. To determine whether the flies exhibited TPR, we repeated the temperature preference behavioral analysis at various points during a span of 24 hr. We found that Drosophila exhibit a daily TPR, which is low in the morning and high in the evening and follows a pattern similar to that of BTR in humans13.
In Drosophila, there are ~150 clock neurons in the brain. The clock neurons that regulate locomotor activity are called M and E oscillators. However, interestingly, M and E oscillators do not regulate TPR, instead, we showed that DN2 clock neurons in the brain regulate TPR but not locomotor activity. These data indicate that TPR is regulated independently from locomotor activity. Notably, mammalian BTR is also independently regulated from locomotor activity. Ablation studies in rats show that BTR is controlled through specific SCN neurons that target a different subset of subparaventricular zone neurons than those that control locomotor activity14. Therefore, our data considers the possibility that the mammalian BTR and the fly TPR are evolutionally conserved3, since both fly TPR and mammalian BTR exhibit circadian clock-dependent temperature rhythms, which are independently regulated from locomotor activity.
Here, we describe the details of how to analyze the TPR behavioral assay in Drosophila. This method allows for the investigation of not only the molecular mechanism and neural circuits of TPR, but also how the brain integrates different environmental cues and inner biological clocks.
1. Preparation of Flies
2. The Apparatus for the Temperature Preference Behavioral Assay
3. Preparation of Apparatus for Use
4. Temperature Preference Behavior Assay
5. Data Analysis
An example of the temperature preference rhythm is shown in Figure 5. If the behavior procedure is successfully done, the flies should exhibit a TPR in which they prefer a low temperature in the morning and higher temperature in the evening. The ~1-1.5 °C increase during the daytime in temperature preference should be observed during the course of the day, regardless of the genetic background, since we showed that w1118, yw and Canton S flies exhibit a similar temperature preference during the daytime3.
Figure 1. A schematic of the fly preparation in DD day. (A) An example of a DD daytime experiment. The light is ON from 1 pm-7 pm and light is OFF from 7 pm-1 pm in the transition incubator. Collect the flies which have been raised in the day incubator. Place the fly vials in the transition incubator sometime between 1 pm-7 pm. The next day before 1 pm, take the fly vials out of the transition incubator in the dark, wrap them with aluminum foil and place them in a box. (B) An example of a DD nighttime experiment. Collect the flies which have been raised in the night incubator either in the dark during 7am to 7pm or in the light during 7pm to 7am. Take the fly vials out of the night incubator in the dark between 7am and 7pm, and wrap them with aluminum foil and place them in a box
Figure 2. Temperature preference behavioral apparatus. (A) Top view. The plexiglass cover is placed on the aluminum plate with six C-clamps. Six temperature probes are attached at various positions on the inside of the cover within one of the lanes. Two rulers are placed on the top and bottom of the plexiglass cover along the edges to determine the temperature gradient. (B) Side view. Four Peltier devices are placed underneath an aluminum plate (44 cm x 22 cm). Each Peltier device is connected to the temperature controllers that generate cold or hot temperatures. To prevent the Peltiers from overheating, the computer cooling system is connected to water tubes, air-cooling fans, and power supplies. Temperature probes are embedded into the edge of the aluminum plate and are connected to the temperature controllers to directly control temperatures on the aluminum plate. For our current apparatus, the cold and hot sides are set at 12 °C and 36 °C, respectively.
Figure 3. A diagram of the apparatus. The temperature probes are employed as a feedback control reading the temperature on the aluminum plate. The Peltier devices are connected to the temperature controllers. To prevent overheating of the Peltiers, the liquid coolers are directly placed underneath the Peltiers. The four liquid coolers are connected by water tubes which connect to the pump and the radiator. The radiator has two fans which cool down the temperature of water. The pump and radiator are connected to the power supply.
Figure 4. The plan of the plexiglass cover. This is the plan for the cover that is made of plexiglass. The cover has four lanes divided by three 0.2 cm thick dividers, and a 0.7 cm diameter hole is located in the center of the top panel on each lane (Figure 2A).
Figure 5. An example of the TPR behavioral data. TPR of w1118 flies over 24 hr. Preferred temperatures were calculated using the distribution of flies in the temperature preference behavior experiments. Data are shown as the mean preferred temperature in each time zone. Numbers represent the number of assays. ANOVA, P < 0.0001. Tukey-Kramer test compared to ZT1-3, ***P < 0.001, **P < 0.01 or *P < 0.05. This figure of the TPR phenotype is adapted from Kaneko et al.3 with permission.
Here, we illustrate the details of the temperature preference behavioral apparatus and analysis of the TPR behavior. Drosophila exhibit the salient, robust, and reproducible features of clock-controlled TPR. However, our data suggests that at least two factors, ambient light and age, significantly disturb the TPR behavioral phenotypes.
We observe that light significantly affects temperature preference in Drosophila. It is consistent with the fact that w1118 flies kept in LD prefer higher temperatures during the daytime than those kept in DD, although the rhythmic changes of preferred temperature are still maintained under LD and DD3. Therefore, light affects the fly's temperature preference independent of the circadian clock. Since it is not clear how much light intensity is required and what mechanisms regulate this light dependent temperature preference, we use the same light intensity (~500-1,000 lux) during the experiments to obtain reproducible results.
The flies' ages also affect temperature preference. We avoid using day 1 flies because the TPR phenotypes of the day 1 flies (one day after hatching) are variable. Although day 4 and older flies show constant TPR behavior, they prefer lower temperatures than 2 or 3 day old flies. Therefore, it is very important not to mix wide-ranging aged flies. We use the day 2-3 flies or the day 4-5 flies group as necessary.
In our current TPR behavior method, we only examine temperature preference behaviors for 30 min. The reason for this is because the flies kept >1 hr in the temperature gradient tend to prefer a lower temperature. This maybe due to the lack of the food and water in the apparatus. Therefore, we discard the flies after each 30 min behavioral experiment. It would be a huge advantage if the TPR behavior could be measured continuously for at least 24 hr, ideally ~15 days. In this case, the TPR behavior assay would be easily done without transferring the fly vials to the different incubators. More importantly, TPR phenotypes would be more efficiently compared to other circadian behaviors such as locomotor activity.
Animals are very sensitive to small changes in the environment. We showed that the flies temperature preference behavior is not only regulated by the clock but is strongly influenced by light. TPR might be a behavioral output that is integrated by all of the environmental cues and internal states. Drosophila is a sophisticated model system to dissect fundamental mechanisms of brain functions by using the variety of genetic tools, relatively simple brain structure and versatile behavioral assays. Therefore, studying temperature preference behavior assays could shed light on the fundamental mechanisms of how the brain integrates different information to produce optimal behaviors.
Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Because the mechanisms controlling sleep in flies are analogous to those controlling mammalian sleep17-20, we believe that further exploration of Drosophila TPR will contribute to a greater understanding of circadian rhythm and sleep behavior.
The authors have nothing to disclose.
We are grateful to Drs. Aravinthan Samuel and Marc Gershow who helped develop the initial version of the behavioral apparatus and Matthew Batie who modified the behavioral apparatus. This research was supported by Trustee Grant from Cincinnati Children’s Hospital, JST/PRESTO, March of Dimes and NIH R01 GM107582 to F.N.H.
Bright Lab Jr. Safelight | Amazon | #B00013J8UY | Red light for dark rooms |
Rain X | SOPUS products | Water repellent: Apply the plexiglass cover | |
C-Clamp | Home Depot | ||
Temperature/hygrometer | Fisher | 15-077-963 | |
Peltier devices | TE Technology, Inc. | HP-127-1.4-1.15-71P | |
Thermometer | Fluke | Fluke 52II | |
Bench top controller | Oven Industries | 5R6-570-15R and 5R6-570-24R | |
Temperature sensor probe | Oven Industries | TR67-32 | |
Generic 480 Watt ATX power supply | computer cooling system | ||
MCR220-QP-RES Dual 120 mm Radiator with reservoir | Swiftech | computer cooling system | |
MCP350 In-Line 12V DC pump | Swiftech | computer cooling system | |
MCW50 graphics Card liquid cooler | Swiftech | computer cooling system | |
Scythe Kaze-Jyuni SY1225SL12SH fan | Crazy PC | computer cooling system |