Name: analysis of circadian photoresponses in drosophila using locomotor activity
Uploaded: 2015-09-22T15:02:49
Description: Circadian rhythms are only beneficial to animals if they can be synchronized by changes of ambient conditions. Light and temperature are two dominant ...

This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20 GM103650, and the University of Nevada Reno. We thank Matthew Gruner and three anonymous reviewers for critically reading the manuscript and helpful comments.

1. Constant light (LL) Experiments
<ol>
	<li>Preparation of experimental flies
	<ol>
		<li>Raise flies in incubators or rooms that have regular light: dark cycles on standard fly food at 25&#176;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...

<ol>
	<li>Mohawk, J.A., Green, CB, Takahashi, JS. Central and peripheral circadian clocks in mammals. Annu Rev Neuroscience. 35, 445- 462 (2012).</li>
	<li>Hardin, PE, Panda, S. Circadian timekeeping and output mechanisms in animals. Curr Opin Neurobiol. 5, 724-731 (2013).</li>
	<li>Zhang, Y, and Emery, P. Molecular and neural control of insect circadian rhythms. Insect Molecular Biology and Biochemistry. 15, 513-551, (2012).</li>
	<li>Dubruille, R, and Emery, P. A plastic clock: how circadian rhythms respond to environmental cues in Drosophila. Mol Neurobiol. 2, 129-145 (2008).</li>
	<li>Emery, P, So, WV, Kaneko, M, Hall, JC, Rosbash, M. CRY, A Drosophila clock and light-regulated cryptochrome, is a major contributor to circadian rhythm resetting and photosensitivity. Cell. 95, 669-679 (1998).</li>
	<li>Stanewsky, R., et al. The cryb mutation identifies cryptochrome as a circadian photoreceptor in Drosophila. Cell. 95, 681-692 (1998).</li>
	<li>Koh, K., Zheng, X., Seghal, A., JETLAG resets the Drosophila circadian clock by promoting light-induced degradation of TIMELESS. Science. 312, 1809-1812 (2006).</li>
	<li>Peschel, N, Chen, KF, Szabo, G, Stanewsky, R. Light-dependent interaction between the Drosophila circadian clock factors cryptochrome, jetlag, and timeless. Curr Biol. 19, 241-237 (2009).</li>
	<li>Lamba, P, Bilodeau-Wentworth, D, Emery, P, Zhang, Y. Morning and evening oscillators cooperate to reset circadian behavior in response to light input. Cell Rep. 7, 601-608 (2014).</li>
	<li>Chiu, JC, Low, KH, Pike, DH, Yildirim, E, Edery, I. Assaying locomotor activity to study circadian rhythms and sleep parameters in Drosophila. J Vis Exp. 43. Pii:2157 (2010).</li>
	<li>Abreu, T, Bragan&#231;a, M. The bipolarity of light and dark: A review on Bipolar Disorder and circadian cycles. J Affect Disord. 185:219-29. (2015).</li>
	<li>Dubruille, R, Murad, A, Rosbash, M, Emery, P. A constant light-genetic screen identifies KISMET as a regulator of circadian photoresponses. PloS Genet. (2009).</li>
	<li>Peschel, N., Veleri, S., Stanewsky R. Veela defines a molecular link between Cryptochrome and Timeless in the light-input pathway to Drosophila&#39;s circadian clock. Proc Natl Acad Sci USA. 103, 17313-17318 (2006).</li>
</ol>

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...

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 loop2,3. Two transcription factors, CLOCK (CLK) and CYCLE (CYC) form a heterodimer and generate rhythmic transcriptions of down stream clock controlled genes. Among these genes, PERIOD (PER) and TIM are two main transcriptional repressors. PER/TIM undergo post-translational modifications, and accumulate in the cytoplasm, then enter the nucleus to repress their own transcription by blocking CLK activity. 
Circadian pacemakers are self-sustained, but environmental cues determine their phase of oscillations. Light and temperature are the most crucial cues for synchronization of the circadian clock4. Compared to temperature, entrainment by light is much better understood. Circadian photo-responses are mainly mediated by CRY input pathways in flies. CRY is the blue light photoreceptor, which changes its conformation after receiving light, and then it is able to bind TIM5,6. After binding with CRY, TIM undergoes proteosomal dependent degradation through E3 ubiquitin ligase JETLAG7-9 (JET). Thus, the degradation of TIM resets the circadian pacemaker.
Flies become arrhythmic in constant light conditions, because of the constant degradation of TIM by CRY. However flies with defects in the CRY pathway still remain rhythmic. Based on this observation, the behavioral rhythmicity in LL is often used to demonstrate circadian photo-responses in flies. A short light pulse in the night will cause transient degradation of TIM, thus shifting the phase of circadian pacemaker5,6. Light pulse at early night will mimic a delayed day, thus called phase delay; while a light pulse at late night will mimic an advanced dawn, which is named as phase advance. This phase response is sensitive to light intensity, which is an important parameter for circadian photo-responses. Phase response is almost abolished in mutants of CRY input pathways. Measuring the phase response by light pulse is also extensively used to examine circadian photo-responses. Here we describe how to perform these two experiments as well as methods to analyze the behavior data.

<table border="0" cellpadding="0" cellspacing="0" width="528">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:101px;">Percival Scientific</td>
			<td style="width:119px;">I-36LL</td>
			<td style="width:119px;"></td>
			<td style="width:189px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:101px;">Trikinetics</td>
			<td style="width:119px;">DAM2</td>
			<td style="width:119px;"></td>
			<td>activity monitor-2&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:101px;">Trikinetics</td>
			<td style="width:119px;">PGT5</td>
			<td style="width:119px;"></td>
			<td>autoclavable</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:101px;">Trikinetics</td>
			<td style="width:119px;">DAMSystem308</td>
			<td style="width:119px;"></td>
			<td>free download</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:101px;">Trikinetics</td>
			<td style="width:119px;">DAMFileScan110X</td>
			<td style="width:119px;"></td>
			<td>activity monitor-2 software&#160;</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:101px;">Centre National de la Recherche Scientifique</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td>Data analysis, Rouyer lab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:101px;">Fishersci</td>
			<td style="width:119px;">S2-500GM</td>
			<td style="width:119px;"></td>
			<td>making fly food for behavior</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:101px;">BD Biosciences</td>
			<td align="right" style="width:119px;">214010</td>
			<td style="width:119px;"></td>
			<td>making fly food for behavior</td>
		</tr>
	</tbody>
</table>

analysis of circadian photoresponses in drosophila using locomotor activity

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.

Under constant light, wild type flies become arrhythmic because of constant degradation of TIM, while circadian photo-response mutants remain rhythmic. Figure 1 shows behavior actograms of flies under constant light. The results can also be presented in a quantitative manner. Table 1 shows the percentage of rhythmic flies, and period in constant light. Normally, very few wild type flies show rhythmicity (0-25%), while majority of flies with defects in circadian photo-response remains rhyt...

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.

Analysis of Circadian Photoresponses in Drosophila Using Locomotor Activity

Circadian rhythms are only beneficial to animals if they can be synchronized by changes of ambient conditions. Light and temperature are two dominant ...

Research

JoVE Journal

Biology

A Rapid In Vivo Bioassay for Developmentally Active Enhancers

A potential regulatory sequence (PRS) can be identified by several approaches including conservation analysis and transcription factor-specific chromatin ...

Indacenodithienothiophene-Based Ternary Organic Solar Cells: Concept, Devices and Optoelectronic Analysis

We report on a novel ternary bulk-heterojunction solar cell by implementing a novel conjugated polymer (ADV-2) containing alternating ...

Experimental Approach for Determining Semiconductor/liquid Junction Energetics by Operando Ambient-Pressure X-ray Photoelectron Spectroscopy

Operando Ambient Pressure X-ray photoelectron spectroscopy (operando AP-XPS) investigation of semiconductor/liquid junctions provides quantitative ...

A Murine Model of Experimental Necrotizing Enterocolitis Using Gavage Feeding, Lipopolysaccharide, and Systemic Hypoxia

This protocol describes a model of experimental necrotizing enterocolitis (NEC) using rats or mice. NEC is a gastrointestinal disease unique to premature ...