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>

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<li>Sen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ultradian+feeding+in+mice+not+only+affects+the+peripheral+clock+in+the+liver%2C+but+also+the+master+clock+in+the+brain%5BTitle%5D)+AND+%22Chronobiology+International%22%5BJournal%5D)">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/pubmed?term=((Natural+food+intake+patterns+do+not+synchronize+peripheral+clocks%5BTitle%5D)+AND+%22BMC+Biology%22%5BJournal%5D)">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/pubmed?term=((Circadian+disruption+of+food+availability+significantly+reduces+reproductive+success+in+mice%5BTitle%5D)+AND+%22Hormones+and+Behavior%22%5BJournal%5D)">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/pubmed?term=((Protocol+for+setup+and+circadian+analysis+of+inverted+feeding+in+mice%5BTitle%5D)+AND+%22STAR+Protocols%22%5BJournal%5D)">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><tbody><tr><td>#6 x 0.75 inch Phillips Pan Head Stainless Steel Sheet Metal Screw (50-Pack)</td><td>Everbilt</td><td>#800172</td><td></td></tr><tr><td>#8 x 1.5 inch Phillips Pan Head Zinc Plated Sheet Metal Screw (100-Pack)</td><td>Everbilt&amp;nbsp;</td><td>#801622</td><td></td></tr><tr><td>0.25 inch gray PVC sheet (24 inch x 48 inch)</td><td>USPlastic</td><td>#45088</td><td></td></tr><tr><td>4 inch PVC pipe (10 ft)</td><td>Home Depot</td><td>#531103</td><td></td></tr><tr><td>45 mg dustless precision pellets</td><td>Bio-Serv</td><td>#F0165</td><td></td></tr><tr><td>6 ft. Extension Cord</td><td>HDX</td><td>HD#145-017</td><td></td></tr><tr><td>Food container (eight-compartment jewelry organizer)&amp;nbsp;</td><td>JewelrySupply</td><td>#PB8301</td><td></td></tr><tr><td>Indoor Basic Timer</td><td>General Electric</td><td>#15119</td><td></td></tr><tr><td>Oatey 4 inch ABS Pipe Test Cap with Knockout</td><td>Home Depot</td><td>#39103D</td><td></td></tr><tr><td>Rodent Diet with 45 kcal% fat (with red dye)</td><td>Research Diets</td><td>#D12451</td><td></td></tr></tbody></table>

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 ...

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Research

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Biology

2.2K 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

Automated, Quantitative Cognitive/Behavioral Screening of Mice: For Genetics, Pharmacology, Animal Cognition and Undergraduate Instruction

We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and pharmacological manipulations on basic mechanisms of cognition and learning in mice. A standard polypropylene mouse housing tub is connected through an acrylic tube to a standard commercial mouse test box. The test box has 3 hoppers, 2 of which are connected to pellet feeders. All are internally illuminable with an LED and monitored for head entries by infrared (IR) beams. Mice live in the environment, which eliminates handling during screening. They obtain their food during two or more daily feeding periods by performing in operant (instrumental) and Pavlovian (classical) protocols, for which we have written protocol-control software and quasi-real-time data analysis and graphing software. The data analysis and graphing routines are written in a MATLAB-based language created to simplify greatly the analysis of large time-stamped behavioral and physiological event records and to preserve a full data trail from raw data through all intermediate analyses to the published graphs and statistics within a single data structure. The data-analysis code harvests the data several times a day and subjects it to statistical and graphical analyses, which are automatically stored in the &#34;cloud&#34; and on in-lab computers. Thus, the progress of individual mice is visualized and quantified daily. The data-analysis code talks to the protocol-control code, permitting the automated advance from protocol to protocol of individual subjects. The behavioral protocols implemented are matching, autoshaping, timed hopper-switching, risk assessment in timed hopper-switching, impulsivity measurement, and the circadian anticipation of food availability. Open-source protocol-control and data-analysis code makes the addition of new protocols simple. Eight test environments fit in a 48 in x 24 in x 78 in&#160;cabinet; two such cabinets (16 environments) may be controlled by one computer.

Fully automated system for measuring physiologically meaningful properties of the mechanisms mediating spatial localization, temporal localization, duration, rate&#160;and probability estimation, risk assessment, impulsivity, and the accuracy and precision of memory, in order to assess the effects of genetic and pharmacological manipulations on foundational mechanisms of cognition in mice.

We describe a high-throughput, high-volume, fully automated, live-in 24/7 behavioral testing system for assessing the effects of genetic and ...

Behavior

A Fully Automated Rodent Conditioning Protocol for Sensorimotor Integration and Cognitive Control Experiments

Rodents have been traditionally used as a standard animal model in laboratory experiments involving a myriad of sensory, cognitive, and motor tasks. Higher cognitive functions that require precise control over sensorimotor responses such as decision-making and attentional modulation, however, are typically assessed in nonhuman primates. Despite the richness of primate behavior that allows multiple variants of these functions to be studied, the rodent model remains an attractive, cost-effective alternative to primate models. Furthermore, the ability to fully automate operant conditioning in rodents adds unique advantages over the labor intensive training of nonhuman primates while studying a broad range of these complex functions.
Here, we introduce a protocol for operantly conditioning rats on performing working memory tasks. During critical epochs of the task, the protocol ensures that the animal's overt movement is minimized by requiring the animal to 'fixate' until a Go cue is delivered, akin to nonhuman primate experimental design. A simple two alternative forced choice task is implemented to demonstrate the performance. We discuss the application of this paradigm to other tasks.

A fully automated protocol for rodent operant conditioning is proposed. The protocol relies on precise temporal control of behavioral events to investigate the extent to which this control influences neural activity underlying sensorimotor integration and cognitive control experiments.

Rodents have been traditionally used as a standard animal model in laboratory experiments involving a myriad of sensory, cognitive, and motor tasks. ...

Study Motor Skill Learning by Single-pellet Reaching Tasks in Mice

Reaching for and retrieving objects require precise and coordinated motor movements in the forelimb. When mice are repeatedly trained to grasp and retrieve food rewards positioned at a specific location, their motor performance (defined as accuracy and speed) improves progressively over time, and plateaus after persistent training. Once such reaching skill is mastered, its further maintenance does not require constant practice. Here we introduce a single-pellet reaching task to study the acquisition and maintenance of skilled forelimb movements in mice. In this video, we first describe the behaviors of mice that are commonly encountered in this learning and memory paradigm, and then discuss how to categorize these behaviors and quantify the observed results. Combined with mouse genetics, this paradigm can be utilized as a behavioral platform to explore the anatomical underpinnings, physiological properties, and molecular mechanisms of learning and memory.

Persistent practice improves the precision of coordinated movements.&#160;Here we introduce a single-pellet reaching task, which is designed to assess the learning and memory of forelimb skill in mice.&#160;

Reaching for and retrieving objects require precise and coordinated motor movements in the forelimb. When mice are repeatedly trained to grasp and ...

The Attentional Set Shifting Task: A Measure of Cognitive Flexibility in Mice

Cognitive impairment, particularly involving dysfunction of circuitry within the prefrontal cortex (PFC), represents a core feature of many neuropsychiatric and neurodevelopmental disorders, including depression, post-traumatic stress disorder, schizophrenia and autism spectrum disorder. Deficits in cognitive function also represent the most difficult symptom domain&#160;to successfully treat, as serotonin reuptake inhibitors and tricyclic antidepressants have only modest effects. Functional neuroimaging studies and postmortem analysis of human brain tissue implicate the PFC as being a primary region of dysregulation in patients with these disorders. However, preclinical behavioral assays used to assess these deficits in mouse models which can be readily manipulated genetically and could provide the basis for studies of new treatment avenues have been underutilized. Here we describe the adaptation of a behavioral assay, the attentional set shifting task (AST), to be performed in mice to assess prefrontal cortex mediated cognitive deficits. The neural circuits underlying behavior during the AST are highly conserved across humans, nonhuman primates and rodents, providing excellent face, construct and predictive validity.

The goal of this protocol is to perform a behavioral assay such as the attentional set shifting task (AST) to assess prefrontal cortex-mediated cognitive flexibility in mice.

Cognitive impairment, particularly involving dysfunction of circuitry within the prefrontal cortex (PFC), represents a core feature of many ...

Using the Activity-based Anorexia Rodent Model to Study the Neurobiological Basis of Anorexia Nervosa

Anorexia nervosa (AN) is a psychiatric illness characterized by excessively restricted caloric intake and abnormally high levels of physical activity. A challenging illness to treat, due to the lack of understanding of the underlying neurobiology, AN has the highest mortality rate among psychiatric illnesses. To address this need, neuroscientists are using an animal model to study how neural circuits may contribute toward vulnerability to AN and may be affected by AN. Activity-based anorexia (ABA) is a bio-behavioral phenomenon described in rodents that models the key symptoms of anorexia nervosa. When rodents with free access to voluntary exercise on a running wheel experience food restriction, they become hyperactive &#8211; running more than animals with free access to food. Here, we describe the procedures by which ABA is induced in adolescent female C57BL/6 mice. On postnatal day 36 (P36), the animal is housed with access to voluntary exercise on a running wheel. After 4 days of acclimation to the running wheel, on P40, all food is removed from the cage. For the next 3 days, food is returned to the cage (allowing animals free food access) for 2 hr daily. After the fourth day of food restriction, free access to food is returned and the running wheel is removed from the cage to allow the animals to recover. Continuous multi-day analysis of running wheel activity shows that mice become hyperactive within 24 hr following the onset of food restriction. The mice run even during the limited time during which they have access to food. Additionally, the circadian pattern of wheel running becomes disrupted by the experience of food restriction. We have been able to correlate neurobiological changes with various aspects of the animals&#8217; wheel running behavior to implicate particular brain regions and neurochemical changes with resilience and vulnerability to food-restriction induced hyperactivity.

Here we present a protocol to induce activity-based anorexia (ABA) in female adolescent mice. ABA is a condition of hyperactivity evoked by imposing food restriction on rodents with access to a running wheel. This phenomenon is being used as a model to study the underlying neurobiology of anorexia nervosa.

Anorexia nervosa (AN) is a psychiatric illness characterized by excessively restricted caloric intake and abnormally high levels of physical activity. A ...

Feeding Experimentation Device (FED): Construction and Validation of an Open-source Device for Measuring Food Intake in Rodents

Food intake measurements are essential for many research studies. Here, we provide a detailed description of a novel solution for measuring food intake in mice: the Feeding Experimentation Device (FED). FED is an open-source system that was designed to facilitate flexibility in food intake studies. Due to its compact and battery powered design, FED can be placed within standard home cages or other experimental equipment. Food intake measurements can also be synchronized with other equipment in real-time via FED's transistor-transistor logic (TTL) digital output, or in post-acquisition processing as FED timestamps every event with a real-time clock. When in use, a food pellet sits within FED's food well where it is monitored via an infrared beam. When the pellet is removed by the mouse, FED logs the timestamp onto its internal secure digital (SD) card and dispenses another pellet. FED can run for up to 5 days before it is necessary to charge the battery and refill the pellet hopper, minimizing human interference in data collection. Assembly of FED requires minimal engineering background, and off-the-shelf materials and electronics were prioritized in its construction. We also provide scripts for analysis of food intake and meal patterns. Finally, FED is open-source and all design and construction files are online, to facilitate modifications and improvements by other researchers.

Feeding Experimentation Device FED: Construction and Validation of an Open-source Device for Measuring Food Intake in Rodents

Feeding Experimentation Device (FED) is an open-source device for measuring food intake in mice. FED can also synchronize food intake measurements with other techniques via a real-time digital output. Here, we provide a step-by-step tutorial for the construction, validation, and usage of FED.

Food intake measurements are essential for many research studies. Here, we provide a detailed description of a novel solution for measuring food intake in ...

Progressive-ratio Responding for Palatable High-fat and High-sugar Food in Mice

Foods that are rich in fat and sugar significantly contribute to over-eating and escalating rates of obesity. The consumption of palatable foods can produce a rewarding effect that strengthens action-outcome associations and reinforces future behavior directed at obtaining these foods. Increasing evidence that the rewarding effects of energy-dense foods play a profound role in overeating and the development of obesity has heightened interest in studying the genes, molecules and neural circuitry that modulate food reward1,2. The rewarding impact of different stimuli can be studied by measuring the willingness to work to obtain them, such as in operant conditioning tasks3. Operant models of food reward measure acquired and voluntary behavioral responses that are directed at obtaining food. A commonly used measure of reward strength is an operant procedure known as the progressive ratio (PR) schedule of reinforcement.4,5 In the PR task, the subject is required to make an increasing number of operant responses for each successive reward. The pioneering study of Hodos (1961) demonstrated that the number of responses made to obtain the last reward, termed the breakpoint, serves as an index of reward strength4. While operant procedures that measure changes in response rate alone cannot separate changes in reward strength from alterations in performance capacity, the breakpoint derived from the PR schedule is a well-validated measure of the rewarding effects of food. The PR task has been used extensively to assess the rewarding impact of drugs of abuse and food in rats (e.g.,6-8), but to a lesser extent in mice9. The increased use of genetically engineered mice and diet-induced obese mouse models has heightened demands for behavioral measures of food reward in mice. In the present article we detail the materials and procedures used to train mice to respond (lever-press) for a high-fat and high-sugar food pellets on a PR schedule of reinforcement. We show that breakpoint response thresholds increase following acute food deprivation and decrease with peripheral administration of the anorectic hormone leptin and thereby validate the use of this food-operant paradigm in mice.

The present report details the protocol employed to measure the rewarding effects of high-fat food in mice using a progressive ratio operant conditioning task.

Foods that are rich in fat and sugar significantly contribute to over-eating and escalating rates of obesity. The consumption of palatable foods can ...

Neuroscience

Investigating Binge Eating Disorder with a Palatable Food Access Mouse Model

Intermittent Binge-Intake Model in Mice

Obesity affects one in eight individuals globally. Overeating, especially high-calorie foods, plays a significant role in obesity. Binge eating disorder (BED) is a complex condition caused by a combination of biological, psychological, and social factors. It involves excessive consumption of high-calorie foods in a short period and is often linked to anxiety and cravings.
Here, we present a protocol employing continuous and intermittent access to a new highly palatable food (HPF) -- M&amp;M&#39;s -- to develop a mouse model for investigating binge eating disorder (BED). The HPF, chosen for its high fat and sugar content, represents an optimal food source to study due to its strong palatability, which makes it particularly suited for examining compulsive eating behaviors. Using C57BL/6 mice, we provided continuous or intermittent access to M&amp;M&#39;s while allowing unrestricted access to standard chow and water. By the 8th day, the mice in the intermittent access group exhibited pronounced binge-eating behaviors, which persisted through the 26th day. These mice also consumed significantly more calories -- predominantly from the confectionary -- than those in the continuous access and control groups. The contrast between continuous and intermittent access underscores the critical role of feeding schedules in promoting the overconsumption of palatable foods.
Furthermore, behavioral assessments revealed intermittent access to the HPF-induced anxiety-like behaviors, highlighting the psychological impact of access patterns on both eating behavior and emotional states. By incorporating these two distinct feeding paradigms, this study offers valuable insights into how the availability of highly palatable foods can exacerbate binge-eating tendencies. This model provides a more realistic approach to studying binge-eating behaviors and their metabolic consequences. It highlights the importance of standardized models in biomedical research. Our findings offer insights into the physiological and neural mechanisms underlying BED, which could pave the way for developing effective therapeutic interventions for BED and obesity.

Author Spotlight: Accessible M&amp;M-Based Mouse Model for Investigating Binge Eating Disorder - Insights into Eating Behaviors, Anxiety, and Neural Mechanisms

A binge-eating model was established in mice using intermittent access to M&amp;M's (a highly palatable food made of fat and sugar). Mice with intermittent access to this high-fat, high-sugar food showed stress-like behaviors, increased caloric intake, and a preference for that food over standard chow compared to mice with continuous or no access to the food.

Obesity affects one in eight individuals globally. Overeating, especially high-calorie foods, plays a significant role in obesity. Binge eating disorder ...

Palatable Western-style Cafeteria Diet as a Reliable Method for Modeling Diet-induced Obesity in Rodents

Obesity is rapidly increasing in incidence in developed and developing countries and is known to induce or exacerbate many diseases.&#160;The health burden of obesity and its comorbid conditions highlight the need for better understanding of its pathogenesis, yet ethical constraints limit studies in humans. To this end externally valid models of obesity in laboratory animals are essential for the understanding of being overweight and obesity.&#160;While many species have been used to model the range of changes that accompany obesity in humans, rodents are most commonly used. Our laboratory has developed a western-style cafeteria diet that consistently leads to considerable weight gain and markers of metabolic disease in rodents. The diet exposes rodents to a variety of highly palatable foods to induce hyperphagia, modeling the modern western food environment. This diet rapidly induces weight gain and body fat accumulation in rats allowing for the study of effects of overeating and obesity. While the cafeteria diet may not provide the same control over macronutrient and micronutrient profile as purified high-fat or high-fat, high-sugar diets, the cafeteria diet typically induces a more severe metabolic phenotype than that observed with purified diets and is more in line with metabolic disturbances observed in the overweight and obese human population.

This protocol describes the use of a highly palatable, western-style cafeteria diet to model overeating and obesity in rodents. Here, we provide a detailed outline of food selection, preparation and measurement, and explain methodological factors that assist in generating a robust and reproducible phenotype.

Obesity is rapidly increasing in incidence in developed and developing countries and is known to induce or exacerbate many diseases.&#160;The health ...

Medicine

Sucrose Preference and Novelty-Induced Hypophagia Tests in Rats using an Automated Food Intake Monitoring System

The prevalence and incidence of depressive disorders are rising worldwide, affecting about 322 million individuals, underlining the need for behavioral studies in animal models. In this protocol, to study depression-like and anhedonic behavior in rats, the established sucrose preference and novelty-induced hypophagia tests are combined with an automated food and liquid intake monitoring system. Prior to testing, in the sucrose preference paradigm, male rats are trained for at least 2 days to consume a sucrose solution in addition to tap water. During the test, rats are again exposed to water and sucrose solution. Consumption is registered every second by the automated system. The ratio of sucrose to total water intake (sucrose preference ratio) is a surrogate parameter for anhedonia. In the novelty-induced hypophagia test, male rats undergo a training period in which they are exposed to a palatable snack. During training, rodents show a stable baseline snack intake. On test day, the animals are transferred from home cages into a fresh, empty cage representing a novel unknown environment with access to the known palatable snack. The automated system records the total intake and its underlying microstructure (e.g., latency to approaching the snack), providing insight into anhedonic and anxious behaviors. The combination of these paradigms with an automated measuring system provides more detailed information, along with higher accuracy by reducing measuring errors. However, the tests use surrogate parameters and only depict depression and anhedonia in an indirect manner.

Presented here is a protocol to study depression-like and anhedonic behavior in rats. It combines two well-established behavioral methods, the sucrose preference and novelty-induced hypophagia tests, with an automated food and liquid intake monitoring system, to indirectly investigate rodent behavior using surrogate parameters.

The prevalence and incidence of depressive disorders are rising worldwide, affecting about 322 million individuals, underlining the need for behavioral ...