Analysis of Circadian Photoresponses in Drosophila Using Locomotor Activity

Summary

Drosophila locomotor activity is a robust and quantitative measurement of circadian photo-responses. We describe protocols for designing behavior experiments for circadian photo-responses and analyzing the data. Studying the circadian photo-responses is important for dissecting the neuronal and molecular mechanisms of light entrainment.

Abstract

Circadian rhythms are only beneficial to animals if they can be synchronized by changes of ambient conditions. Light and temperature are two dominant environmental parameters that synchronize animal circadian clocks. In Drosophila circadian photo-responses are mediated by a solely blue light photoreceptor named CRYPTOCHROME (CRY). Upon photoreception, CRY changes its conformation and initiates the proteosomal dependent degradation of TIMELESS (TIM). TIM is an important pacemaker protein, thus degradation of TIM will reset the circadian clock. Under constant light conditions (LL), wild type flies quickly become arrhythmic because of the constant degradation of TIM, while flies bearing defects with circadian photo-responses will still be rhythmic. Thus LL triggered arrhythmicity has been used for screening of components in circadian light input pathways. A brief short light pulse in the night can also dramatically shift phases of circadian rhythms. As expected, this phase shift response is reduced in flied with defects in circadian photoresponse. Thus analyzing locomotion behavior rhythmicity under LL or phase changes after short light pulse in constant darkness (DD) are two major methods to study circadian photoresponse. Here we describe how to design and analyze LL and phase response experiments. LL arrhythmicity is suitable for screening light input pathways mutants, whereas phase response validates the results and provide further information for light sensitivity.

Introduction

Most organisms, from cyanobacteria to mammals, use circadian clocks to anticipate daily environmental changes. Circadian clocks synchronize most bodily functions of animals, from metabolic level, to rest/activity cycles and other behaviors¹. Circadian rhythms are self-sustained, which can be maintained even in constant conditions for several days. Circadian rhythm is generated by a molecular pacemaker, which is highly conserved among organisms. In fruit flies, the core of this circadian clock, is a transcriptional-translational feedback loop^2,3. Two transcription factors, CLOCK (CLK) and CYCLE (CYC) form a heterodimer and generate rhythmic transc....

Protocol

1. Constant light (LL) Experiments

1. Preparation of experimental flies
   1. Raise flies in incubators or rooms that have regular light: dark cycles on standard fly food at 25°C. Collect female and male virgin flies for crosses.
      NOTE: Unlike mated flies newly emerged virgin female flies have larger white abdomens. There is also a dark dot on their abdomen, which is only for virgin flies.). For RNAi screen, collect driver lines with GAL4 driven by circadianly regulated promote.......

Discussion

Circadian rhythms exist in most organisms on earth. Animals utilize circadian clocks to coordinate their bodily functions with daily changes. Since environmental conditions are variable, the circadian clock is only beneficial if it can be adjusted by different changes. For flies, light is the primary environmental cue used to synchronize and shift the circadian clock. Studying circadian photo-response is important for understanding how light is processed and regulates circadian behavior. Disruptions of circadian rhyth.......

Materials

Name	Company	Catalog Number	Comments
Percival Scientific	I-36LL
Trikinetics	DAM2		activity monitor-2
Trikinetics	PGT5		autoclavable
Trikinetics	DAMSystem308		free download
Trikinetics	DAMFileScan110X		activity monitor-2 software
Centre National de la Recherche Scientifique			Data analysis, Rouyer lab
Fishersci	S2-500GM		making fly food for behavior
BD Biosciences	214010		making fly food for behavior