This work was funded by a NARSAD Independent Investigator Award and an IMHRO Rising Star Depression Research Award in Memory of George Largay to SCD.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of the University of California, San Diego.
1. Mice
<ol>
	<li>Choose the appropriate mouse strain for the study.</li>
	<li>Purchase mice from a supplier or amplify a line to obtain appropriate experimental numbers.</li>
	<li>Group-house purchased mice in the animal facility for at least 1 week prior to beginning the study to allow for an ample acclimation period.</li>
	<li>Begin the pre-experimental acclimation phase when mice are 8 weeks old.</li>
</ol>
2. Housing
<ol>
	<li>Choose cages that are large enough to contain an un-obstructed running wheel, food receptacle, and a water bottle.</li>
	<li>Regulate room temperature and humidity to complement the hypothesis being tested. 
	NOTE: Higher temperatures will reduce the development of ABA28.</li>
</ol>
3. Running Wheels
<ol>
	<li>Choose wheels with wireless communication to avoid potential distractions and entanglements within the cage, see the Table of Materials for an example.</li>
</ol>
4. Water Bottles
<ol>
	<li>Select a water bottle that will not compete for space with the running wheel or food receptacle in the home cage.</li>
	<li>If providing drug treatment in the drinking water, duct tape the bottle to create a light-sensitive environment.</li>
</ol>
5. Food
<ol>
	<li>Select a low-fat chow appropriate for the hypothesis being tested (e.g., Envigo&#39;s Rodent Diet 8604). 
	NOTE: Diets high in fat and sugar can reduce the development of ABA29.</li>
	<li>Use a small glass jar to provide chow, approximately 65 cm in diameter x 50 cm in height. 
	NOTE: An automated feeder may be used instead of a glass jar, which may reduce stress by limiting investigator interference. This may be most advantageous if performing ABA studies with drug manipulations.</li>
</ol>
6. Pre-experimental Acclimation Phase
<ol>
	<li>Set up the experimental area by choosing a cage rack close to the laptop and wheel hubs. This will limit issues with data transmission.</li>
	<li>Turn on the laptop and wireless hubs.</li>
	<li>Open the wheel management software on the laptop.
	<ol>
		<li>Look at the software to ensure both hubs are active.</li>
		<li>Open &#34;tools&#34; and select &#34;delete wheels.&#34;&#160;Repeat this step 5 times.</li>
		<li>Close the software, then re-open it.</li>
	</ol>
	</li>
	<li>Place 3 AAA batteries into the battery pack of a wheel base.
	<ol>
		<li>Connect the battery pack wires to the hardware of the wheel base. This will be disconnected prior to use. Close the battery pack into the wheel base.</li>
	</ol>
	</li>
	<li>Check the software to ensure the wheel base is listed with the ID &#34;1&#34; under the appropriate wireless hub. If the base is not listed as ID &#34;1&#34;, unhook the battery pack, and start over at step 6.2.</li>
	<li>Change the wheel ID from &#34;1&#34; to the mouse ID under the name column.</li>
	<li>Adhere the running wheel&#39;s base to the bottom of the cage with duct tape to ensure stability. Do not allow any tape to stick out, as mice will chew on exposed tape.</li>
	<li>Choose a running wheel disc without a corroded magnetic piece.</li>
	<li>Place the wheel on the base and rotate the wheel to verify clearance from the cage walls and grating.</li>
	<li>Spin the disc a specific number of rotations to ensure the wheel running software is properly counting rotations.</li>
	<li>Prevent pieces of chow from being lost in the bedding or getting wet by duct-taping the underside of the food jar to the cage floor, away from the running wheel and the water bottle. 
	NOTE: Wet chow increases food intake compared to dry chow30.</li>
	<li>Place 5 pieces of chow into the jar. 
	NOTE: The chow during the pre-experimental phase is available ad libitum and does not need to be weighed. Exposure to running wheels during the acclimation period may intensify ABA21,31.</li>
	<li>Place a full water bottle in the cage and provide water ad libitum.</li>
	<li>Repeat until all cages have a wirelessly connected running wheel, food, and water.</li>
	<li>Individually place mice into their appropriate cages.</li>
	<li>In the wheel managing software, go to &#34;file&#34; and click &#34;start acquisition.&#34;</li>
	<li>Watch the software update to ensure wheel rotations are being counted and the system is working well.</li>
	<li>Acclimate mice to the experimental housing conditions for 2 total days.</li>
	<li>Assess mice during this phase to ensure they are healthy enough to handle the ABA experiment.</li>
	<li>Check cages for any flooding from water bottles each day. If bottles have flooded, replace the old cage with a new cage and repeat the necessary steps to adhere wheels and food to the cage without interference.</li>
	<li>On day 2, check cages for food levels. If food is low, refill with 2 pieces of chow. Do this without disturbing the mice. 
	NOTE: If during the pre-experimental acclimation phase any mouse is exhibiting hypophagia or is underweight, remove the mouse from the study.</li>
</ol>
7. Experimental Baseline Phase - Day 1
<ol>
	<li>Select &#34;end&#34;, and then &#34;start acquisition&#34; in the wheel management software.</li>
	<li>Measure out 9 g chow in a small plastic beaker on a scale with 0.00 g sensitivity. Record the exact weight.</li>
	<li>Gently remove and weigh the mouse in a large plastic beaker on a scale with 0.0 g sensitivity. Record weight. 
	NOTE: It is crucial to limit the stress of mice while handling, therefore handle them patiently.</li>
	<li>Dispose of all acclimation food.</li>
	<li>Search bedding for any food that may have been removed from the jar and dispose of that as well.</li>
	<li>Leave the water bottle in place, but ensure it is adequately full.</li>
	<li>Clean soiled running wheels with 70% isopropanol and paper towels.</li>
	<li>If cleaning was necessary, after the isopropanol dries, return the mouse to its cage and replace the cage on the rack.</li>
	<li>Repeat these steps until every mouse is weighed and 9 g of food is measured out and placed in cages.</li>
</ol>
8. Experimental Baseline Phase - Days 2 - 7
<ol>
	<li>Return to the animal facility at the same time every day. Do not vary in taking measurements by over or under 10 min.</li>
	<li>Measure out 9 g chow in a small plastic beaker on a scale with 0.00 g sensitivity. Record the exact weight.</li>
	<li>Gently remove and weigh the mouse in a large plastic beaker on a scale with 0.0 g sensitivity. Record weight.</li>
	<li>Pull the food jar out of the cage and dump all the food into a small beaker on the 0.00 g scale.
	<ol>
		<li>If feces and bedding are in the food jar with the food, pick out all of the bedding and feces. Use a small strainer to rub powdered food into the weighing beaker. Remove any feces before doing this with tweezers (if necessary). 
		&#8203;NOTE: Bedding will not break up into smaller pieces to fit through the strainer.</li>
		<li>Search for any food remnants in the cage and add them to the beaker to be weighed. Record the amount of food remaining. 
		NOTE: If the bedding and cage must be searched, it is best to do so while the mouse is in the weigh beaker or transfer cage to reduce stress.</li>
		<li>Dispose of all food.</li>
	</ol>
	</li>
	<li>Wipe jar clean and place back into the cage. Replace tape if necessary.</li>
	<li>Place the previously measured 9 g of new food into the jar.</li>
	<li>Clean soiled running wheels with 70% isopropanol, then dry the wheels. Do this one cage at a time and do not accidentally interchange wheels.</li>
	<li>End and restart acquisition in wheel running program every other day.</li>
	<li>Repeat these steps until the start of the restriction phase.</li>
</ol>
9. Experimental Restriction Phase - Day 1
<ol>
	<li>End and start acquisition in the wheel management software.</li>
	<li>Weigh mice, old, and new food per baseline protocol.</li>
	<li>Do not clean wheels now. This may distract mice during their limited feeding period.</li>
	<li>Allow access to new food, 9 g of chow, for 6 h. Record the time food is offered. 
	NOTE: The time during the light cycle that food is available will affect the development of ABA. Since mice normally sleep during the light cycle, giving access to food during this time exacerbates the development of ABA, since mice do not usually consume the majority of their daily food at this time. Conversely, mice given food access during the dark cycle will develop ABA more slowly32. Adjusting duration of food access will result in longer or shorter survival times (Figure 2). Food anticipatory activity (FAA) is hyperactivity, which occurs immediately before food is offered32,33,34. FAA can affect the development of ABA, as it has been shown that the denial of wheel running access during FAA ameliorates ABA behavior32.
	<ol>
		<li>Remove food exactly 6 h later for each mouse and measure the quantity remaining. Record the time food is removed.</li>
	</ol>
	</li>
	<li>Clean wheels now, if necessary, and attempt to do so without getting behind on pulling food from subsequent cages.</li>
	<li>Weigh food and calculate the difference in food to determine the amount of food eaten per mouse.</li>
	<li>Calculate the dropout weight for each mouse. 
	NOTE: Dropout weight is when a mouse must be removed from the experiment when it reaches 75% of its final measurement of baseline body weight. For example, if a mouse weighs 20 g at the beginning of the restriction phase, when it reaches 15 g it must be removed from the experiment.</li>
	<li>Keep a copy of the dropout weights on hand in the animal facility. 
	NOTE: Experiments evaluating recovery from ABA may be attempted; food is either provided ad libitum, or wheels are locked to study the process by which mice return to a healthy body weight34.</li>
</ol>
10. Experimental Restriction Phase - Days 2 to 14
<ol>
	<li>Repeat all measurement steps in the Restriction Day 1 protocol. Do not restart wheel acquisition.</li>
	<li>If a mouse reaches drop weight (75% of its baseline body weight), remove the wheel and food jar from the cage and provide ample food on the bottom of the cage, then proceed with the other mice. Once all cages have been attended to, euthanize all mice that have reached drop weight.</li>
	<li>Leave the wheel on for each mouse for the duration of the experiment.</li>
	<li>Check each mouse daily for any injuries. If necessary, clip damaged toenails, treat sores, etc. Ask animal care personnel for advice if unsure about how to proceed with an injury.</li>
</ol>
11. Ending the Study
<ol>
	<li>If the study has lasted 14 days, weigh all the mice on day 15. Restriction day 1 weight is considered a baseline weight. 
	NOTE: The length of the study can be modified to address the hypothesis being tested. Longer duration food access periods may be used to extend study length.</li>
	<li>Euthanize mice as necessary.</li>
	<li>Take dirty equipment to appropriate rooms.</li>
	<li>End wheel count acquisition on day 15 to provide 10 full days of data.</li>
	<li>Turn off wireless wheel hubs.</li>
	<li>Extract data.</li>
	<li>Analyze data.</li>
</ol>

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</ol>

The authors have nothing to disclose.

The ABA experiment can be modified by experimenters to test different strains, ages, drugs, and various other variables of AN. Bearing in mind genetic variability, adjustments to the running wheel or food access period would increase or decrease the severity of symptoms and rate of dropout. This can serve to increase or decrease the length of the experiment, depending on the experimental question of interest. 
Accurate measurement of food intake is a critical part of the ABA paradigm, but can ...

Since 1953, rodents have been reported to show a paradoxical hyperactivity on running wheels when they are given free access to the wheels while undergoing voluntary hypophagia when food availability is restricted1. Conversely, rodents do not rapidly drop in body weight when offered food on a schedule without running wheels or when housed with running wheels and offered food ad libitum1,2,3. The ABA model reliably results in dramatic declines in body weight, hypophagia, hypothermia, loss of estrus, and increased stimulation of the HPA axis4. Ultimately, ABA results in death, unless the subject is removed from the paradigm5. The ABA paradigm provides researchers with an animal model of AN, a complex eating disorder which features severe dysregulation of appetite-behavior, affecting approximately 1 in 100 females and a smaller percentage of males6. Patients suffering from AN frequently exhibit hyperactivity, consisting of extreme amounts of exercise, and/or general restlessness7,8. With a mortality rate of approximately 10%, AN has the highest mortality rate among all psychiatric disorders9. Current treatment for AN is limited to cognitive therapies, as there are no approved pharmacological treatments for those suffering from AN10,11.
AN has typically been regarded as a disorder affecting primarily females. As a diagnosis that is 10 times more likely in females than males, female subjects are traditionally the focus in AN12. However, special consideration should be taken in excluding males from studies. While the diagnosis of AN remains lower in males, muscle dysmorphia (MD) is a condition that has many similarities to AN, in that body image is distorted and diet is often disordered. There is support for the notion that MD and AN may be classified in a similar manner13,14,15. This may suggest that some cases of MD represent the &#34;male version&#34; of AN. In the context of animal models, some reports have suggested that males are more susceptible than females to the ABA paradigm. For example, a recent study showed a higher mortality rate and decreased food intake compared to females in C57Bl/6 mice16. One predictor of susceptibility to ABA is spontaneous physical activity (SPA). Rats with higher or lower SPA are more likely to lose weight in the ABA paradigm, with male rats showing a stronger effect than females17. Conversely, female rodents have been observed to exercise more than males during the restriction phase of ABA18. Additionally, studies with Balb/cJ mice have shown the opposite effect of C57Bl/6 mice, where female mice have a higher mortality rate and decreased food intake compared to males (Figure 1)6. With varying results between the sexes in the ABA paradigm and increasing awareness of males with disordered eating patterns, both male and female subjects should be tested.
Aside from sex differences in the ABA paradigm, age and strain must be considered when choosing subjects. Adolescent mice may more accurately model AN, since AN typically emerges in adolescence, as observed with rats and mice19,20,21,22. Strains that are more active than others at a baseline level have a higher rate of susceptibility and severity of ABA23. Strains known to have higher levels of anxiety, such as DBA/2, have increased wheel running activity, which would indicate a faster dropout rate in the ABA paradigm24. Depending on the experimental design, the strain of choice can be tailored to maximize the duration of ABA.
The ABA paradox is not unique to mice. Other mammals including rats, hamsters, gerbils, pigs, chipmunks, and guinea pigs have demonstrated this phenomenon6. The conservation of the ABA phenomenon across mammalian species suggests that the ABA paradigm can provide a translational tool to investigate the mechanisms underlying anorexia-like behavior in humans. Mice in particular are well-suited for the study of mechanisms underlying ABA. Mice can be densely housed and generation time is relatively short. Mice have a fully sequenced genome, and numerous inbred, outbred, and special strains, such as congenics, are available. A vast number of genetically manipulated lines have been generated, making them ideal for studies assessing genetic influences on disorders such as AN. Depending on the question at hand, researchers may manipulate complex neural circuitry and/or gene expression to assess behavior in the ABA paradigm, potentially answering questions about genetic influence that are not possible when studying humans.
A limited number of animal models of AN currently exist. Stress models induce hypophagia in rodents using tail pinching, novelty-induced hypophagia, cold swimming, and brain stimulation. By inducing stress, changes in the HPA-axis decrease appetite resulting in reduced body weight25. However, the HPA-axis is also potently stimulated by ABA, which also incorporates additional features of AN such as hyperactivity. Another model to consider in studying AN is the chronic food restriction model. By limiting food to a range from 40 to 60% of ad libitum, one can mimic the physiological response to malnourishment26. Though this method is effective for studying the effects of inadequate feeding, it does not reproduce a core issue of AN, which is voluntary food restriction. In the ABA paradigm, animals are deprived of food access for part of the day, but also voluntarily reduce food intake if a wheel is also present. Genetic models have also been used to examine the etiology of AN. Researchers have found neurochemical and genetic factors implicated in AN, such as the gene BDNF and neurotransmitters dopamine and serotonin27. The use of genetic models is crucial to understand the neural mechanisms behind AN. However, genome wide association studies for AN have not yet yielded significant hits, and no rare variants in AN have been identified. Future studies should combine a genetic approach with the ABA model to increase understanding of AN-related phenotypes.
Developing animal models for entire psychiatric disorders is virtually impossible due to the complexity and heterogeneity of human disorders. However, by modeling specific, well-defined components of a psychiatric disorder, unique insights into the underlying neurobiology or pathophysiology may be obtained. Such biological insights may then be used to identify novel treatments. The rodent ABA paradigm therefore provides a preclinical tool for studying the mechanisms underlying AN-like behavior that cannot ethically be studied in humans, such as the effects of genetic manipulations, perturbations to neural circuits, and the consequences of certain environmental factors.

<table><tbody><tr><td>Wireless Low Profile Running Wheels, Hubs, Software</td><td>Med Associates</td><td>ENV-047</td><td></td></tr><tr><td>Standard Teklad Rodent Chow</td><td>Envigo</td><td>8604</td><td></td></tr><tr><td>8 Week Old Mice</td><td>Jackson Laboratories</td><td>Balb/cJ</td><td>Strain used in our study - Can use other strains to assess ABA</td></tr><tr><td>Scout Pro Scale 200 g</td><td>Ohaus</td><td>SPE202</td><td>Used to weigh mice</td></tr><tr><td>Scout Pro Scale 400 g</td><td>Ohaus</td><td>SPE402</td><td>Used to weight food</td></tr></tbody></table>

assessing activity-based anorexia in mice

Rodents develop activity-based anorexia (ABA) when exposed to a restricted feeding schedule and allowed free access to a running wheel. These conditions lead to a life-threatening reduction in body weight. However, rodents exposed to only one of these conditions ultimately adapt to re-establish normal body weight. Although increased running coupled with reduction in voluntary food intake appear paradoxical under ABA conditions, ABA behavior is observed across numerous mammalian species. 
The ABA paradigm provides an animal model for anorexia nervosa (AN), an eating disorder with severe dysregulation of appetite-behavior. Subjects are singly housed with free access to a running wheel. Each day, the subject is offered food for a limited amount of time. During the course of the experiment, a subject's body weight decreases from high activity and low caloric intake. The duration of the study varies based on how long food is offered daily, the type of food offered, the strain of mouse, if drugs are being tested, and environmental factors.
A lack of effective pharmacological treatments for AN patients, their low quality of life, high cost of treatment, and their high mortality rate indicate the urgency to further research AN. We provide a basic outline for performing ABA experiments with mice, offering a method to investigate AN-like behavior in order to develop novel therapies. This protocol is optimized for use in Balb/cJ mice, but can easily be manipulated for other strains, providing great flexibility in working with different questions, especially related to genetic factors of ABA.

Brain-derived neurotrophic factor (BDNF), a protein that contributes to the regulation of feeding as well as weight maintenance, is reduced in the serum of patients with AN35. This experiment assessed the effects of scheduled feeding, running wheel access, or both on BDNF expression within the hippocampus (HPC), ventral tegmental area (VTA), nucleus accumbens (NAc), and medial prefrontal cortex (mPFC). Expression of neuronal cell adhesion molecule 1 (NCAM1) was als...

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Assessing Activity-based Anorexia in Mice

Mice individually housed with a running wheel while given limited access to food develop reductions in food consumption and increase activity on the running wheel. This experimental phenomenon is called activity-based anorexia. This paradigm provides an experimental tool for studying the neurobiology and behaviors underlying aspects of anorexia nervosa.

Rodents develop activity-based anorexia (ABA) when exposed to a restricted feeding schedule and allowed free access to a running wheel. These conditions ...

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Research

JoVE Journal

Behavior

12.5K Views.  University of California, San Diego. Mice individually housed with a running wheel while given limited access to food develop reductions in food consumption and increase activity on the running wheel. This experimental phenomenon is called activity-based anorexia. This paradigm provides an experimental tool for studying the neurobiology and behaviors underlying aspects of anorexia nervosa.

Video: Assessing Activity-based Anorexia in Mice

Trace Fear Conditioning in Mice

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings between a neutral stimulus and an unconditioned stimulus. There is a 20 sec trace period that separates each conditioning trial. On the following day freezing is measured during presentation of the conditioned stimulus (CS) and trace period. On the third day there is an 8 min test to measure contextual memory. The representative results are from mice that were presented with the aversive unconditioned stimulus (shock) compared to mice that received the tone presentations without the unconditioned stimulus. Trace fear conditioning has been successfully used to detect subtle learning and memory deficits and enhancements in mice that are not found with other fear conditioning methods. This type of fear conditioning is believed to be dependent upon connections between the medial prefrontal cortex and the hippocampus. One current controversy is whether this method is believed to be amygdala-independent. Therefore, other fear conditioning testing is needed to examine amygdala-dependent learning and memory effects, such as through the delay fear conditioning.

In the following experiment we describe a protocol for trace fear conditioning in mice. This type of associative memory includes a trace period that separates the neutral stimulus and the unconditioned stimulus.

In this experiment we present a technique to measure learning and memory. In the trace fear conditioning protocol presented here there are five pairings ...

Assessing Functional Performance in the Mdx Mouse Model

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder for which no cure is available. Nevertheless, several potential pharmaceutical compounds and gene therapy approaches have progressed into clinical trials. With improvement in muscle function being the most important end point in these trials, a lot of emphasis has been placed on setting up reliable, reproducible, and easy to perform functional tests to pre clinically assess muscle function, strength, condition, and coordination in the mdx mouse model for DMD. Both invasive and noninvasive tests are available. Tests that do not exacerbate the disease can be used to determine the natural history of the disease and the effects of therapeutic interventions (e.g. forelimb grip strength test, two different hanging tests using either a wire or a grid and rotarod running). Alternatively, forced treadmill running can be used to enhance disease progression and/or assess protective effects of therapeutic interventions on disease pathology. We here describe how to perform these most commonly used functional tests in a reliable and reproducible manner. Using these protocols based on standard operating procedures enables comparison of data between different laboratories.

Assessing Functional Performance in the Mdx Mouse Model

The primary outcome measure in clinical trials for neuromuscular disorders is generally improved muscle function. Therefore, assessing the effect of potential therapeutic compounds on muscle performance pre clinically in mouse models is of great importance. We here describe several functional tests to address this.

Duchenne muscular dystrophy (DMD) is a severe and progressive muscle wasting disorder for which no cure is available. Nevertheless, several potential ...

Operant Procedures for Assessing Behavioral Flexibility in Rats

Executive functions consist of multiple high-level cognitive processes that drive rule generation and behavioral selection. An emergent property of these processes is the ability to adjust behavior in response to changes in one&#8217;s environment (i.e., behavioral flexibility). These processes are essential to normal human behavior, and may be disrupted in diverse neuropsychiatric conditions, including schizophrenia, alcoholism, depression, stroke, and Alzheimer&#8217;s disease. Understanding of the neurobiology of executive functions has been greatly advanced by the availability of animal tasks for assessing discrete components of behavioral flexibility, particularly strategy shifting and reversal learning. While several types of tasks have been developed, most are non-automated, labor intensive, and allow testing of only one animal at a time. The recent development of automated, operant-based tasks for assessing behavioral flexibility streamlines testing, standardizes stimulus presentation and data recording, and dramatically improves throughput. Here, we describe automated strategy shifting and reversal tasks, using operant chambers controlled by custom written software programs. Using these tasks, we have shown that the medial prefrontal cortex governs strategy shifting but not reversal learning in the rat, similar to the dissociation observed in humans. Moreover, animals with a neonatal hippocampal lesion, a neurodevelopmental model of schizophrenia, are selectively impaired on the strategy shifting task but not the reversal task. The strategy shifting task also allows the identification of separate types of performance errors, each of which is attributable to distinct neural substrates. The availability of these automated tasks, and the evidence supporting the dissociable contributions of separate prefrontal areas, makes them particularly well-suited assays for the investigation of basic neurobiological processes as well as drug discovery and screening in disease models.

The ability to assess executive functions such as behavioral flexibility in rats is useful for investigating the neurobiology of cognition in both intact animals and disease models. Here we describe automated tasks for assessing strategy shifting and reversal learning, which are particularly sensitive to disruptions in prefrontal cortical networks.

Executive functions consist of multiple high-level cognitive processes that drive rule generation and behavioral selection. An emergent property of these ...

A Protocol for Housing Mice in an Enriched Environment

Environmental enrichment (EE) is a housing environment for mice that boosts mental and physical health compared to standard laboratory housing. Our recent studies demonstrate that environmental enrichment decreases adiposity, increases energy expenditure, resists diet induced obesity, and causes cancer remission and inhibition in mice. EE typically consists of larger living space, a variety of &#8216;toys&#8217; to interact with, running wheels, and can include a number of other novel environmental changes. All of this fosters a more complex social engagement, cognitive and physical stimulations. Importantly, the toy location and type of toy is changed regularly, which encourages the mice to adapt to a frequently changing and novel environment. Many variables can be manipulated in EE to promote health effects in mice. Thus these approaches are difficult to control and must be properly managed to successfully replicate the associated phenotypes. Therefore, the goal of this video is to demonstrate how EE is properly set up and maintained to assure a complex, challenging, and controlled environment so that other researchers can easily reproduce the protective effects of EE against obesity and cancer.

Environmental enrichment for mice requires a complex and challenging setup, as well as comprehensive husbandry and handling techniques to assure robust metabolic and anti-cancer effects in the mice. This protocol provides detailed procedures to reproduce the above mentioned effects in mice.

Environmental enrichment (EE) is a housing environment for mice that boosts mental and physical health compared to standard laboratory housing. Our recent ...

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 ...

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 ...

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate utilization, and behaviors such as food intake and physical activity can provide important information regarding the underlying mechanisms of disease. Given the importance of body composition and metabolism to the development of obesity and its subsequent sequelae, it is necessary to make accurate measures of these parameters in the pre-clinical research setting. Advances in technology over the past few decades have made it possible to derive these measures in rodent models in a non-invasive and longitudinal fashion. Consequently, these metabolic measures have proven useful when assessing the response of genetic manipulations (for example knockout or transgenic mice, viral knock-down or overexpression of genes), experimental drug/compound screening and dietary, behavioral or physical activity interventions. Herein, we describe the protocols used to measure body composition and metabolic parameters using an animal monitoring system in chow-fed and high fat diet-fed mice.

This protocol describes the use of a body composition analyzer and metabolic animal monitoring system to characterize body composition and metabolic parameters in mice. An obesity model induced by high-fat feeding is used as an example for the application of these techniques.

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate ...

Medicine

Assessment of the Metabolic Effects of Isocaloric 2:1 Intermittent Fasting in Mice

Intermittent fasting (IF), a dietary intervention involving periodic energy restriction, has been considered to provide numerous benefits and counteract metabolic abnormalities. So far, different types of IF models with varying durations of fasting and feeding periods have been documented. However, interpreting the outcomes is challenging, as many of these models involve multifactorial contributions from both time- and calorie-restriction strategies. For example, the alternate day fasting model, often used as a rodent IF regimen, can result in underfeeding, suggesting that health benefits from this intervention are likely mediated via both caloric restriction and fasting-refeeding cycles. Recently, it has been successfully demonstrated that 2:1 IF, comprising 1 day of fasting followed by 2 days of feeding, can provide protection against diet-induced obesity and metabolic improvements without a reduction in overall caloric intake. Presented here is a protocol of this isocaloric 2:1 IF intervention in mice. Also described is a pair-feeding (PF) protocol required to examine a mouse model with altered eating behaviors, such as hyperphagia. Using the 2:1 IF regimen, it is demonstrated that isocaloric IF leads to reduced body weight gain, improved glucose homeostasis, and elevated energy expenditure. Thus, this regimen may be useful to investigate the health impacts of IF on various disease conditions.

The current article describes a detailed protocol for isocaloric 2:1 intermittent fasting to protect and treat against obesity and impaired glucose metabolism in wild-type and ob/ob mice.

Intermittent fasting (IF), a dietary intervention involving periodic energy restriction, has been considered to provide numerous benefits and counteract ...

Biology

Determining Basal Energy Expenditure and the Capacity of Thermogenic Adipocytes to Expend Energy in Obese Mice

Energy expenditure measurements are necessary to understand how changes in metabolism can lead to obesity. Basal energy expenditure can be determined in mice by measuring whole-body oxygen consumption, CO2 production, and physical activity using metabolic cages. Thermogenic brown/beige adipocytes (BA) contribute significantly to rodent energy expenditure, particularly at low ambient temperatures. Here, measurements of basal energy expenditure and total BA capacity to expend energy in obese mice are described in two detailed protocols: the first explaining how to set up the assay to measure basal energy expenditure using analysis of covariance (ANCOVA), a necessary analysis given that energy expenditure co-varies with body mass. The second protocol describes how to measure BA energy expenditure capacity in vivo in mice. This procedure involves anesthesia, needed to limit expenditure caused by physical activity, followed by the injection of beta3-adrenergic agonist, CL-316,243, which activates energy expenditure in BA. These two protocols and their limitations are described in sufficient detail to allow a successful first experiment.

This manuscript describes a protocol to measure the basal metabolic rate and the oxidative capacity of thermogenic adipocytes in obese mice.

Energy expenditure measurements are necessary to understand how changes in metabolism can lead to obesity. Basal energy expenditure can be determined in ...

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.

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 ...

Assessing Activity-based Anorexia in Mice

Summary

Explore More Videos

Trace Fear Conditioning in Mice

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

Intermittent Binge-Intake Model in Mice

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

Assessment of the Metabolic Effects of Isocaloric 2:1 Intermittent Fasting in Mice

Determining Basal Energy Expenditure and the Capacity of Thermogenic Adipocytes to Expend Energy in Obese Mice

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