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&#8217;s Hospital, JST/PRESTO, March of Dimes and NIH R01 GM107582 to F.N.H.

1. Preparation of Flies
<ol>
	<li>Light Dark (LD) Experiments
	<ol>
		<li>Raise flies in incubators (25 &#176;C/40-60%relative humidity (RH)) under light 12 hr/dark 12 hr (LD) cycles. The light intensity of the incubators is ~500-1,000 lux.</li>
		<li>Two incubators are necessary to complete the behavior assays over a 24 hr period. Both incubators should have a programmable light with ON OFF functions. They should also have solid doors that are not permeable to light (i.e. no glass or plexigla...

<ol>
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There is nothing to disclose.

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</em...

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 &#176;C, the flies avoid both warm and cold temperatures and finally choose a preferred temperature of 25 &#176;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&#160;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.

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design and analysis of temperature preference behavior and its circadian rhythm 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&#160;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.

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 &#176;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&#160;Canton S flies exhibit a simi...

Watch this Scientific Journal Video about Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila at JoVE.com

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

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&#160;we describe the design and analysis of TPR in Drosophila.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

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8.0K Views.  Cincinnati Childrens Hospital Medical Center. 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.