This work was financially supported by the grant R01DK128133 from NIH/NIDDK (to J.S.M) and startup funds from Texas A&#38;M University.

All the animal subjects are used in accordance with the guidelines set forth by the Institutional Animal Care and Use Committee of Texas A&#38;M University (AUP #2022-0050). Both male and female C57BL/6 mice between 2-4 months of age are used here. The procedure for building the feeding system is described below, and the raw materials required to build the apparatus are referenced in the Table of Materials.
1. Construction of the feeding system
<ol>
	<li>Constructing a poly vinyl chloride (PVC) base
	<ol>
		<li>Procure four pieces of 0.25-inch PVC sheets cut as per the following dimensions: 4.875 inch x 4.5 inch; 4.875 inch x 2.125 inch; 9.5 inch x 2.125 inch (two pieces). Drill four holes in the 4.875-inch x 4.5-inch base for attaching a 24 h timer using the measurements provided in Figure 1A. Glue the four pieces of PVC to obtain the base as in Figure 1A.</li>
	</ol>
	</li>
	<li>Securing the timer on the PVC base
	<ol>
		<li>Open the timer to remove the plug. Make a connection using a standard extension/electric cord (Figure 1B).</li>
		<li>Place the timer on the PVC base to align it with the holes drilled on the base in step 1.1.1. Use 1.5-inch screws to fix the timer on the PVC base. Make sure the timer lies flat and is stable on the PVC base.</li>
		<li>Drill four holes on top of the timer and fix 0.75-inch screws to hold the eight-compartment food container (Figure 1B). The base for the system is complete and looks like Figure 1C. 
		CAUTION: Do not drill the holes through the entire timer.</li>
	</ol>
	</li>
	<li>Cage setup
	<ol>
		<li>Cut a 4-inch PVC tube (external diameter of 4.5 inch) at a 3.125-inch height. Drill a hole of about 0.5 inch at the bottom of the pipe to let the electric cable through.</li>
		<li>Sand the top of the pipe (e.g., with a router or a rotary tool) to allow the cap to be removed easily when changing the food container.</li>
		<li>Use a mouse cage having a width greater than 4.5 inch and cut a 4.5-inch hole at the bottom of the cage using a hole saw. 
		NOTE: The location of the hole depends on the cage setup (e.g., location of the cage top and water bottle, addition of a running wheel, etc.).</li>
	</ol>
	</li>
	<li>Feeding container setup
	<ol>
		<li>Make a food dispenser out of a 4-inch eight-compartment jewelry organizer as shown in Figure 1D. Cut the rim of the container such that it fits well into the pipe.</li>
		<li>Use a 4-inch PVC pipe cap and cut a hole corresponding to the size of a single dispenser compartment (e.g., with a rotary tool) to create an opening that exposes only one of the eight compartments at a time. As the timer moves, the opening exposes a new compartment every 3 h. The feeding system components are now ready to be used. Once all the cages are set, the final setup resembles the one shown in Figure 1E.</li>
		<li>Transport of many food containers at a time can be cumbersome. To facilitate easier transport, take three pieces of 0.25-inch PVC pipe. Drill a 0.625-inch hole in the center of two pieces and glue them together. Then, use a narrow 0.625-inch PVC pipe that fits in the hole and through the center of the food cups to stack food cups to facilitate their transport, as shown in Figure 1F.</li>
		<li>Test the timers prior to introducing the mice by plugging the setup into power outlets, placing a piece of nestlet in one compartment at a recorded time, and monitoring the position of the nestlet 12 h later to ensure that the timer turns promptly.</li>
	</ol>
	</li>
</ol>
2. Application of the feeding system
<ol>
	<li>Continuous measurement of food intake in mice
	<ol>
		<li>Transfer mice to the experimental room and acclimate them to the light-dark (LD) cycle set in the room for at least 1 week, and for 2 weeks if the light-dark cycle is shifted by more than 3 h. For this experiment, data is collected from male and female C57BL/6 mice between 2-4 months of age exposed to LD 12:12 (n = 7 males and 4 females).</li>
		<li>Record mice weight prior to individually housing them in the feeder cages (weights were recorded at 3:00 pm, i.e., at the time food changeouts were carried out). Make sure that mice have ad libitum access to water and enough bedding and nestlets.</li>
		<li>Add 1.5 g of food (routinely used 45 mg of dustless precision pellets) to all eight compartments of the feeder cup. Place the feeder cup on the timer. Then, put the lid on the feeder cup such that only one compartment is exposed, and note the time of food presentation. Four compartments represent nighttime points and the other four represent day time points. 
		NOTE: Due to hoarding behavior observed upon excess food presentation, 1.5 g was optimized as the starting food weight. Mice tend to hoard pellets either on top of food cup lids or in the bedding within the cage. This skews the data leading to its misinterpretation. Male mice do not hoard pellets when fed with 1.5 g or less per compartment. Females tend to hoard food more than males, but this is mouse-specific and can be attenuated if providing 1 g of pellet per compartment.</li>
		<li>Change food every day at the same time and count the number of pellets remaining in each compartment to calculate the amount of food consumed. Monitor the feeding profile for a week to get a baseline feeding profile of mice fed ad libitum.</li>
		<li>Based on the food consumed in every compartment, calculate the average daily food consumption for each mouse (Figure 2).</li>
	</ol>
	</li>
	<li>High fat diet (HFD) treatment 
	NOTE: This feeding system can also be used to study the effect of HFD on metabolic diseases and eventually be used for time-restricted feeding schedules. HFD is not commercially available as precise size and weight pellets, and the pellets for feeding are usually procured as 0.5-inch pellets.
	<ol>
		<li>Place the HFD pellets on a clean surface or transparent film and cut them into 6-7 smaller pieces of even size using a razor blade. Cut the pellets small enough so that they resemble normal chow pellets as used in section 2.1 above. 
		NOTE: Mice tend to hoard bigger pellets in their cages, leading to miscalculation of food consumed.</li>
		<li>Weigh out 1.5 g of cut HFD pieces and place in each of the 8 food compartments. For food changeout every other day, 1.5 g of HFD per compartment is sufficient.</li>
		<li>Change the food every day or every other day as per experimental needs and record the weight of the food that remains.</li>
		<li>Calculate the amount of food consumed by subtracting the remaining weight of food from the initial amount of food given. Repeat this process over a period of 1 week to obtain a baseline of HFD food intake (Figure 2).</li>
	</ol>
	</li>
	<li>Acclimating male mice to a night restricted (NR) diet
	<ol>
		<li>Follow steps 2.1.1-2.1.4 to obtain a baseline of ad libitum feeding. For this experiment, data is collected from male C57BL/6 mice between 2-4 months of age exposed to LD 12:12 (n = 18 males).</li>
		<li>After 3-7 days of ad libitum diet, put the mice on a transition diet by gradually reducing the number of pellets in the day compartments. To do this, have five pellets per compartment on day 1 of transition (0.225 g per compartment), 3 pellets on day 2 (0.135 g per compartment), 1 pellet on day 3 (0.045 g per compartment), and none thereafter to completely transition the mice on a night restricted diet. 
		NOTE: Ensure that mice are not calorie restricted. Average the daily food consumption of mice per compartment based on the ad libitum baseline and give them the same amount of food, distributing it only across the four-night compartments.</li>
		<li>Keep monitoring the food intake for 2 weeks after the mice have adapted to the night restricted regimen. During this period, adjust the amount of food given to each mouse to better suit its total food consumption (Figure 3A). Typically add food pellets (1 pellet for each of the four night compartments) when mice eat all their food for two consecutive nights.</li>
		<li>Weigh the mice at the end of the 2-week period to monitor any change of weight due to the feeding regimen. At the end of this period, anesthetize the mice with isoflurane and euthanize them by decapitation. Collect tissues and analyze them for daily changes due to the feeding paradigm.</li>
	</ol>
	</li>
	<li>Acclimating male mice to an arrhythmic (AR) diet
	<ol>
		<li>Follow steps 2.1.1-2.1.4 to obtain a baseline of ad libitum feeding. For this experiment, data is collected from male C57BL/6 mice between 2-4 months of age exposed to LD 12:12 (n = 18 males).</li>
		<li>After a week of ad libitum diet, calculate the average food consumption per day and divide that number by 8 to obtain the amount of food to be provided in each compartment. Achieve AR feeding by ensuring that mice get an equal amount of food in all eight compartments throughout the day.</li>
		<li>Then, put the mice on a transition diet by gradually reducing, over 3-5 days, the amount of food given per compartment to ultimately abolish any rhythm of food intake (as shown in Figure 3B). When on an AR diet, ensure that the mice have access to 1/8 of their daily food intake in each of the eight compartments, and thus food access at every 3 h. Ensure that mice are not calorie restricted.</li>
		<li>Maintain the mice on AR diet for 2 weeks or more (Figure 3B).</li>
		<li>During AR diet, adjust the food every day to make sure that mice leave only a few pellets behind (typically less than 5). This ensures that mice are getting just the right amount of food and are not calorie restricted. Make adjustments by reducing or adding pellets in either all the eight compartments or by reducing or adding pellets in two opposite compartments, to not induce any rhythms of food intake. 
		NOTE: Mice fed under an AR diet leave food almost exclusively between ZT3 and ZT9 (between 3 h and 9 h after light on) yet are hungry at night and bite the food dispenser to access the next compartment. Nevertheless, AR fed mice are not calorie restricted, and in fact gain more weight than NR fed mice over time.</li>
		<li>Weigh the mice at the end of the 2-week period to monitor any change of weight due to the feeding regimen. At the end of this period, anesthetize the mice with isoflurane and euthanize them by decapitation. Collect tissues and analyze them for daily changes due to the feeding paradigm.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Boothroyd, C. E., Wijnen, H., Naef, F., 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=Integration+of+light+and+temperature+in+the+regulation+of+circadian+gene+expression+in+Drosophila.">Integration of light and temperature in the regulation of circadian gene expression in Drosophila.</a> PLoS Genetics. 3 (4), 0492-0507 (2007).</li><li>Brown, S. A., Zumbrunn, G., Fleury-Olela, F., Preitner, N., Schibler, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rhythms+of+mammalian+body+temperature+can+sustain+peripheral+circadian+clocks.">Rhythms of mammalian body temperature can sustain peripheral circadian clocks.</a> Current Biology. 12 (18), 1574-1583 (2002).</li><li>Buhr, E. D., Yoo, S. H., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+as+a+universal+resetting+cue+for+mammalian+circadian+oscillators.">Temperature as a universal resetting cue for mammalian circadian oscillators.</a> Science. 330 (6002), 379-385 (2010).</li><li>Kawamoto, T., 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=Effects+of+fasting+and+re-feeding+on+the+expression+of+Dec,+Per1,+and+other+clock-related+genes.">Effects of fasting and re-feeding on the expression of Dec, Per1, and other clock-related genes.</a> Journal of Biochemistry. 140 (3), 401-408 (2006).</li><li>Lamia, K. A., Storch, K. F., Weitz, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+significance+of+a+peripheral+tissue+circadian+clock.">Physiological significance of a peripheral tissue circadian clock.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (39), 15172-15177 (2008).</li><li>Oosterman, J. E., Kalsbeek, A., La Fleur, S. E., Belsham, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+nutrients+on+circadian+rhythmicity.">Impact of nutrients on circadian rhythmicity.</a> American Journal of Physiology - Regulatory Integrative and Comparative Physiology. 308 (5), 337-350 (2015).</li><li>Pitts, S. N., Perone, E., Silver, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Food-entrained+circadian+rhythms+are+sustained+in+arrhythmic+Clk/Clk+mutant+mice.">Food-entrained circadian rhythms are sustained in arrhythmic Clk/Clk mutant mice.</a> American Journal of Physiology - Regulatory Integrative and Comparative Physiology. 285, 57-67 (2003).</li><li>Sheward, W. J., 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=Entrainment+to+feeding+but+not+to+light:+Circadian+phenotype+of+VPAC+2+receptor-null+mice.">Entrainment to feeding but not to light: Circadian phenotype of VPAC 2 receptor-null mice.</a> Journal of Neuroscience. 27 (16), 4351-4358 (2007).</li><li>Gekakis, N., 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=Role+of+the+CLOCK+protein+in+the+mammalian+circadian+mechanism.">Role of the CLOCK protein in the mammalian circadian mechanism.</a> Science. 280 (5369), 1564-1569 (1998).</li><li>King, D. 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=Positional+cloning+of+the+mouse+circadian+Clock+gene.">Positional cloning of the mouse circadian Clock gene.</a> Cell. 89 (4), 641-653 (1997).</li><li>Kume, K., 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=mCRY1+and+mCRY2+are+essential+components+of+the+negative+limb+of+the+circadian+clock+feedback+loop.">mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop.</a> Cell. 98 (2), 193-205 (1999).</li><li>Shearman, L. 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=Interacting+molecular+loops+in+the+mammalian+circadian+clock.">Interacting molecular loops in the mammalian circadian clock.</a> Science. 288 (5468), 1013-1019 (2000).</li><li>Beytebiere, J. R., 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=Tissue-specific+BMAL1+cistromes+reveal+that+rhythmic+transcription+is+associated+with+rhythmic+enhancer-enhancer+interactions.">Tissue-specific BMAL1 cistromes reveal that rhythmic transcription is associated with rhythmic enhancer-enhancer interactions.</a> Genes and Development. 33 (5-6), 294-309 (2019).</li><li>Menet, J. S., Pescatore, S., 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=CLOCK:+BMAL1+is+a+pioneer-+like+transcription+factor.">CLOCK: BMAL1 is a pioneer- like transcription factor.</a> Genes and Development. 28 (1), 8-13 (2014).</li><li>Koike, N., 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=Transcriptional+architecture+and+chromatin+landscape+of+the+core+circadian+clock+in+mammals.">Transcriptional architecture and chromatin landscape of the core circadian clock in mammals.</a> Science. 338 (6105), 349-354 (2012).</li><li>Mure, L. S., 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=Diurnal+transcriptome+atlas+of+a+primate+across+major+neural+and+peripheral+tissues.">Diurnal transcriptome atlas of a primate across major neural and peripheral tissues.</a> Science. 359 (6381), (2018).</li><li>Ruben, M. D., 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=A+database+of+tissue-specific+rhythmically+expressed+human+genes+has+potential+applications+in+circadian+medicine.">A database of tissue-specific rhythmically expressed human genes has potential applications in circadian medicine.</a> Science Translational Medicine. 10 (458), 1-8 (2018).</li><li>Zhang, R., Lahens, N. F., Ballance, H. I., Hughes, M. E., Hogenesch, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+circadian+gene+expression+atlas+in+mammals:+Implications+for+biology+and+medicine.">A circadian gene expression atlas in mammals: Implications for biology and medicine.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (45), 16219-16224 (2014).</li><li>Menet, J. S., Rodriguez, J., Abruzzi, K. 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=Nascent-Seq+reveals+novel+features+of+mouse+circadian+transcriptional+regulation.">Nascent-Seq reveals novel features of mouse circadian transcriptional regulation.</a> eLife. 2012 (1), 1-25 (2012).</li><li>Miller, B. H., 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=Circadian+and+CLOCK-controlled+regulation+of+the+mouse+transcriptome+and+cell+proliferation.">Circadian and CLOCK-controlled regulation of the mouse transcriptome and cell proliferation.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (9), 3342-3347 (2007).</li><li>Cailotto, C., 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+suprachiasmatic+nucleus+controls+the+daily+variation+of+plasma+glucose+via+the+autonomic+output+to+the+liver:+Are+the+clock+genes+involved.">The suprachiasmatic nucleus controls the daily variation of plasma glucose via the autonomic output to the liver: Are the clock genes involved.</a> European Journal of Neuroscience. 22 (10), 2531-2540 (2005).</li><li>Eckel-Mahan, K. 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=Coordination+of+the+transcriptome+and+metabolome+by+the+circadian+clock.">Coordination of the transcriptome and metabolome by the circadian clock.</a> Proceedings of the National Academy of Sciences of the United States of America. 109 (14), 5541-5546 (2012).</li><li>Damiola, F., 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=Restricted+feeding+uncouples+circadian+oscillators+in+peripheral+tissues+from+the+central+pacemaker+in+the+suprachiasmatic+nucleus.">Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus.</a> Genes and Development. 14 (23), 2950-2961 (2000).</li><li>Saini, C., 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=Real-time+recording+of+circadian+liver+gene+expression+in+freely+moving+mice+reveals+the+phase-setting+behavior+of+hepatocyte+clocks.">Real-time recording of circadian liver gene expression in freely moving mice reveals the phase-setting behavior of hepatocyte clocks.</a> Genes and Development. 27 (13), 1526-1536 (2013).</li><li>Stokkan, K. A., Yamazaki, S., Tei, H., Sakaki, Y., Menaker, M. <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+circadian+clock+in+the+liver+by+feeding.">Entrainment of the circadian clock in the liver by feeding.</a> Science. 291 (5503), 490-493 (2001).</li><li>Atger, F., 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=Circadian+and+feeding+rhythms+differentially+affect+rhythmic+mRNA+transcription+and+translation+in+mouse+liver.">Circadian and feeding rhythms differentially affect rhythmic mRNA transcription and translation in mouse liver.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (47), 6579-6588 (2015).</li><li>Greenwell, B. J., 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=Rhythmic+food+intake+drives+rhythmic+gene+expression+more+potently+than+the+hepatic+circadian+clock+in+mice.">Rhythmic food intake drives rhythmic gene expression more potently than the hepatic circadian clock in mice.</a> Cell Reports. 27 (3), 649-657 (2019).</li><li>Izumo, M., 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=Differential+effects+of+light+and+feeding+on+circadian+organization+of+peripheral+clocks+in+a+forebrain+Bmal1+mutant.">Differential effects of light and feeding on circadian organization of peripheral clocks in a forebrain Bmal1 mutant.</a> eLife. 3, 04617 (2014).</li><li>Mange, F., 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=Diurnal+regulation+of+RNA+polymerase+III+transcription+is+under+the+control+of+both+the+feeding-fasting+response+and+the+circadian+clock.">Diurnal regulation of RNA polymerase III transcription is under the control of both the feeding-fasting response and the circadian clock.</a> Genome Research. 27 (6), 973-984 (2017).</li><li>Van Der Veen, D. R., 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=Impact+of+behavior+on+central+and+peripheral+circadian+clocks+in+the+common+vole+Microtus+arvalis,+a+mammal+with+ultradian+rhythms.">Impact of behavior on central and peripheral circadian clocks in the common vole Microtus arvalis, a mammal with ultradian rhythms.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (9), 3393-3398 (2006).</li><li>Vollmers, C., 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=Time+of+feeding+and+the+intrinsic+circadian+clock+drive+rhythms+in+hepatic+gene+expression.">Time of feeding and the intrinsic circadian clock drive rhythms in hepatic gene expression.</a> Proceedings of the National Academy of Sciences of the United States of America. 106 (50), 21453-21458 (2009).</li><li>Chaix, A., Lin, T., Le, H. D., Chang, M. W., 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=Time-Restricted+feeding+prevents+obesity+and+metabolic+syndrome+in+mice+lacking+a+circadian+clock.">Time-Restricted feeding prevents obesity and metabolic syndrome in mice lacking a circadian clock.</a> Cell Metabolism. 29 (2), 303-319 (2019).</li><li>Hatori, M., 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=Time-restricted+feeding+without+reducing+caloric+intake+prevents+metabolic+diseases+in+mice+fed+a+high-fat+diet.">Time-restricted feeding without reducing caloric intake prevents metabolic diseases in mice fed a high-fat diet.</a> Cell Metabolism. 15 (6), 848-860 (2012).</li><li>Acosta-Rodr&#237;guez, V. A., de Groot, M. H. M., Rijo-Ferreira, F., Green, C. B., Takahashi, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mice+under+caloric+restriction+self-impose+a+temporal+restriction+of+food+intake+as+revealed+by+an+automated+feeder+system.">Mice under caloric restriction self-impose a temporal restriction of food intake as revealed by an automated feeder system.</a> Cell Metabolism. 26 (1), 267-277 (2017).</li><li>Chung, H., 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=Time-restricted+feeding+improves+insulin+resistance+and+hepatic+steatosis+in+a+mouse+model+of+postmenopausal+obesity.">Time-restricted feeding improves insulin resistance and hepatic steatosis in a mouse model of postmenopausal obesity.</a> Metabolism: Clinical and Experimental. 65 (12), 1743-1754 (2016).</li><li>Sen, S., 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=Ultradian+feeding+in+mice+not+only+affects+the+peripheral+clock+in+the+liver,+but+also+the+master+clock+in+the+brain.">Ultradian feeding in mice not only affects the peripheral clock in the liver, but also the master clock in the brain.</a> Chronobiology International. 34 (1), 17-36 (2017).</li><li>Xie, 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=Natural+food+intake+patterns+do+not+synchronize+peripheral+clocks.">Natural food intake patterns do not synchronize peripheral clocks.</a> BMC Biology. 18 (160), 1-11 (2020).</li><li>Swamy, S., 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=Circadian+disruption+of+food+availability+significantly+reduces+reproductive+success+in+mice.">Circadian disruption of food availability significantly reduces reproductive success in mice.</a> Hormones and Behavior. 105, 177-184 (2018).</li><li>Xin, H., 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=Protocol+for+setup+and+circadian+analysis+of+inverted+feeding+in+mice.">Protocol for setup and circadian analysis of inverted feeding in mice.</a> STAR Protocols. 2 (3), 100701 (2021).</li></ol>

All the authors declare having no conflicts of interest.

Extensive research has been carried out over the last few decades on the manipulation of feeding rhythms and their effect on physiology. The construction and utilization of the feeding system described here can be used as an efficient method for manipulating food intake. The protocol utilizes a common 24 h timer and a food cup designed as an eight-compartment organizer as key components of the system. The cages can be constructed with ease using just a few easily accessible tools, and the handling of the system is user-friendly. Some of the key aspects of the protocol for adapting the system to manipulate rhythmic food intake include daily changing of food cups since the timer turns across a 24 h period, manual counting or weighing of remaining food, and daily adjustment of the number of pellets for AR feeding. Typically, plastic shavings are seen when mice are hungry and do not get adequate food. This issue can be fixed by adding a few more food pellets abiding by the feeding regimen until no plastic shavings are seen. In the case of AR feeding, where daily food needs to be adjusted, care should be taken not to induce the rhythm of food intake (Figure 3B). Hence, it is preferable to either add or subtract pellets in opposite compartments throughout to keep mice arrhythmically fed.
This system can be further improved by coating the food cups with a layer of epoxy to prevent the mice from biting the plastic, and thus help prolong the life of the food cups. The surface of the timer for food cup placement can also be modified to help the food cup sit flat and stable on the timer. This could prevent the accidental stopping of the timer caused by an unevenly placed timer. A few of the cage components, such as food cups, can also be 3D printed to reduce cost and custom-made to the liking of the researcher. This can include food cups with more than eight compartments, which can give a better time resolution than the current 3 h window.
Although very efficient, this system has some limitations, such as being labor intensive, with the researcher still needing to change food cups every 24 h and requiring them to manually count/weigh the remaining food. In addition, the timers need to be monitored from time to time to identify potential issues and/or if they stopped working. This can be achieved while counting the food pellets remaining after feeding (e.g., by determining whether some mice ate food only in a few compartments and left some compartments untouched). Another limitation of this system is that it may not work as well with female mice, since the few experiments performed with females showed that they tend to hoard food and chew the plastic more than male mice.
Nevertheless, this feeding system is very effective in manipulating food intake, is easy to construct, operate, maintain, and is inexpensive compared to the expensive automated feeders existing in the market. It can be easily adapted and modified to suit the researcher's requirements and does not need any special training to operate the system. Importantly, timers only produce a low amount of constant white noise, which prevents mice from associating any sound with food availability.
In summary, this paper describes an innovative feeding system that can be used to monitor daily food consumption in mice and can be adapted to feed mice on different paradigms such as time-restricted feeding, arrhythmic feeding, and feeding on high-fat diet. This system adds to the list of tools that can be used to address important questions in the field of rhythmic food intake and its effect on physiology.

The circadian clock is found ubiquitously across species and provides a time-keeping mechanism that helps organisms to adapt to their rhythmically changing environment. The master circadian pacemaker is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN is primarily entrained by the environmental light-dark cycle, and synchronizes peripheral clocks present in nearly every cell of the body via multiple cues, including neuronal and hormonal signals, feeding, and body temperature1,2,3,4,5,6,7,8. In mammals, the molecular circadian clock relies on the heterodimeric transcription factor CLOCK: BMAL19,10, which controls the expression of the core clock genes named Period (Per1, Per2, and Per3) and Cryptochrome (Cry1 and Cry2) to initiate a transcriptional feedback loop that is critical for the generation of circadian rhythms9,11,12. The molecular clock also regulates the rhythmic transcription of thousands of genes that control the rhythmicity of virtually every biological function13,14,15. More than 50% of the genome in mammals is rhythmically expressed in at least one tissue type16,17,18, and tissues such as the liver in mice have about 25%-30% of their transcriptome expressed rhythmically18,19. Rhythmic gene expression is crucial to activate important biological processes such as cell cycle control20, glucose homeostasis21, and amino acid metabolism22 at the right time of the day in order to increase organism fitness.
Over the past few decades, there has been increasing evidence suggesting that food intake can act as a potent synchronizing cue for entraining rhythms in gene expression in multiple tissues, including the liver23,24. Importantly, feeding has been shown to entrain rhythms in the liver independently of the SCN or of the light-dark cycle25, and rhythmic feeding can drive rhythmic gene expression without involving the molecular clock26,27,28,29,30,31. Feeding restricted to the inactive period of mice (daytime) inverts the phase of expression of the core clock genes and of many rhythmic genes31. Time-restricted feeding (TRF), which is a nutritional intervention where the daily caloric intake is restricted to a period of 8-10 h, has been shown to protect against obesity, hyperinsulinemia, hepatic steatosis, and metabolic syndrome32,33. All the above experiments involving manipulation of food intake require the experimenter to make use of effective methods to deliver food at the right time of day.
Different methods of food delivery have been developed, bearing several advantages and disadvantages29,34,35,36,37,38,39 (Table 1). Some automated feeders have been designed to operate based on a software that controls the amount, duration, and timing of food availability while recording feeding and voluntary wheel-running activity in mice34. A few other methods involve mice being placed in different cages for different feeding conditions, with the experimenter manually adding food pellets at the prerequisite time38,39. Another system uses an automated feeder system controlled by a computer where a pneumatic-driven shield prevents access to food and which can be controlled either by time intervals or mass of food35. All these methods either require utilization and setup of a computerized software that can be expensive and require some training for proper operation of the instrument or are labor intensive because the experimenter needs to be present at specific times to manually change feeding conditions. Computerized systems also come with their share of issues, including malfunctioning of levers or doors that let the food out, food pellets getting stuck in the outlets, and software breakdown. Moreover, the sound that may be produced during the opening of doors or levers presents the risk of conditioning mice to associate these with food delivery, thereby compromising the interpretation of effects of food manipulation as being strictly due to food access or due to effects on other behavioral rhythms such as sleep/wake cycle. The overall goal of this study was to develop an affordable and efficient system to manipulate long-term rhythmic food intake that would help alleviate many of these aforementioned issues. First and foremost, the feeding apparatus that was developed and is described below can be constructed at a very minimal cost compared to the automated machines (Table 2) and does not require sophisticated training for handling, operation, and maintenance. Secondly, the feeding system only produces a background white noise and no loud sounds during food delivery, thereby preventing Pavlovian conditioning. Altogether, this feeding system is economic, more accessible, and reliable for researchers while still being efficient in the manipulation of rhythmic food intake.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>#6 x 0.75 inch Phillips Pan Head Stainless Steel Sheet Metal Screw (50-Pack)</td>
			<td>Everbilt</td>
			<td>#800172</td>
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			<td>#8 x 1.5 inch Phillips Pan Head Zinc Plated Sheet Metal Screw (100-Pack)</td>
			<td>Everbilt&#160;</td>
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			<td>0.25 inch gray PVC sheet (24 inch x 48 inch)</td>
			<td>USPlastic</td>
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			<td>4 inch PVC pipe (10 ft)</td>
			<td>Home Depot</td>
			<td>#531103</td>
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			<td>45 mg dustless precision pellets</td>
			<td>Bio-Serv</td>
			<td>#F0165</td>
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			<td>6 ft. Extension Cord</td>
			<td>HDX</td>
			<td>HD#145-017</td>
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			<td>Food container (eight-compartment jewelry organizer)&#160;</td>
			<td>JewelrySupply</td>
			<td>#PB8301</td>
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			<td>Indoor Basic Timer</td>
			<td>General Electric</td>
			<td>#15119</td>
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			<td>Oatey 4 inch ABS Pipe Test Cap with Knockout</td>
			<td>Home Depot</td>
			<td>#39103D</td>
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			<td>Rodent Diet with 45 kcal% fat (with red dye)</td>
			<td>Research Diets</td>
			<td>#D12451</td>
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manipulation of rhythmic food intake in mice using a custom-made feeding system

Rhythmic gene expression is a hallmark of the circadian rhythm and is essential for driving the rhythmicity of biological functions at the appropriate time of day. Studies over the last few decades have shown that rhythmic food intake (i.e., the time at which organisms eat food during the 24 h day), significantly contributes to the rhythmic regulation of gene expression in various organs and tissues throughout the body. The effects of rhythmic food intake on health and physiology have been widely studied ever since and have revealed that restricting food intake for 8 h during the active phase attenuates metabolic diseases arising from a variety of obesogenic diets. These studies often require the use of controlled methods for timing the delivery of food to animals. This manuscript describes the design and use of a low-cost and efficient system, built in-house for measuring daily food consumption as well as manipulating rhythmic food intake in mice. This system entails the use of affordable raw materials to build cages suited for food delivery, following a user-friendly handling procedure. This system can be used efficiently to feed mice on different feeding regimens such as ad libitum, time-restricted, or arrhythmic schedules, and can incorporate a high-fat diet to study its effect on behavior, physiology, and obesity. A description of how wild-type (WT) mice adapt to the different feeding regimens is provided.

The feeding system described above can be used for long term manipulation of rhythmic food intake in mice. This system essentially exposes a new food compartment to the mouse every 3 h enabling the researcher to specifically manipulate food in every compartment. One application was to analyze the profile of food intake over the 24 h period. The data indicate that WT mice fed normal chow ad libitum eat about 75% of their food during the night (Figure 2A). Moreover, most of the food eaten during the day occurs within the 3 h before light off.
Mice fed HFD ad libitum ate more food on the first 2 days of exposure, likely because of the novelty of HFD (Figure 2A). After 2 days, the HFD intake remained rhythmic, yet with a decreased amplitude compared to when fed&#160;normal chow ad libitum. While both male and female WT mice were fed HFD, it was found that female mice hoarded a large amount of food on the lid of the feeding apparatus and in the cage, whereas males did not show any noticeable hoarding. As mentioned above, hoarding food can result in miscalculating food consumption and lead to data misinterpretation. In addition, female mice bit into the plastic rims of the food cups more frequently, especially in the night compartments. Male mice showed significant weight gain after 1 week of ad libitum normal chow and after 1 week of HFD (Figure 2E). A similar trend was observed with female mice but did not reach significant p-values, likely in part because of the lower number of females used compared to males.
Mice transitioned to a NR diet eat their daily total calories only at night, without a significant decrease in calorie intake for the first 3-5 weeks (Figure 3A). Longer exposure to NR schedule decreases the daily average calorie intake by 10%-15% compared to mice fed ad libitum, as described elsewhere34. Mice transitioned to an AR diet consumed their daily total calories in equal quantities across the day, leading to a dramatic dampening of the daily rhythm of food intake (Figure 3B). As for the NR feeding schedule, the daily average of calorie intake is not affected by the AR feeding schedule for the first 3-5 weeks of exposure but decreases with longer exposure. Mice showed weight gain after the NR (Figure 3C) and AR schedules (Figure 3C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64624/64624fig01.jpg" /> 
Figure 1: Design and construction of the feeding apparatus system. (A) The dimensions of the PVC base for the feeding system, and description of where holes need to be made for fixing the timer. (B) A 24 h timer before and after repurposing the wire and drilling with screws for placement of the food cup. (C) The assembled gray base and timer along with a 4-inch PVC pipe. (D) The eight-compartment food cup after trimming outer edges. (E) The final setup of cages with the food cup covered with a 4-inch cap such that only one compartment is accessible at a time. (F) The transportation of multiple food cups during an experiment. <a href="https://www.jove.com/files/ftp_upload/64624/64624fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64624/64624fig02.jpg" /> 
Figure 2: Feeding profiles under different dietary regimes. (A) The feeding profile of male WT mice fed ad libitum with normal chow (NC) for 7 days and high-fat diet (HFD) for an additional 7 days. Colored lines represent individual mouse profiles (n = 7) and the black line indicates the average &#177; SEM of seven mice. (B) The high-fat diet before and after slicing. (C) The daily average of food intake every 3 h &#177;&#160;SEM (n = 7). The average was calculated over the last 5 days of either the NC or the HFD feeding schedule. (D) The average (left) and the percentage (right) of food intake during the day and night for mice fed with NC or HFD. The values represent the average of seven mice &#177; SEM and were calculated using the food intake data over the last 5 days of either NC or HFD feeding schedules. * p &#60; 0.05 between the two groups (paired t-test). (E) The average body weights of mice used in the experiment after 1 week of NC and 1 week of HFD. The data for males (left) and females (right) is shown with * p &#60; 0.05 between the two groups (paired t-test). <a href="https://www.jove.com/files/ftp_upload/64624/64624fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64624/64624fig03.jpg" /> 
Figure 3: Manipulation of the daily rhythm of food intake. (A) The feeding profile of male WT mice fed with normal chow ad libitum for 2 days, transitioned to a night-restricted (NR) feeding regime for 3 days, and maintained under NR feeding for 8 nights. The colored lines represent individual mouse profiles (n = 18) and the black line indicates the average &#177;&#160;SEM of 18 mice. The gray asterisk indicates malfunctioning of the timer for that mouse on that single day where the timer stopped turning. (B) Feeding profile of male WT mice fed with normal chow ad libitum for 2 days, transitioned to an arrhythmic (AR) feeding regime for 1 day, and maintained under AR feeding for 8 nights. The colored lines represent individual mouse profiles (n = 18) and the black line indicates the average &#177; SEM of the 18 mice. (C) The average body weights of mice used in the experiment after 2 weeks of exposure to NR and AR diets. Data is shown with * p &#60; 0.05 between the two groups (paired t-test). <a href="https://www.jove.com/files/ftp_upload/64624/64624fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Table 1: Advantages and disadvantages of the existing feeding systems. A table highlighting different feeding systems used for manipulation of food intake, with a brief description of the pros and cons of each system. <a href="https://www.jove.com/files/ftp_upload/64624/Table1_JoVE_Sahasrabudhe et al_R1_RE.xlsx" target="_blank">Please click here to download this Table.</a>
Table 2: Cost of materials required to construct the feeding system. A table listing the cost of items required for the construction of the feeding system described in this paper along with an estimate of the cost of construction per cage. <a href="https://www.jove.com/files/ftp_upload/64624/Table2_JoVE_Sahasrabudhe et al_RE.xls" target="_blank">Please click here to download this Table.</a>

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Manipulation of Rhythmic Food Intake in Mice Using a Custom-Made Feeding System

Restricting the timing of food intake has emerged as a promising intervention to attenuate diet-induced metabolic diseases. This manuscript details the construction and use of an efficient system built in-house for measuring and manipulating rhythmic food intake in mice.

Rhythmic gene expression is a hallmark of the circadian rhythm and is essential for driving the rhythmicity of biological functions at the appropriate ...

manipulation-rhythmic-food-intake-mice-using-custom-made-feeding

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2.1K Views.  Texas A&M University. Restricting the timing of food intake has emerged as a promising intervention to attenuate diet-induced metabolic diseases. This manuscript details the construction and use of an efficient system built in-house for measuring and manipulating rhythmic food intake in mice.

Video: Manipulation of Rhythmic Food Intake in Mice Using a Custom-Made Feeding System

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Evaluation of Respiratory System Mechanics in Mice using the Forced Oscillation Technique

The forced oscillation technique (FOT) is a powerful, integrative and translational tool permitting the experimental assessment of lung function in mice in a comprehensive, detailed, precise and reproducible manner. It provides measurements of respiratory system mechanics through the analysis of pressure and volume signals acquired in reaction to predefined, small amplitude, oscillatory airflow waveforms, which are typically applied at the subject's airway opening. The present protocol details the steps required to adequately execute forced oscillation measurements in mice using a computer-controlled piston ventilator (flexiVent; SCIREQ Inc, Montreal, Qc, Canada). The description is divided into four parts: preparatory steps, mechanical ventilation, lung function measurements, and data analysis. It also includes details of how to assess airway responsiveness to inhaled methacholine in anesthetized mice, a common application of this technique which also extends to other outcomes and various lung pathologies. Measurements obtained in na&iuml;ve mice as well as from an oxidative-stress driven model of airway damage are presented to illustrate how this tool can contribute to a better characterization and understanding of studied physiological changes or disease models as well as to applications in new research areas.

The present protocol provides a detailed step-by-step description of the procedures required to execute measurements of respiratory system mechanics as well as the assessment of airway responsiveness to inhaled methacholine in mice using the forced oscillation technique (flexiVent; SCIREQ Inc, Montreal, Qc, Canada).

The  forced oscillation technique (FOT) is a powerful, integrative and translational  tool permitting the experimental assessment of lung function in mice ...

A High-throughput Method for Measurement of Glomerular Filtration Rate in Conscious Mice

The measurement of glomerular filtration rate (GFR) is the gold standard in kidney function assessment. Currently, investigators determine GFR by measuring the level of the endogenous biomarker creatinine or exogenously applied radioactive labeled inulin (3H or 14C). Creatinine has the substantial drawback that proximal tubular secretion accounts for ~50% of total renal creatinine excretion and therefore creatinine is not a reliable GFR marker. Depending on the experiment performed, inulin clearance can be determined by an intravenous single bolus injection or continuous infusion (intravenous or osmotic minipump). Both approaches require the collection of plasma or plasma and urine, respectively. Other drawbacks of radioactive labeled inulin include usage of isotopes, time consuming surgical preparation of the animals, and the requirement of a terminal experiment. Here we describe a method which uses a single bolus injection of fluorescein isothiocyanate-(FITC) labeled inulin and the measurement of its fluorescence in 1-2 &mu;l of diluted plasma. By applying a two-compartment model, with 8 blood collections per mouse, it is possible to measure GFR in up to 24 mice per day using a special work-flow protocol. This method only requires brief isoflurane anesthesia with all the blood samples being collected in a non-restrained and awake mouse. Another advantage is that it is possible to follow mice over a period of several months and treatments (i.e. doing paired experiments with dietary changes or drug applications). We hope that this technique of measuring GFR is useful to other investigators studying mouse kidney function and will replace less accurate methods of estimating kidney function, such as plasma creatinine and blood urea nitrogen.

Measurement of glomerular filtration rate (GFR) is the gold standard for kidney function assessment. Here we describe a high-throughput method which allows the determination of GFR in conscious mice by using a single bolus injection, determination of fluorescein isothiocyanate (FITC)-inulin in plasma and calculation of GFR by a two-phase exponential decay model.

The measurement of  glomerular filtration rate (GFR) is the gold standard in kidney function  assessment. Currently, investigators determine GFR by ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Measuring Glutathione-induced Feeding Response in Hydra

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. The movement of prey organisms causes mechanosensory discharge of the stinging cells called nematocysts from hydra, which are inserted into the prey. The feeding response in hydra, which includes curling of the tentacles to bring the prey towards the mouth, opening of the mouth and consequent engulfing of the prey, is triggered by GSH present in the fluid released from the injured prey. To be able to identify the molecular mechanism of the feeding response in hydra which is unknown to date, it is necessary to establish an assay to measure the feeding response. Here, we describe a simple method for the quantitation of the feeding response in which the distance between the apical end of the tentacle and mouth of hydra is measured and the ratio of such distance before and after the addition of GSH is determined. The ratio, called the relative tentacle spread, was found to give a measure of the feeding response. This assay was validated using a starvation model in which starved hydra show an enhanced feeding response in comparison with daily fed hydra.

Here we describe a simple assay for the quantification of the feeding response in hydra induced by the reduced form of glutathione. This assay relies on measuring the distance between the apical end of the tentacle and mouth of hydra.

Hydra is among the most primitive organisms possessing a nervous system and chemosensation for detecting reduced glutathione (GSH) for capturing the prey. ...

High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich meal, which consists of an emulsion of chicken egg yolk liposomes created by sonicating egg yolk in embryo media. Detailed instructions are provided to screen larvae for egg yolk consumption so that larvae that fail to feed will not confound experimental results. The chicken egg yolk liposomes can be spiked with fluorescent lipid analogs, including fatty acids and cholesterol, enabling both systemic and subcellular visualization of dietary lipid processing. Several methods are described to mount larvae that are conducive to short- and long-term live imaging with both upright and inverted objectives at high and low magnification. Additionally presented is an assay to quantify larval food intake by extracting the lipids of larvae fed fluorescent lipid analogs, spotting the lipids on a thin layer chromatography plate, and quantifying the fluorescence. Finally, critical aspects of the procedures, important controls, options for modifying the protocols to address specific experimental questions, and potential limitations are discussed. These techniques can be applied not only to focused, hypothesis driven inquiries, but also to a variety of screens and live imaging techniques to study dietary lipid metabolism and the control of food intake.

Zebrafish are emerging as a valuable model of dietary lipid processing and metabolic disease. Described are protocols of lipid-rich larval feeds, live imaging of dietary fluorescent lipid analogs, and quantification of food intake. These techniques can be applied to a variety of screening, imaging, and hypothesis driven inquiry techniques.

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich ...

A Method to Assess Bacteriocin Effects on the Gut Microbiota of Mice

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the factors that contribute to maintaining the population balance. However, there are limited available methodologies to evaluate these factors. Bacteriocins are antimicrobial peptides produced by many bacteria that may confer a competitive advantage for food acquisition and/or niche establishment. Many probiotic lactic acid bacteria (LAB) strains have great potential to promote human and animal health by preventing the growth of pathogens. They can also be used for immuno-modulation, as they produce bacteriocins. However, the antagonistic activity of bacteriocins is normally determined by laboratory bioassays under well-defined but over-simplified conditions compared to the complex gut environment in humans and animals, where bacteria face multifactorial influences from the host and hundreds of microbial species sharing the same niche. This work describes a complete and efficient procedure to assess the effect of a variety of bacteriocins with different target specificities in a murine system. Changes in the microbiota composition during the bacteriocin treatment are monitored using compositional 16S rDNA sequencing. Our approach uses both the bacteriocin producers and their isogenic non-bacteriocin-producing mutants, the latter giving the ability to distinguish bacteriocin-related from non-bacteriocin-related modifications of the microbiota. The fecal DNA extraction and 16S rDNA sequencing methods are consistent and, together with the bioinformatics, constitute a powerful procedure to find faint changes in the bacterial profiles and to establish correlations, in terms of cholesterol and triglyceride concentration, between bacterial populations and health markers. Our protocol is generic and can thus be used to study other compounds or nutrients with the potential to alter the host microbiota composition, either when studying toxicity or beneficial effects.

Bacteriocins are believed to play a key role in defining microbial diversity in different ecological niches. Here, we describe an efficient procedure to assess how bacteriocins affect gut microbiota composition in an animal model.

Very intriguing questions arise with our advancing knowledge on gut microbiota composition and the relationship with health, particularly relating to the ...

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...