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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G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+excessive+running+in+the+development+of+activity+anorexia.">An analysis of excessive running in the development of activity anorexia.</a> Physiol Behav. 58 (3), 451-457 (1995).</li><li>Dixon, D. P., Ackert, A. M., Eckel, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of,+and+recovery+from,+activity-based+anorexia+in+female+rats.">Development of, and recovery from, activity-based anorexia in female rats.</a> Physiol Behav. 80 (2-3), 273-279 (2003).</li><li>Klenotich, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olanzapine,+but+not+fluoxetine,+treatment+increases+survival+in+activity-based+anorexia+in+mice.">Olanzapine, but not fluoxetine, treatment increases survival in activity-based anorexia in mice.</a> Neuropsychopharmacology. 37 (7), 1620-1631 (2012).</li><li>Ho, E. V., Klenotich, S. J., McMurray, M. S., Dulawa, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-Based+Anorexia+Alters+the+Expression+of+BDNF+Transcripts+in+the+Mesocorticolimbic+Reward+Circuit.">Activity-Based Anorexia Alters the Expression of BDNF Transcripts in the Mesocorticolimbic Reward Circuit.</a> PLoS ONE. 11 (11), 0166756 (2016).</li></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...

Watch this Scientific Journal Video about Assessing Activity-based Anorexia in Mice at JoVE.com

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

12.4K 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

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 ...

Assessing Burrowing, Nest Construction, and Hoarding in Mice

Deterioration in the ability to perform "Activities of daily living" (ADL) is an early sign of Alzheimer's disease (AD). Preclinical behavioural screening of possible treatments for AD currently largely focuses on cognitive testing, which frequently demands expensive equipment and lots of experimenter time. However, human episodic memory (the most severely affected aspect of memory in AD) is different to rodent memory, which seems to be largely non-episodic. Therefore the present ways of screening for new AD treatments for AD in rodents are intrinsically unlikely to succeed. A new approach to preclinical screening would be to characterise the ADL of mice. Fortuitously, several such assays have recently been developed at Oxford, and here the three most sensitive and well-characterised are presented.
Burrowing was first developed in Oxford13. It evolved from a need to develop a mouse hoarding paradigm. Most published rodent hoarding paradigms required a distant food source to be linked to the home cage by a connecting passage. This would involve modifying the home cage as well as making a mouse-proof connecting passage and food source. So it was considered whether it would be possible to put the food source inside the cage. It was found that if a container was placed on the floor it was emptied by the next morning., The food pellets were, however, simply deposited in a heap at the container entrance, rather than placed in a discrete place away from the container, as might be expected if the mice were truly hoarding them. Close inspection showed that the mice were performing digging ("burrowing") movements, not carrying the pellets in their mouths to a selected place as they would if truly hoarding them.6



Food pellets are not an essential substrate for burrowing; mice will empty tubes filled with sand, gravel, even soiled bedding from their own cage. Moreover, they will empty a full tube even if an empty one is placed next to it8.



Several nesting protocols exist in the literature. The present Oxford one simplifies the procedure and has a well-defined scoring system for nest quality5. 



A hoarding paradigm was later developed in which the mice, rather than hoarding back to the real home cage, were adapted to living in the "home base" of a hoarding apparatus. This home base was connected to a tube made of wire mesh, the distal end of which contained the food source. This arrangement proved to yield good hoarding behaviour, as long as the mice were adapted to living in the "home base" during the day and only allowed to enter the hoarding tube at night.

Burrowing, nesting, and hoarding are species-typical activities that mice readily perform in the laboratory. This article describes how they can be easily and cheaply assessed. These protocols are extremely sensitive to mouse strain, brain lesions and diseases. Moreover they constitute &#x201C;environmental enrichment&#x201D; for the mice, and embody the &#x201C;Refinement&#x201D; aspect of the &#x201C;3 Rs&#x201D;18.

Deterioration in the ability to perform "Activities of daily living" (ADL) is an early sign of Alzheimer's disease (AD). Preclinical behavioural screening ...

Neuroscience

Assessment of Social Transmission of Food Preferences Behaviors

Olfactory recognition deficits are suggested to be able to serve as clinical marker to differentiate Alzheimer&#39;s disease (AD) subjects from healthy aging groups. For example, olfactory dysfunction in AD can present as impairment in olfactory recognition, emerging during early stages of the disease and worsening while the disease progresses. The social transmission of food preferences (STFP) task is based on a rudimentary form of communication between rodents concerning distant foods dependent on the transmission of olfactory cues. Healthy wild-type mice would prefer to eat a novel, flavored food that was previously cued by a conspecific, and this food preference would be hampered in transgenic AD mice, such as the APP/PS1 model. Indeed, a strong preference for the cued food in C57Bl6/J mice of 3 months of age was found, and this was reduced in 3 months old transgenic APP/PS1 mice. In summary, STFP task could be a powerful measure to be integrated in present subclinical detection assays of AD.

This paper presents a protocol for the investigation of social transmission of food preference in mice. The advantages and possible applications for this procedure, for instance, in detecting early changes in AD mouse models, are highlighted. To conclude, interpretation of the results in light of critical details are discussed.

Olfactory recognition deficits are suggested to be able to serve as clinical marker to differentiate Alzheimer&#39;s disease (AD) subjects from healthy ...

Systematic Assessment of Well-Being in Mice for Procedures Using General Anesthesia

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal experimentation whenever possible. When there is no alternative to animal experimentation, the total number of laboratory animals used should be the minimum needed to obtain valuable data. Moreover, appropriate refinement measures should be applied to minimize pain, suffering, and distress accompanying the experimental procedure. The categories used to classify the degree of pain, suffering, and distress are non-recovery, mild, moderate, or severe (EU Directive 2010/63). To determine which categories apply in individual cases, it is crucial to use scientifically sound tools.
The well-being-assessment protocol presented here is designed for procedures during which general anesthesia is used. The protocol focuses on home cage activity, the Mouse Grimace Scale, and luxury behaviors such as burrowing and nest building behavior as indicators of well-being. It also uses the free exploratory paradigm for trait anxiety-related behavior. Fecal corticosterone metabolites as indicators of acute stress are measured over the 24-h post-anesthetic period.
The protocol provides scientifically solid information on the well-being of mice following general anesthesia. Due to its simplicity, the protocol can easily be adapted and integrated in a planned study. Although it does not provide a scale to classify distress in categories according to the EU Directive 2010/63, it can help researchers estimate the degree of severity of a procedure using scientifically sound data. It provides a way to improve the assessment of well-being in a scientific, animal-centered manner.

We developed a protocol to assess well-being in mice during procedures using general anesthesia. A series of behavioral parameters indicating levels of well-being as well as glucocorticoid metabolites were analyzed. The protocol can serve as a general aid to estimate the degree of severity in a scientific, animal-centered manner.

In keeping with the 3R Principle (Replacement, Reduction, Refinement) developed by Russel and Burch, scientific research should use alternatives to animal ...

Behavioral Assays for Autism Spectrum Disorder in Mouse Models

Strategies for Assessing Autistic-Like Behaviors in Mice

Autism spectrum disorder (ASD) is a neurobiologically complex condition with a heterogeneous genetic etiology. Clinically, ASD is diagnosed by social communication impairments and restrictive or repetitive behaviors, such as hand flapping or lining up objects. These behavioral patterns can be reliably observed in mouse models with ASD-linked genetic mutations, making them highly useful tools for studying the underlying cellular and molecular mechanisms in ASD. Understanding how genetic changes affect the neurobiology and behaviors observed in ASD will facilitate the development of novel targeted therapeutic compounds to ameliorate core behavioral impairments. Our lab has employed several protocols encompassing well-described training and testing procedures that reflect a wide range of behavioral deficits related to ASD. Here, we detail two assays to study the core features of ASD in mouse models: self-grooming (a measure of repetitive behavior) and the three-chamber social interaction test (a measure of social interaction approach and preference for social novelty).

Rodent models are valuable tools for studying core behaviors related to autism spectrum disorder (ASD). In this article, we expound on two behavioral tests for modeling the core features of ASD in mice: self-grooming, which assesses repetitive behavior, and the three-chamber social interaction test, which documents social impairments.

Autism spectrum disorder (ASD) is a neurobiologically complex condition with a heterogeneous genetic etiology. Clinically, ASD is diagnosed by social ...

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 ...