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

Summary

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.

Abstract

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms^1,2. 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 flies^5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Introduction

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 behavior^6,7. 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, TRPA1^6,9. The cold temperature sensors are located in the 3rd antenna....

Protocol

1. Preparation of Flies

1. Light Dark (LD) Experiments
   1. Raise flies in incubators (25 °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.
   2. 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 plexiglass).
      Note: One incubator should be designated a “day” incubator and set to a LD cycle of 12 hr light and 12 hr dark. The....

Results

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 temper.......

Discussion

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

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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