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-e...

<ol>
	<li>Hall, J. F., Hanford, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+as+a+function+of+a+restricted+feeding+schedule.">Activity as a function of a restricted feeding schedule.</a> J Comp Physiol Psychol. 47 (5), 362-363 (1954).</li><li>Hall, J. F., Smith, K., Schnitzer, S. B., Hanford, P. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevation+of+activity+level+in+the+rat+following+transition+from+ad+libitum+to+restricted+feeding.">Elevation of activity level in the rat following transition from ad libitum to restricted feeding.</a> J Comp Physiol Psychol. 46 (6), 429-433 (1953).</li><li>Routtenberg, A., Kuznesof, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-starvation+of+rats+living+in+activity+wheels+on+a+restricted+feeding+schedule.">Self-starvation of rats living in activity wheels on a restricted feeding schedule.</a> J Comp Physiol Psychol. 64 (3), 414-421 (1967).</li><li>Taksande, B. G., Chopde, C. T., Umekar, M. J., Kotagale, N. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agmatine+attenuates+hyperactivity+and+weight+loss+associated+with+activity-based+anorexia+in+female+rats.">Agmatine attenuates hyperactivity and weight loss associated with activity-based anorexia in female rats.</a> Pharmacol Biochem Behav. 132, 136-141 (2015).</li><li>Pierce, D. W., Epling, W. F., Boulton, A. A., Baker, G. B., Iverson, M. T. -. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity+anorexia.">Activity anorexia.</a> Neuromethods, 18, Animal Models in Psychiatry. 1, 267-311 (1991).</li><li>Klenotich, S. J., 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=The+activity-based+anorexia+mouse+model.">The activity-based anorexia mouse model.</a> Methods Mol Biol. 829, 377-393 (2012).</li><li>Kron, L., Katz, J. L., Goryzynski, G., Weiner, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hyperactivity+in+anorexia+nervosa:+a+fundamental+clinical+feature.">Hyperactivity in anorexia nervosa: a fundamental clinical feature.</a> Compr Psychiatry. 19 (5), 433-440 (1978).</li><li>Hebebrand, 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=Hyperactivity+in+patients+with+anorexia+nervosa+and+in+semistarved+rats:+evidence+for+a+pivotal+role+of+hypoleptinemia.">Hyperactivity in patients with anorexia nervosa and in semistarved rats: evidence for a pivotal role of hypoleptinemia.</a> Physiol Behav. 79 (1), 25-37 (2003).</li><li>Birmingham, C. L., Su, J., Hlynsky, J. A., Goldner, E. M., Gao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mortality+rate+from+anorexia+nervosa.">The mortality rate from anorexia nervosa.</a> Int J Eat Disord. 38 (2), 143-146 (2005).</li><li>Walsh, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluoxetine+after+weight+restoration+in+anorexia+nervosa:+a+randomized+controlled+trial.">Fluoxetine after weight restoration in anorexia nervosa: a randomized controlled trial.</a> JAMA. 295 (22), 2605-2612 (2006).</li><li>Kaye, W. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Double-blind+placebo-controlled+administration+of+fluoxetine+in+restricting-+and+restricting-purging-type+anorexia+nervosa.">Double-blind placebo-controlled administration of fluoxetine in restricting- and restricting-purging-type anorexia nervosa.</a> Biol Psychiatry. 49 (7), 644-652 (2001).</li><li>Jagielska, G., Kacperska, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Outcome,+comorbidity+and+prognosis+of+anorexia+nervosa.">Outcome, comorbidity and prognosis of anorexia nervosa.</a> Psychiatr Pol. 51 (2), 205-218 (2017).</li><li>Murray, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+eating,+exercise,+shape,+and+weight+related+symptomatology+in+males+with+muscle+dysmorphia+and+anorexia+nervosa.">A comparison of eating, exercise, shape, and weight related symptomatology in males with muscle dysmorphia and anorexia nervosa.</a> Body Image. 9 (2), 193-200 (2012).</li><li>Nieuwoudt, J. E., Zhou, S., Coutts, R. A., Booker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Symptoms+of+muscle+dysmorphia,+body+dysmorphic+disorder,+and+eating+disorders+in+a+nonclinical+population+of+adult+male+weightlifters+in+Australia.">Symptoms of muscle dysmorphia, body dysmorphic disorder, and eating disorders in a nonclinical population of adult male weightlifters in Australia.</a> J Strength Cond Res. 29 (5), 1406-1414 (2015).</li><li>Griffiths, S., Mond, J. M., Murray, S. B., Touyz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positive+beliefs+about+anorexia+nervosa+and+muscle+dysmorphia+are+associated+with+eating+disorder+symptomatology.">Positive beliefs about anorexia nervosa and muscle dysmorphia are associated with eating disorder symptomatology.</a> Aust N Z J Psychiatry. 49 (9), 812-820 (2015).</li><li>Achamrah, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+differences+in+response+to+activity-based+anorexia+model+in+C57Bl/6+mice.">Sex differences in response to activity-based anorexia model in C57Bl/6 mice.</a> Physiol Behav. 170, 1-5 (2017).</li><li>Perez-Leighton, C. E., Grace, M., Billington, C. J., Kotz, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+spontaneous+physical+activity+in+prediction+of+susceptibility+to+activity+based+anorexia+in+male+and+female+rats.">Role of spontaneous physical activity in prediction of susceptibility to activity based anorexia in male and female rats.</a> Physiol Behav. 135, 104-111 (2014).</li><li>Pirke, K. M., Broocks, A., Wilckens, T., Schweiger, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Starvation-induced+hyperactivity+in+the+rat:+the+role+of+endocrine+and+neurotransmitter+changes.">Starvation-induced hyperactivity in the rat: the role of endocrine and neurotransmitter changes.</a> Neurosci Biobehav Rev. 17 (3), 287-294 (1993).</li><li>Doerries, L. E., Epling, W. F., Pierce, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gender+differences+in+activity+anorexia:+predictable,+paradoxical,+or+enigmatic.">Gender differences in activity anorexia: predictable, paradoxical, or enigmatic.</a> Activity anorexia: Theory, research, and treatment. , 69-77 (1996).</li><li>Woods, D. J., Routtenberg, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term="Self-starvation"+in+activity+wheels:+developmental+and+chlorpromazine+interactions.">"Self-starvation" in activity wheels: developmental and chlorpromazine interactions.</a> J Comp Physiol Psychol. 76 (1), 84-93 (1971).</li><li>Pare, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+food+consumption+and+running+activity+on+the+activity-stress+ulcer+in+the+rat.">The influence of food consumption and running activity on the activity-stress ulcer in the rat.</a> Am J Dig Dis. 20 (3), 262-273 (1975).</li><li>Boakes, R. A., Mills, K. J., Single, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+differences+in+the+relationship+between+activity+and+weight+loss+in+the+rat.">Sex differences in the relationship between activity and weight loss in the rat.</a> Behav Neurosci. 113 (5), 1080-1089 (1999).</li><li>Pjetri, E., 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=Identifying+predictors+of+activity+based+anorexia+susceptibility+in+diverse+genetic+rodent+populations.">Identifying predictors of activity based anorexia susceptibility in diverse genetic rodent populations.</a> PLoS One. 7 (11), (2012).</li><li>Gelegen, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Difference+in+susceptibility+to+activity-based+anorexia+in+two+inbred+strains+of+mice.">Difference in susceptibility to activity-based anorexia in two inbred strains of mice.</a> Eur Neuropsychopharmacol. 17 (3), 199-205 (2007).</li><li>Shimizu, N., Oomura, Y., Kai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress-induced+anorexia+in+rats+mediated+by+serotonergic+mechanisms+in+the+hypothalamus.">Stress-induced anorexia in rats mediated by serotonergic mechanisms in the hypothalamus.</a> Physiol Behav. 46 (5), 835-841 (1989).</li><li>Siegfried, Z., Berry, E. M., Hao, S., Avraham, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+in+the+investigation+of+anorexia+nervosa.">Animal models in the investigation of anorexia nervosa.</a> Physiol Behav. 79 (1), 39-45 (2003).</li><li>Kim, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+eating+disorders.">Animal models of eating disorders.</a> Neuroscience. 211, 2-12 (2012).</li><li>Gutierrez, E., Cerrato, M., Carrera, O., Vazquez, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+reversal+of+activity-based+anorexia:+implications+for+the+treatment+of+anorexia+nervosa.">Heat reversal of activity-based anorexia: implications for the treatment of anorexia nervosa.</a> Int J Eat Disord. 41 (7), 594-601 (2008).</li><li>Brown, A. J., Avena, N. M., Hoebel, B. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-fat+diet+prevents+and+reverses+the+development+of+activity-based+anorexia+in+rats.">A high-fat diet prevents and reverses the development of activity-based anorexia in rats.</a> Int J Eat Disord. 41 (5), 383-389 (2008).</li><li>Boakes, R. A., Juraskova, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+drinking+in+the+suppression+of+food+intake+by+rodent+activity.">The role of drinking in the suppression of food intake by rodent activity.</a> Behav Neurosci. 115 (3), 718-730 (2001).</li><li>Boakes, R. A., Dwyer, D. 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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. 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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 border="0" cellpadding="0" cellspacing="0" style="width:881px;" width="881">
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	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">Wireless Low Profile Running Wheels, Hubs, Software</td>
			<td style="width:139px;">Med Associates</td>
			<td style="width:119px;">ENV-047</td>
			<td style="width:419px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">Standard Teklad Rodent Chow</td>
			<td style="width:139px;">Envigo</td>
			<td style="width:119px;">8604</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:205px;">8 Week Old Mice</td>
			<td style="width:139px;">Jackson Laboratories</td>
			<td style="width:119px;">Balb/cJ</td>
			<td>Strain used in our study - Can use other strains to assess ABA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">Scout Pro Scale 200 g</td>
			<td style="width:139px;">Ohaus</td>
			<td>SPE202</td>
			<td>Used to weigh mice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:205px;">Scout Pro Scale 400 g</td>
			<td style="width:139px;">Ohaus</td>
			<td style="width:119px;">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 ...

The overall goal of this experiment is to examine the neurobiology and behavior underlying anorexia nervosa. This method can help answer key questions in the field of psychiatry related to eating disorders such as what are the neural underpinnings of anorexia nervosa, and what brain circuits are involved. The main advantage of this technique is that it can be used to study some underlying mechanisms of anorexia, which can't be assessed in humans.Setup an experimental area by choosing a cage rack close to the laptop and wheel hubs. This will limit issues with data transmission. Now activate the connection between the wheel hubs and the software.First open the wheel management software to ensure both wireless hubs are active. From the software reset the default wheel connections. Open tools and select delete wheels.Repeat this step five times then close the software and reopen it. Next, provide power to the wheels. Connect the battery pack wires to the wheel hardware and insert the batteries into the wheel base.Then have a look at the software. The wheelbase should be listed with the ID of one under the appropriate wireless hub. If not, unhook the battery pack, delete the wheel in the software, and try again.If the wheel is recognized, replace the ID with the appropriate animal's ID.Next, adhere the running wheel'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. Next, place a running wheel disc without a corroded magnetic on the base and rotate the wheel to verify clearance from the cage walls and grating.Spin the disc and check that each revolution registers properly with the software. Next, use duct tape to secure the food jar to the cage. Position it away from the running wheel and water bottle.Then place two pieces of chow in the jar, attach full water bottle to the cage, and add bedding to the cage bottom. After all the cages have been prepared in this manner, individually place mice into their appropriate cages. Once all the mice are in their cages, go to the software and click on file, and then start acquisition.Throughout the experiment start new acquisitions to breakup the trial phases. Now let the mice acclimate to the housing conditions for two full days. During this phase, decide if each mouse is healthy enough to handle the ABA experiment.On the third day of the experiment, measure out about nine grams chow and record the weight to 1/1000 of a gram. Next, carefully remove the mouse from the cage and weigh it in a large plastic beaker to and nearest tenth of a gram. Do this with minimal stress to the mouse.With the mouse out of the cage, search out and remove all of the food in the cage. Besides removing the food in the jar, look through the bedding for scattered food. Next, if needed, clean the running wheel with 70%isopropanol and paper towels.Before returning the mouse to its cage, be sure that the isopropanol has dried. Then load the mouse and the nine grams of chow into the cage. On the next day return to the animal facility within 10 minutes of the last visit.Again load nine grams of chow in a small plastic beaker and record the exact weight. Again gently remove the mouse and weigh it with minimal stress to the mouse. Now remove the food jar out of the cage and investigate it.Remove any feces with tweezers and use a strainer over the beaker to remove bedding. Crumble the larger pieces of food so they pass through the filter leaving just bedding in the strainer. Then search the cage bedding for food remnants and add them to the beaker.Then record the mass of the food that remained in the cage. Next, wipe the jar clean and return it to the cage empty. Apply new tape if necessary.Then load the jar with the prepared nine gram food ration. As before, clean any soiled running wheels and return them to the cages. Reset all the cages in this manner everyday.On days five, seven, and nine, restart the computer data acquisition in wheel running program. After collecting the baseline data, on day 11 restart the data acquisition and begin the restriction phase. Basically, follow the baseline protocol with a few changes.First, do not clear the running wheels when changing the food. Second, limit the animals'access to their nine grams food ration to just six hours. The feeding period can be diurnal or nocturnal.Both choices have merit. After six hours, weigh the food remaining in the jar and cage. At this point, if needed, clean the wheel, but do so quickly so the next cage can be rapidly attended to.On each and every day of the restriction phase, repeat this procedure. Now it becomes very important to monitor the animals'weight loss. If a mouse drops to 75%of its baseline body weight, remove it from the experiment.Provide these mice unlimited food from the cage floor and no access to a running wheel. After all the cages are assessed, euthanize the dropped mice unless their recovery is to be studied. Using the described experiment, a group of activity based anorexia mice were assessed along with three control groups.Following the described procedure, the food was restricted to six diurnal hours. When a mouse was dropped from the study due to excessive weight loss, a randomly selected control mouse was as well. Body weight was reduced on days one through five in both groups exposed to scheduled feeding, and this correlated logically with reduced food intake.Curiously, weight loss had no effect on wheel running activity. After the behavior study, brains were harvested and frozen for biochemical analysis. One analysis of tissue from the ventral tegmental area showed that the wheel running significantly increased BDNF expression while food restriction increased NCAM1 MRNA expression but did not alter BDNF MRNA expression.While attempting this procedure, it's important to remember to limit stress on the mice, be consistent in timing your measurements, be patient so you acquire accurate readings, and consistently check on the wheel running software for any technical issues. Following this procedure, other methods like immunohistochemistry and radioligand binding can be performed on mouse brains in order to answer additional questions pertaining to virus function in the anorexia paradigm.

assessing-activity-based-anorexia-in-mice

Research

JoVE Journal

Behavior

12.3K visualizações.  University of California, San Diego. The overall goal of this experiment is to examine the neurobiology and behavior underlying anorexia nervosa. This method can help answer key questions in the field of psychiatry related to eating disorders such as what are the neural underpinnings of anorexia nervosa, and what brain circuits are involved. The main advantage of this technique is that it can be used to study some underlying mechanisms of anorexia, which can't be assessed in humans.Setup an experimental area by choosing a...