Special thanks to the University of Missouri-Kansas City and the Jeffrey L. Price laboratory.

1. Fly food production
<ol>
	<li>Per every 1 L of water, prepare fly food consisting of 4.69 g of dried molasses, 19.70 g of dry active yeast, 87.22 g of corn meal and 7.83 g of agar.</li>
	<li>Combine the contents listed above in a crockpot and turn the heat to 250 &#176;F. Mix well as ingredients are added.</li>
	<li>Keep the lid on the crockpot as the fly food is heating while also mixing the contents every 10 min until it reaches a rolling boil. Allow the rolling boil to continue for 20 min before turning off the heat.</li>
	<li>Add 83.60 mL of water and keep the lid of the crockpot off as the food cools.</li>
	<li>Mix and record the temperature of the food every 10 min with a glass thermometer. Avoid letting a layer of film settle on top of the food.</li>
	<li>Once the food has cooled to 60 &#176;C, add 10.44 mL of Tegosept and 5.51 mL of propionic acid per liter of water added (see step 1.1).</li>
	<li>Mix well and turn heat up to 60 &#176;C to prevent the food from becoming too cool.</li>
	<li>Pump the fly food as needed using the Droso-filler.</li>
	<li>For narrow vials, pump 10 mL and for 6 oz square bottom bottles pump 60 mL.</li>
</ol>
2. Collecting flies of defined age
<ol>
	<li>Monitor bottles of wild type flies stored at 25 &#176;C for 5-7 days until large amounts of pupa (about 200 pupa) are attached to the side of the bottle. The bottles used are 6 oz Drosophila stock bottles with square bottoms.</li>
	<li>Clear existing adults from the bottle. Either tip the adults into a new bottle or place them in 70% ethanol. Use the dull end of a #0 paintbrush to push any remaining adults into the fly food. Be sure to wipe the paint brush down with 70% ethanol before and after uses.</li>
	<li>Allow cleared bottles to sit for 3 days in a 25 &#176;C incubator to allow for the next generation to eclose. These flies will be between 0 and 3 days old.</li>
</ol>
3. Fly separation
<ol>
	<li>After 3 days, separate males from females and collect the desired number for each sex for each of the four time points. Flies can be differentiated by sex by examining genitalia; males have dark rounded genitalia whereas females have lighter, more pointed genitalia. Females are also much larger than male flies.
	<ol>
		<li>Use a CO2 anesthesia pad to effectively separate the flies and differentiate between the sexes. Move the flies with paint brushes to avoid killing them.</li>
		<li>Collect 100 males and 100 females for each time point, with 50 males per vial and 100 females per vial. Males tend to be more aggressive and their social interactions lead to a lot of deaths when there are 100 individuals in a vial. The females-up to 100 individuals- are unaffected while contained in the vial.</li>
	</ol>
	</li>
	<li>Perform the collections at the following time points: ZT1, ZT7, ZT13 and ZT19 (Table 1). Note that collections done in the dark (ZT12-ZT24) are light sensitive whereas ZT0-ZT11 collections are done during lights-on times and room lights can be on. Please note that two separate incubators are used with inverse 12 hour light patterns to allow for all collections to occur during the day and not overnight.</li>
	<li>Use excess flies to create new bottles of flies for future entrainment. Place 25 females with 5-7 males in each new bottle and place in the incubator at 25 &#176;C.</li>
</ol>
4. Fly incubation
<ol>
	<li>Allow the flies to stay in light regulated incubators for 3-5 days to allow circadian entrainment to occur. Ensure that incubators are light-tight because even small amounts of light pollution will disturb entrainment.</li>
</ol>
5. Immunofluorescence fixation
<ol>
	<li>After entrainment, prepare new vials of fixation solution for samples that will be used for immunofluorescence. Prior to removing the flies from the incubator, add 4.8 mL of 4% formaldehyde diluted in 1x PBS + 0.1% Tween-20 to each new narrow vial for every 100 flies that are to be fixated. Each vial will house 100 male or 100 female Circadian entrained flies. Place the narrow vials in ice.</li>
</ol>
6. Immunofluorescence collection
<ol>
	<li>When collecting the flies from the incubator for immunofluorescence, remove the bottle cap and quickly invert the bottle into the funnel. Gently tap the flies into the solution via the funnel to help guide the flies into the vial; combine two tubes of the 50 males for a total of 100 males in one vial and use a single tube of 100 females for the other vial.</li>
	<li>Perform ZT13 and ZT19 collections in the dark; use a red light in order to see as drosophila are far less sensitive to these light wavelengths and are therefore less prone to light pollution13. Cryptochrome protein, in particular, is especially sensitive to blue light, which must be avoided14.
	<ol>
		<li>To reduce light exposure, ensure the room of collection is light tight with any sources of light blocked out or covered. Wrap these vials in foil so that ZT13 and ZT19 flies are not exposed to light when placed on the nutating mixer in the following step. ZT1 and ZT7 flies are not light sensitive and can be placed on the mixer without foil covering.</li>
	</ol>
	</li>
</ol>
7. Nutating mixer and storage
<ol>
	<li>After the flies have been collected, tape the top of the vials containing fixative to avoid spillage and place them on the nutating mixer at 165 RPM at 4 &#176;C for 4 h. The flies are no longer light sensitive after this fixation step, so foil may be removed to verify the solution is moving and flies are being submerged in the fixative.</li>
	<li>After removing the fly containing vials from the nutating mixer remove the formaldehyde and wash three times with 3,000 &#181;L of 1x PBS, inverting the vial with each wash.</li>
	<li>Store the vials bearing immunofluorescence samples at 4 &#176;C to await future immunofluorescence15.</li>
</ol>
8. Collection for protein extraction
<ol>
	<li>Prepare four 50 mL tubes and a Dewar containing liquid nitrogen for preservation of samples for protein extraction</li>
	<li>For collecting flies for protein or nucleic acid extraction, transfer flies from the bottles to the tube in the same manner as in step 6.1 and quickly cap the tube to prevent the release of flies and place the tube into liquid nitrogen to snap freeze.</li>
</ol>
9. Storage for protein extraction
<ol>
	<li>Store snap frozen samples for protein extraction at -80 &#176;C. These can be processed according to the protein extraction protocol appropriate for downstream analysis, including immunoblotting16.</li>
</ol>

<ol>
	<li>Halberg, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+physiological+and+clinical+aspects+of+24-hour+periodicity.">Some physiological and clinical aspects of 24-hour periodicity.</a> The Journal-Lancet. 73 (1), 20-32 (1953).</li><li>Smarr, B. L., Jennings, K. J., Driscoll, J. R., Kriegsfeld, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+time+to+remember:+the+role+of+circadian+clocks+in+learning+and+memory.">A time to remember: the role of circadian clocks in learning and memory.</a> Behavioral Neuroscience. 128 (3), 283-303 (2014).</li><li>Leng, Y., Musiek, E. S., Hu, K., Cappuccio, F. P., Yaffe, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+between+circadian+rhythms+and+neurodegenerative+diseases.">Association between circadian rhythms and neurodegenerative diseases.</a> The Lancet. Neurology. 18 (3), 307-318 (2019).</li><li>Hood, S., Amir, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurodegeneration+and+the+Circadian+Clock.">Neurodegeneration and the Circadian Clock.</a> Frontiers in aging Neuroscience. 9, 170 (2017).</li><li>Sulli, G., Lam, M. T. Y., Panda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+Circadian+Clock+and+Cancer:+New+Frontiers+for+Cancer+Treatment.">Interplay between Circadian Clock and Cancer: New Frontiers for Cancer Treatment.</a> Trends in Cancer. 5 (8), 475-494 (2019).</li><li>Zehring, W. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P-element+transformation+with+period+locus+DNA+restores+rhythmicity+to+mutant,+arrhythmic+Drosophila+melanogaster.">P-element transformation with period locus DNA restores rhythmicity to mutant, arrhythmic Drosophila melanogaster.</a> Cell. 39 (2 Pt 1), 369-376 (1984).</li><li>Bargiello, T. A., Jackson, F. R., Young, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+circadian+behavioural+rhythms+by+gene+transfer+in+Drosophila.">Restoration of circadian behavioural rhythms by gene transfer in Drosophila.</a> Nature. 312 (5996), 752-754 (1984).</li><li>Siwicki, K. K., Eastman, C., Petersen, G., Rosbash, M., Hall, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibodies+to+the+period+gene+product+of+Drosophila+reveal+diverse+tissue+distribution+and+rhythmic+changes+in+the+visual+system.">Antibodies to the period gene product of Drosophila reveal diverse tissue distribution and rhythmic changes in the visual system.</a> Neuron. 1 (2), 141-150 (1988).</li><li>Hardin, P. E., Hall, J. C., Rosbash, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feedback+of+the+Drosophila+period+gene+product+on+circadian+cycling+of+its+messenger+RNA+levels.">Feedback of the Drosophila period gene product on circadian cycling of its messenger RNA levels.</a> Nature. 343 (6258), 536-540 (1990).</li><li>Liu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+period+gene+encodes+a+predominantly+nuclear+protein+in+adult+Drosophila.">The period gene encodes a predominantly nuclear protein in adult Drosophila.</a> Journal of Neuroscience. 12 (7), 2735-2744 (1992).</li><li>Vosshall, L. B., Price, J. L., Sehgal, A., Saez, L., Young, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Block+in+nuclear+localization+of+period+protein+by+a+second+clock+mutation,+timeless.">Block in nuclear localization of period protein by a second clock mutation, timeless.</a> Science (New York, N.Y). 263 (5153), 1606-1609 (1994).</li><li>Price, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=double-time+is+a+novel+Drosophila+clock+gene+that+regulates+period+protein+accumulation.">double-time is a novel Drosophila clock gene that regulates period protein accumulation.</a> Cell. 94 (1), 83-95 (1998).</li><li>Hanai, S., Hamasaka, Y., Ishida, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+entrainment+to+red+light+in+Drosophila:+requirement+of+Rhodopsin+1+and+Rhodopsin+6.">Circadian entrainment to red light in Drosophila: requirement of Rhodopsin 1 and Rhodopsin 6.</a> Neuroreport. 19 (14), 1441-1444 (2008).</li><li>Vinayak, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exquisite+light+sensitivity+of+Drosophila+melanogaster+cryptochrome.">Exquisite light sensitivity of Drosophila melanogaster cryptochrome.</a> PLoS Genetics. 9 (7), e1003615 (2013).</li><li>Kelly, S. M., Elchert, A., Kahl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+and+Immunofluorescent+Staining+of+Mushroom+Body+and+Photoreceptor+Neurons+in+Adult+Drosophila+melanogaster+Brains.">Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains.</a> Journal of Visualized Experiments. (129), e56174 (2017).</li><li>Au-Eslami, A., Au-Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+Blotting:+Sample+Preparation+to+Detection.">Western Blotting: Sample Preparation to Detection.</a> Journal of Visualized Experiments. (44), e2359 (2010).</li><li>Fan, J. Y., Muskus, M. J., Price, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entrainment+of+the+Drosophila+circadian+clock:+more+heat+than+light.">Entrainment of the Drosophila circadian clock: more heat than light.</a> Science's signal Transduction Knowledge Environment. (413), pe65 (2007).</li><li>Miyasako, Y., Umezaki, Y., Tomioka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Separate+sets+of+cerebral+clock+neurons+are+responsible+for+light+and+temperature+entrainment+of+Drosophila+circadian+locomotor+rhythms.">Separate sets of cerebral clock neurons are responsible for light and temperature entrainment of Drosophila circadian locomotor rhythms.</a> Journal of Biological Rhythms. 22 (2), 115-126 (2007).</li><li>Edery, I., Zwiebel, L. J., Dembinska, M. E., Rosbash, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+phosphorylation+of+the+Drosophila+period+protein.">Temporal phosphorylation of the Drosophila period protein.</a> Proceedings of the National Academy of Sciences of the United States of America. 91 (6), 2260-2264 (1994).</li><li>Helfrich-Forster, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+neuroarchitecture+of+the+circadian+clock+in+the+brain+of+Drosophila+melanogaster.">The neuroarchitecture of the circadian clock in the brain of Drosophila melanogaster.</a> Microscopy Research and Technique. 62 (2), 94-102 (2003).</li><li>Price, J. 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Researchers utilize this entrainment protocol with success and consistency. This procedure allows the fixation of a large sampling pool that can be stored for future analysis. Additionally, this strategy preserves the neurological patterns induced by entrainment for future examination.
Fixation for storage is a major component of the entrainment process as it helps to stabilize brain tissue and it allows for more time to analyze each brain from the data pool thus minimizing waste from brains that lose viability due to age21. The main goal is to circadian entrain as many flies as possible so that there is continuous inventory available for head dissections and ultimately immunofluorescence or protein extraction to observe the findings and determine if results are of high confidence. To ensure that circadian entrainment is preserved through fixation, it is integral that any source of light pollution is eliminated. The fixation process allows for Drosophila to be stored while maintaining its neurological &#34;timestamp&#34; so that they can be dissected later and analyzed with no noticeable differences to flies that are dissected and have undergone immunofluorescence immediately after entrainment. For the purposes of fixation prior to immunofluorescence, the lab has determined with consistency that flies are viable at least up to 1 month. Fixations for western blot protein extraction render the brains viable indefinitely when stored at -80 &#176;C.
Another critical protocol step is the sexing of the flies. It is important that this step is done accurately as having both sexes in the same vial prior to fixation can lead to mating, which will yield new flies that are of younger age and corrupt protein analysis if males are accidentally examined instead of females or vice versa. Additionally, when sexing it is important to remove larvae specimens that are at times attached to females. This prevents the development of new progeny inside the female vial that could potentially corrupt results.
The next step for the entrainment protocol may be with items related to data analysis. The focus of the protocol is protein localization, but if there are other variables that are impacted by circadian entrainment, they must be explored through new avenues, often requiring protein or nucleic acid extraction. Additionally, there are other proteins of the brain that may still be analyzed via this protocol. The experiments associated with the protocol analyzed certain proteins but the list of genes and proteins that play a role in circadian biology has not been exhausted. The protocol is effective in accomplishing the goal of establishing a circadian rhythm, however, the applications are wide-ranging.

Almost all organisms on Earth, from the largest down to single celled, have an internal biological clock with a cycle of about one day. This is known as the Circadian rhythm (coined in 1953 by Franz Halberg from the Latin terms circa = about/approximately and &#34;dies&#34; = day)1. Although components of the core clock are known and their rudimentary mechanisms of function conceptualized, there is still much to understand about how biological rhythms are maintained throughout the body. Importantly, misregulation of biological rhythms is associated with poor health outcomes including poor memory formation, sleep disorders, seasonal affect disorder, depression, bipolar disorder, diabetes, obesity, neurodegeneration, and cancer2,3,4,5.
Drosophila is a well-established model for investigation of circadian biology. Genetically and biochemically tractable, large numbers are easily entrained (as will be shown). In fact, all seven publications cited as key publications of support in awarding the Nobel Prize for the discovery of circadian rhythms leveraged these strengths of the Drosophila model6,7,8,9,10,11,12.
Additionally, we show effective strategies for collecting entrained flies for the purposes of either immunofluorescence, nucleic acid, or protein extraction-based analysis. Using these strategies, one may process and store larger amounts of samples for analysis in the future. These methods are very advantageous in that they are reproducible and can yield hundreds of entrained flies that can be a part of a large data pool.

<table><tbody><tr><td>100-1000uL pipette</td><td>Eppendorf</td><td>ES-1000</td><td></td></tr><tr><td>10-100uL pipette</td><td>Eppendorf</td><td>ES-100</td><td></td></tr><tr><td>16% Paraformaldehyde Solution</td><td>15710</td><td></td><td></td></tr><tr><td>1X PBS</td><td>Caisson Labs</td><td>PBL01-6X100ML</td><td></td></tr><tr><td>Agar</td><td>Fisher Scientific</td><td>BP1423500</td><td></td></tr><tr><td>Anesthesia Filter Connection Kit</td><td>World Precision Instruments</td><td>EZ-251A</td><td></td></tr><tr><td>Corn meal</td><td>Genesee Scientific</td><td>62-100</td><td></td></tr><tr><td>Dried Molasses</td><td>Food Service Direct</td><td>OT280504</td><td></td></tr><tr><td>Droso-filler Food Pump</td><td>geneseesci.com</td><td>59-169</td><td></td></tr><tr><td>Drosophila Stock bottles, 6 oz square bottom w/ Flugs</td><td>geneseesci.com</td><td>32-130BF</td><td></td></tr><tr><td>Drosophila vials, Narrow K-Resin super bulk</td><td>geneseesci.com</td><td>32-118SB</td><td></td></tr><tr><td>Dry active yeast</td><td>Genesee Scientific</td><td>62-103</td><td></td></tr><tr><td>Ethanol</td><td>IBI Scientific</td><td>IB15720</td><td></td></tr><tr><td>EZ Basic Anesthesia System</td><td>World Precision Instruments</td><td>EZ-175</td><td></td></tr><tr><td>Falcon Centrifuge tubes</td><td>Corning</td><td>352097</td><td></td></tr><tr><td>Falcon round bottom tubes</td><td>Corning</td><td>352057</td><td></td></tr><tr><td>Fine point Sharpie marker</td><td>Sharpie</td><td>30001</td><td></td></tr><tr><td>Fisherbrand Nutating Mixer</td><td>Fisher Scientific</td><td>88-861-043</td><td></td></tr><tr><td>Flugs-Narrow Plastic Vials</td><td>Genesee Scientific</td><td>49-102</td><td></td></tr><tr><td>Glass Thermometer</td><td>Cole-Palmer</td><td>EW-08008-12</td><td></td></tr><tr><td>Liquid nitrogen hose</td><td>Thermo Scientific</td><td>398202</td><td></td></tr><tr><td>Liquid nitrogen tank-Dewar</td><td>Cooper Surgical Inc</td><td>900109-1</td><td></td></tr><tr><td>Liquid nitrogen transfer vessel</td><td>Electron Mircoscopy Sciences</td><td>61891-02</td><td></td></tr><tr><td>Paintbrushes(Red Sable) Size #0</td><td>Electro Microscopy Sciences</td><td>66100-00</td><td>This is used to separate the flies via sex without causing injury.</td></tr><tr><td>Plastic funnel</td><td>Plews and Edelmann</td><td>570-75-062</td><td></td></tr><tr><td>Polarizing light microscope</td><td>Microscope Central</td><td>1100100402241</td><td>Used to more clearly view Drosophila during sexing</td></tr><tr><td>ProPette Pipette Tips</td><td>MTC Bio Incorporated</td><td>P5200-100U</td><td></td></tr><tr><td>ProPette Pipette Tips</td><td>MTC Bio Incorporated</td><td>P5200-1M</td><td></td></tr><tr><td>ProPette Pipette Tips</td><td>MTC Bio Incorporated</td><td>P5200-5M</td><td></td></tr><tr><td>Propionic Acid</td><td>Sigma Aldrich</td><td>P1386-1L</td><td></td></tr><tr><td>Rayon Balls</td><td>Genesee Scientific</td><td>51-100</td><td></td></tr><tr><td>Reynolds wrap standard aluminum foil</td><td>Staples</td><td>1381273</td><td></td></tr><tr><td>Roaster Oven (Crockpot)</td><td>Hamilton Beach</td><td>32950</td><td></td></tr><tr><td>Scotch 810 Magic Tape</td><td>Electron Microscopy Sciences</td><td>77300</td><td></td></tr><tr><td>Spray bottle with trigger</td><td>US Plastic</td><td>66446</td><td>Used to spray ethanol to clean work bend areas</td></tr><tr><td>Tegosept</td><td>Genesee Scientific</td><td>20-258</td><td></td></tr><tr><td>Thermo Scientific Drosophila Incubator</td><td>Thermo Scientific</td><td>3990FL</td><td></td></tr><tr><td>Thermo Scientific Revco 4 degree Lab fridge</td><td>ThermoFisher Scientific</td><td>REL7504D</td><td></td></tr><tr><td>Thermo Scientific Revco Lab Freezer</td><td>ThermoFisher Scientific</td><td>REL7504A</td><td></td></tr><tr><td>Tween 20</td><td>Anatrace</td><td>T1003-1-GA</td><td></td></tr></tbody></table>

circadian entrainment of drosophila melanogaster

Nearly universal among organisms, circadian rhythms coordinate biological activity to earth&#39;s orbit around the sun. To identify factors creating this rhythm and to understand the resulting outputs, entrainment of model organisms to defined circadian time-points is required. Here we detail a procedure to entrain many Drosophila to a defined circadian rhythm. Furthermore, we detail post-entrainment steps to prepare samples for immunofluorescence, nucleic acid, or protein extraction-based analysis.

Controlled circadian entrainment allows researchers to examine biology at specific time points throughout the circadian day using the ZT1-ZT19 timing schedules or to add time-points as necessary. Here we use light and darkness to entrain flies to circadian cycles and verify entrainment by immunoblotting and immunofluorescence analysis of the period protein, a marker for circadian entrainment (Figure 1). Upon correct entrainment, period proteins should have a characteristic intensity and mobility pattern (Figure 1A) and should be visible at specific locations in the ZT1 brain (Figure 1B). Although other variables, including food and temperature, can influence circadian entrainment, light is most simple and reliable to control17. For the purpose of these methods, incubator temperatures are kept constant, relying on cerebral clock neurons that are influenced by light for entrainment18.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61176/61176fig1.jpg" /> 
Figure 1: Verification of entrainment. (A) Immunoblotting of whole cell extracts prepared from heads of entrained flies shows canonical patterns of period protein mobility and intensity19. 1.4 female heads from each of the indicated Zeitgeber times (ZT) were analyzed using an anti-Per antibody. (B) Immunofluorescence of entrained brains collected at ZT, where the Period protein is found in a characteristic pattern (bottom panels, recreated from Helfrich-Forster20). Shown are images taken from different sections of the brain, capturing all neurons expected to contain Period protein at ZT1. Scale bar is 40 &#181;m. <a href="https://www.jove.com/files/ftp_upload/61176/61176fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ZT1</td>
			<td>ZT7</td>
			<td>ZT13</td>
			<td>ZT19</td>
		</tr>
		<tr>
			<td>Light between 10 am - 10 pm</td>
			<td>Light between 10 am - 10 pm</td>
			<td>Dark between 9 am - 9 pm</td>
			<td>Dark between 9 am - 9 pm</td>
		</tr>
		<tr>
			<td>Collected at 11 am after 1 hour in light</td>
			<td>Collected at 5 pm after 7 hours in light</td>
			<td>Collected at 10 am after 1 hour in dark</td>
			<td>Collected at 4 pm after 7 hours in dark</td>
		</tr>
	</tbody>
</table>
Table 1: ZT1-ZT19 Circadian Rhythm Timing Schedules.

Watch this Scientific Journal Video about Circadian Entrainment of Drosophila Melanogaster at JoVE.com

Circadian Entrainment of Drosophila Melanogaster

Here, we detail how to synchronize Drosophila to a circadian day. This is the first, and most important step necessary for studying biological rhythms and chronobiology.

Nearly universal among organisms, circadian rhythms coordinate biological activity to earth&#39;s orbit around the sun. To identify factors creating this ...

circadian-entrainment-of-drosophila-melanogaster

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4.3K Views.  University of Missouri - Kansas City. Het synchroniseren van Drosophila om circadiane tijden te definiëren is de eerste stap in het bestuderen van gebeurtenissen die ofwel controle of reageren op de biologische klok. Synchronisatie van alle modellen volgt dezelfde basisbenadering. Er zijn vele stappen om dit praktisch te doen en het kan overweldigend lijken om ze allemaal samen te stellen.Verwacht een paar keer te proberen voordat het natuurlijk aanvoelt. Houd de temperaturen stabiel en ik wil het licht uit tijdens de dri...

Video: Circadian Entrainment of Drosophila Melanogaster

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) ...

Neuroscience

A Computational Method to Quantify Fly Circadian Activity

In most animals and plants, circadian clocks orchestrate behavioral and molecular processes and synchronize them to the daily light-dark cycle. Fundamental mechanisms that underlie this temporal control are widely studied using the fruit fly Drosophila melanogaster as a model organism. In flies, the clock is typically studied by analyzing multiday locomotor recording. Such a recording shows a complex bimodal pattern with two peaks of activity: a morning peak that happens around dawn, and an evening peak that happens around dusk. These two peaks together form a waveform that is very different from sinusoidal oscillations observed in clock genes, suggesting that mechanisms in addition to the clock have profound effects in producing the observed patterns in behavioral data. Here we provide instructions on using a recently developed computational method that mathematically describes temporal patterns in fly activity. The method fits activity data with a model waveform that consists of four exponential terms and nine independent parameters that fully describe the shape and size of the morning and evening peaks of activity. The extracted parameters can help elucidate the kinetic mechanisms of substrates that underlie the commonly observed bimodal activity patterns in fly locomotor rhythms.

A method to quantify the main temporal features seen in fly circadian locomotor rhythms is presented. The quantification is achieved by fitting fly activity with a multi-parametric model waveform. The model parameters describe the shape and size of the morning and evening peaks of daily activity.

In most animals and plants, circadian clocks orchestrate behavioral and molecular processes and synchronize them to the daily light-dark cycle. ...

Behavior

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

The FlyBar: Administering Alcohol to Flies

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian clock modulates alcohol sensitivity, but not the formation of tolerance. Here, we describe our protocol in detail. Alcohol is administered to the flies using the FlyBar. In this setup, saturated alcohol vapor is mixed with humidified air in set proportions, and administered to the flies in four tubes simultaneously. Flies are reared under standardized conditions in order to minimize variation between the replicates. Three-day old flies of different genotypes or treatments are used for the experiments, preferably by matching flies of two different time points (e.g., CT 5 and CT 17) making direct comparisons possible. During the experiment, flies are exposed for 1 hr&#160;to the pre-determined percentage of alcohol vapor and the number of flies that exhibit the Loss of Righting reflex (LoRR) or sedation are counted every 5 min. The data can be analyzed using three different statistical approaches. The first is to determine the time at which 50% of the flies have lost their righting reflex and use an Analysis of the Variance (ANOVA) to determine whether significant differences exist between time points. The second is to determine the percentage flies that show LoRR after a specified number of minutes, followed by an ANOVA analysis. The last method is to analyze the whole times series using multivariate statistics. The protocol can also be used for non-circadian experiments or comparisons between genotypes.

Drosophila has emerged as a significant model system for dissecting the cellular and molecular underpinnings of behavioral responses to alcohol. Here we present a protocol for the collection of alcohol sensitivity data in a circadian context that can be easily applied to other experiments and is well-suited for undergraduate research.

Fruit flies (Drosophila melanogaster) are an established model for both alcohol research and circadian biology. Recently, we showed that the circadian ...

Conditions Affecting Social Space in Drosophila melanogaster

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a straightforward manner. As we previously demonstrated 1, in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing 1,2. Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior 1,3. Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans 4.

Conditions Affecting Social Space in Drosophila melanogaster

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. The molecular clock within each pacemaker neuron generates circadian rhythms in gene expression, which underlie the rhythmic neuronal functions essential to the operation of the circuit. Investigation of the properties of the individual molecular oscillators in different subclasses of pacemaker neurons and their interaction with neuronal signaling yields a better understanding of the circadian pacemaker circuit. Here, we present a time-lapse fluorescent microscopy approach developed to monitor the molecular clockwork in clock neurons of cultured Drosophila larval brain. This method allows the multi-day recording of the rhythms of genetically encoded fluorescent circadian reporters at single-cell resolution. This setup can be combined with pharmacological manipulations to closely analyze real-time response of the molecular clock to various compounds. Beyond circadian rhythms, this multipurpose method in combination with powerful Drosophila genetic techniques offers the possibility to study diverse neuronal or molecular processes in live brain tissue.

Single-cell Resolution Fluorescence Live Imaging of Drosophila Circadian Clocks in Larval Brain Culture

The goal of this protocol is to establish ex vivo Drosophila larval brain culture optimized to monitor circadian molecular rhythms with long-term fluorescence time-lapse imaging. The application of this method to pharmacological assays is also discussed.

The circadian pacemaker circuit orchestrates rhythmic behavioral and physiological outputs coordinated with environmental cues, such as day/night cycles. ...

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.

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.

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

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

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.

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.

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

Drosophila Activity Monitor (DAM): A Method to Measure Locomotor Activity in Flies

Source: Chiu, J. C., et al. <a href="https://www.jove.com/video/2157/" target="_blank">Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila</a>. J. Vis. Exp. (2010).
This video describes the Drosophila activity monitor (DAM) system used to track locomotor activity. Researchers use activity data collected from the DAM to study circadian rhythms in fruit flies. The featured protocol clip shows how to load flies in the device and record activity data for circadian experiments.

Source: Chiu, J. C., et al. Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila. J. Vis. Exp. (2010).
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Circadian Entrainment of Drosophila Melanogaster

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