We want to express our gratitude to the Biomedical Sciences PhD Program at the National Autonomous University of Mexico and to the support provided by Consejo Nacional de Humanidades, Ciencias y Tecnolog&#237;as (CONAHCYT), No. CVU: 11025222. This work was partially supported by the UNAM-DGAPA-PAPIIT grant IN207423. We thank Andrea Mondrag&#243;n Garc&#237;a and Jos&#233; Enrique Ram&#237;rez S&#225;nchez for their technical support.

All experiments were conducted according to the Mexican Official Standard of Technical Specifications for the Production and Use of Laboratory Animals (NOM-062-ZOO-1999) and in full compliance with the guidelines set forth by the Internal Committee for the Care and Use of Laboratory Animals (CICUAL) at the National Autonomous University of Mexico (UNAM).
1. Animals
<ol>
	<li>To follow this protocol, individually house 27 C57BLC/6 male mice, aged 1 month and weighing 16-20 g, in cages under controlled environmental conditions: a 12-12 h light-dark cycle (06:00 on, 18:00 off), maintaining the temperature at 21 &#177; 22 &#176;C and humidity levels at 70 &#177; 10%. 
	NOTE: In this study, we exclusively used male mice to eliminate the effects of female hormones during binge-type intake. However, in another study18, we applied the same model to female mice and observed no differences in binge intake.</li>
</ol>
2. Habituation
<ol>
	<li>Provide the 30-day-old mice with ad libitum access to standard chow and water. Place them in individual cages to facilitate measuring water and food consumption.</li>
	<li>Provide an enriched environment, including a paper tube, to all the mice and provide wood shavings as bedding.</li>
	<li>Measure body weight, food, and water consumption every 24 h for 7 days.</li>
</ol>
3. Binge eating model
<ol>
	<li>On day 7 of the habituation period, randomly assign the rodents (N= 27) into one of the following three groups.</li>
	<li>Provide all subjects with access to the HPF (nutritional content per 100: 480 kcal, 19g of fat, 70 g of carbohydrates, 5.1 g of protein, 0.13 g of salt) for 2 h (from 16:00 to 18:00) to minimize neophobia toward the HPF.</li>
	<li>Continue the specified feeding conditions for each group consistently for a period of 26 days.
	<ol>
		<li>Chow Group (CG) n = 9: provide ad libitum access to standard chow and water.</li>
		<li>Continuous Group (CoG) n = 9; provide ad libitum access to standard chow, HPF, and water.</li>
		<li>Intermittent Group (IG) n = 9: provide ad libitum access to standard chow and water but give them HPF only on specific days (1, 3, 5, 8, 10, 12, 15, 17, 19, 22, 24, and 26) for 2 h (from 16:00 to 18:00 h).</li>
	</ol>
	</li>
	<li>From the first session to session 26, measure body weight, standard chow intake, palatable food intake, and water consumption for all subjects at 16:00 h and again at 18:00 h.</li>
</ol>
4. Specifical instructions
<ol>
	<li>Place the standard chow on the cage racks and the HPF in small plastic containers. In each session, randomly place these containers at the corners of the cages (left posterior, right posterior, right anterior, and left anterior).</li>
	<li>Replenish water (280-300 mL) and standard chow (10-15 g) on Mondays, Wednesdays, and Fridays.</li>
	<li>Replace the HPF (4-4.5 g) daily for the CoG. For the IG, place the HPF (4-4.5 g) on Mondays, Wednesdays, and Fridays at 16:00 h. Randomly distribute the HPF by color (brown, blue, red, orange, green, and yellow), with no preference observed for any color.</li>
	<li>On day 7 of habituation, give the mice assigned to the CoG group the HPF at 16:00 h. Measure all the animals for standard chow intake, body weight, and water consumption.</li>
</ol>
5. Data analysis
<ol>
	<li>For 24 h intake measurements, record the values at 16:00 h on the previous day and 16:00 h on the current day. For instance, to evaluate the consumption of day 2, subtract the values for standard chow, the HPF, water, and body weight from day 1 from those of day 2 for the same parameters.</li>
	<li>For 2 h intake measurements, record the values at 16:00 h and 18:00 h on the same day. For example, subtract the values for standard chow, the HPF, water, and body weight at 16:00 h from those at 18:00 h for the same parameters.</li>
	<li>Collect all data daily at two time points (16:00 h and 18:00 h), input them into a spreadsheet, and perform kcal calculations.
	<ol>
		<li>To calculate the kcal, convert the grams consumed of standard chow and HPF to kcal based on the energy value of each food using a proportion (e.g., standard chow: 3.02 kcal/g; HPF: 4.80 kcal/g).</li>
	</ol>
	</li>
	<li>Analyze and plot the data using the software of choice.
	<ol>
		<li>For statistical analysis, use the Kruskal-Wallis and Mann-Whitney U tests.</li>
	</ol>
	</li>
</ol>
6. Open field test
NOTE: A 22-day, 14:00 h, open field test was conducted according to the protocol described by Kraeuter and colleagues19. This protocol consists of placing the animals in the center of a black acrylic sheet box. The floor of the arena was white, and the grid was black. Behavior was recorded and quantified with a video tracking system. The system recorded the distance, speed, and time the animals spent in the center or periphery of the arena.
<ol>
	<li>Preparation of the test room and open field apparatus
	<ol>
		<li>For the arena, use a square chamber of 60 cm (length) x 60 cm (width) x 20 cm (height) made of black nonporous plastic.</li>
		<li>Clean the device before and between uses with 70% vol/vol ethanol to remove odor cues.</li>
		<li>Place the camera above the apparatus for optimal exploration view.</li>
		<li>To minimize stress, maintain temperature and humidity like the normal housing conditions. If the mice are moved from one housing room to another for the experiment, acclimate them to the new room for at least 1 h each day before use.</li>
	</ol>
	</li>
	<li>Performing the open field test
	<ol>
		<li>To remove a single mouse from the cage, gently grasp its tail and place it in the center of the open field maze. Activate the video tracking software using the autostart feature, which detects the investigator and begins recording as soon as the investigator is no longer in focus of the camera.</li>
		<li>Allow the mouse to move freely along the maze for 5 min. During this time, the tracking software will record the movement.</li>
		<li>At the end of the trial period, remove the animal from the arena and return it to its home cage.</li>
		<li>Before cleaning the maze, visually count the feces deposited and record them for later analysis.</li>
		<li>Remove all fecal pellets and clean any urine stains. Spray the apparatus floor and walls with 70% ethanol, then wipe with a clean paper towel. Allow the ethanol solution to dry completely.</li>
		<li>After testing all animals, return to the vivarium, clean the arena, and organize all used materials.</li>
	</ol>
	</li>
	<li>Data analysis 
	NOTE: The video tracking software facilitates the extraction of a range of data, such as the animals&#39; average speed, the distance traveled, and the time spent in the center and periphery of the apparatus.
	<ol>
		<li>Select the distance traveled and the time spent by the animals in the center and the periphery of the apparatus.</li>
		<li>Export the data from the tracking software into a spreadsheet or other preferred format for further statistical analysis.</li>
		<li>Analyze the data with the software of choice.</li>
	</ol>
	</li>
</ol>

Investigating Binge Eating Disorder with a Palatable Food Access Mouse Model

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The authors have no conflicts of interest to declare.

We used the binge-eating model proposed by Corwin in 2006 as a basis for developing this protocol. However, the present study modified the variable of providing palatable food for 2 h for the CoG8. Instead, we offered palatable food ad libitum to CoG, as it better simulates a reality where we have 24 h access to any food. We also used M&#38;M&#39;s as an HPF because they combine fats and sugars, unlike other binge-eating models that use either fat or sugar separately8</s...

According to data from the World Health Organization1, one in eight people worldwide is obese. Although eating is a physiological necessity, overeating without a physiological need is known to be a factor associated with overweight and obesity2. It is well known that humans prefer palatable foods high in calories, such as fats and sugars, over healthier foods3.
From the perspective of complexity science, eating disorders, particularly binge eating disorder (BED), are multifaceted phenomena that emerge from the interaction of various biological, psychological, and social factors exhibiting emerging behaviors such as cyclic binging patterns, complex compensatory behaviors, and dysfunctional interpersonal relationships4. BED is characterized by the excessive intake of high-calorie foods within a short period, approximately 2 h, according to the Diagnostic and Statistical Manual of Mental Disorders, 5th ed.5 (DSM-V). Additionally, BED is associated with behavioral comorbidities such as anxiety and cravings6,7. The complexity of these disorders requires better experimental models that can capture this interplay of factors and provide a more comprehensive understanding of binge eating behavior and its implications for obesity.
The most used models for binge-like behavior in basic research have primarily been rats. These models typically use limited and intermittent access to high-fat or high-sugar foods to induce binge-like behaviors8,9,10. An alternative approach involves providing palatable food for a short period (2 h) every 8 days, known as a cyclical model, using a highly palatable food (HPF) paste containing a mix of fats and sugars11. Another method presents the HPF as cookies unpredictably12. In mouse models, binge-like behaviors are often studied using the same cyclical model13. Another study with mice uses limited access to sucrose to induce binge-like behaviors14.
Despite extensive research on binge-like eating behaviors in rats, models for binge eating in mice remain limited. This study aims to propose a binge-like eating model using M&#38;M&#39;s (hereafter referred to as &#34;HPF&#34;) as an HPF according to the nutritional criteria of HPF proposed by Fazzino and colleagues15. Additionally, we aim to evaluate the rewarding properties of the HPF based on whether they are presented continuously or intermittently. In our research, we aimed to observe similarities between specific diagnostic DSM-V criteria for binge eating in humans within a murine model: consuming food within a short period (2 h), ingesting a large amount of food than usual, eating substantial amount despite being in a state of satiety, and experiencing binge episodes at the least twice a week.
The use of laboratory mouse models is an invaluable tool in biomedical research, especially for studying human diseases, as mice share 99% of their genes with humans16. Utilizing mice also offers significant advantages in understanding transcriptomic information and modifying therapeutic targets related to cellular functions and neural circuits associated with various diseases17.
Therefore, this study proposes a continuous and intermittent access model to M&#38;M&#39;s (an HPF made of fat and sugar) to observe binge-like eating in C57BL/6 mice without standard chow or water deprivation. This model is novel and necessary for biomedical research, as it represents a better approach to studying these complex problems. Standardizing animal models is essential to obtain reproducible and comparable results, which is fundamental to advancing translational research and developing effective therapeutic interventions for BED and obesity. The approach described here will expand the scope of binge-like eating behavior research in future studies, facilitating the testing of various drugs as potential therapeutic targets for BED. The implementation of more realistic and standardized models in mice will allow for a better understanding of the complex dynamics involved in eating disorders and their metabolic consequences, thus offering new alternatives for the treatment and prevention of BED.

<table><tbody><tr><td>ANY-maze Video Tracking Software</td><td>Stoelting Co.</td><td>4.73 version</td><td></td></tr><tr><td>C270 HD Webcam</td><td>Logitech</td><td>C270 HD</td><td>It is not necessary to use a specific video recorder; any webcam or camcorder with satisfactory image quality and resolution will suffice.</td></tr><tr><td>Gloves Ambiderm (black)</td><td>Ambiderm</td><td>7.50222E+12</td><td></td></tr><tr><td>GraphPad Prism 9</td><td>GraphPad</td><td></td><td></td></tr><tr><td>Laboratory Rodent Diet 5001</td><td>LabDiet</td><td>1320</td><td>https://di.facmed.unam.mx/comisiones/Composici%C3%B3n%20del%20alimento%20Laboratory%20Rodent%20Diet%205001.pdf</td></tr><tr><td>M&amp;amp;Ms 43.8g (Brown packaging)</td><td>M&amp;amp;M&amp;acute;s&amp;nbsp;</td><td></td><td>Nutritional content per 100 : 480 kcal, 19g of fat, 70 g of carbohydrates, 5.1 g of protein, 0.13 g of saltIngredients:&amp;nbsp;sugar, cocoa mass, skimmed&amp;nbsp;milk&amp;nbsp;powder, cocoa butter,&amp;nbsp;lactose, starch,&amp;nbsp;milk&amp;nbsp;fat, palm fat, glucose syrup, shea fat, stabiliser (gum arabic), dextrin, glazing agents (beeswax, carnauba wax), colours (E100, carmine, E132, E133, E150a, E150c, E150d, E153, E160a, E160e, E162, E163, E170), beetroot concentrate, emulsifiers (soya&amp;nbsp;lecithin, E445), salt, flavourings, palm kernel oil, antioxidant (E306). (May contain:&amp;nbsp;peanut, hazelnut, almond). Milk chocolate contains milk solids 14% minimum.&amp;nbsp;Milk chocolate contains vegetable fats in addition to cocoa butter.</td></tr><tr><td>Open Field maze&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Polypropylene Mice Cage</td><td>Orchid Scientific</td><td>SMP 01</td><td></td></tr><tr><td>Rodent drinkers</td><td>Sunnypet</td><td>SP-3656</td><td></td></tr><tr><td>Uline Balanza-220 g x 0.01 g</td><td>unike.mx</td><td>H-9884</td><td></td></tr><tr><td>UniMask4</td><td>Uniseal</td><td></td><td></td></tr><tr><td>Plastic bottles</td><td>unike.mx</td><td>S-14487</td><td></td></tr><tr><td>Sanitary Bed, Sawdust for Animals</td><td>BIOINVERT</td><td>No number</td><td>10 kg</td></tr></tbody></table>

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.

Using HPF, we successfully established a binge-eating model in C57BL/6 mice. By day 8, binge-eating behavior (consuming excessive food for 2 h) was firmly established and maintained through day 26 (Figure 1A). During the intermittent sessions, the IG group exhibited significantly higher total kcal consumption (from both standard chow and the HPF than the CG and CoG) groups (Figure 1A, ### p &#60; 0.0001, *** p &#60; 0.0001, ** p &#60; 0.001, Kruskal-Wallis test)...

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.

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 ...

Research

JoVE Journal

Behavior

632 Views.  Universidad Nacional Autónoma de México (UNAM). A binge-eating model was established in mice using intermittent access to M&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.

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

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 ...

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station (INBEST)

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of chronic CNS disorders, there is an increasing demand for murine models with high validity at the behavioral level. However, multiple pathogenic mechanisms and complex functional deficits often impose challenges to reliably measure and interpret behavior of chronically sick mice. Therefore, the assessment of peripheral pathology and a behavioral profile at several time points using a battery of tests are required. Video-tracking, behavioral spectroscopy, and remote acquisition of physiological measures are emerging technologies that allow for comprehensive, accurate, and unbiased behavioral analysis in a home-base-like setting. This report describes a refined phenotyping protocol, which includes a custom-made monitoring apparatus (Integrated Behavioral Station, INBEST) that focuses on prolonged measurements of basic functional outputs, such as spontaneous activity, food/water intake and motivated behavior in a relatively stress-free environment. Technical and conceptual improvements in INBEST design may further promote reproducibility and standardization of behavioral studies.

Behavioral Phenotyping of Murine Disease Models with the Integrated Behavioral Station INBEST

Prolonged and comprehensive monitoring of mice in a home-cage environment provides a deeper understanding of aberrant behavior in murine models of brain diseases. This paper describes the Integrated Behavioral Station (INBEST) as the key component of contemporary behavioral analysis.

Due to rapid advances in genetic engineering, small rodents have become the preferred subjects in many disciplines of biomedical research. In studies of ...

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 ...

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

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

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

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

Neuroscience

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.

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 ...

Murine Drinking Models in the Development of Pharmacotherapies for Alcoholism: Drinking in the Dark and Two-bottle Choice

Alcohol Use Disorder (AUD) is a major problem with more than an estimated 76 million people worldwide meeting the diagnostic criteria. Current treatments are limited to three FDA-approved medications that are largely ineffective even when combined with psychosocial intervention, as is evident by the high relapse rate. As such, the search for more novel treatments represents an important public health goal. To this end, the following protocol utilizes two simple rodent drinking models to assess the preclinical efficacy of lead anti-alcohol compounds: two-bottle choice (TBC) and drinking in the dark (DID). The former allows mice to voluntary drink in moderation while the latter induces mice to voluntary consume a large amount of alcohol in a short period that mimics binge drinking. The simple and high throughput nature of both of these paradigms allow for rapid screening of pharmacological agents or for identifying strains of mice that exhibit certain voluntary drinking behavior.

Alcohol Use Disorder (AUD) is a major national health problem and the development of more effective treatments is&#160;required to offset the needs of this patient population. To this end, the following protocol utilizes two simple rodent drinking models to assess the preclinical efficacy of lead anti-alcohol compounds.

Alcohol Use Disorder (AUD) is a major problem with more than an estimated 76 million people worldwide meeting the diagnostic criteria. Current treatments ...

Medicine

Modeling Alcohol Use Disorder Using Chronic Intermittent Ethanol Vapor Exposure

Chronic Intermittent Ethanol Vapor Exposure Paired with Two-Bottle Choice to Model Alcohol Use Disorder

Alcohol use disorder (AUD) is a chronic alcohol-related disorder that typically presents as uncontrolled drinking and preoccupation with alcohol. A key component of AUD research is using translationally relevant preclinical models. Over the past several decades, a variety of animal models have been used to study AUD. One prominent model of AUD is the chronic intermittent ethanol vapor exposure (CIE) model, which is a well-established approach for inducing alcohol dependence in rodents through repeated cycles of ethanol exposure via inhalation. To model AUD in mice, the CIE exposure is paired with a voluntary two-bottle choice (2BC) of alcohol drinking and water to measure the escalation of alcohol drinking. The 2BC/CIE procedure involves alternating weeks of 2BC drinking and CIE, which repeat until the escalation of alcohol drinking is achieved. In the present study, we outline the procedures for performing 2BC/CIE, including the daily use of the CIE vapor chamber, and provide an example of escalated alcohol drinking in C57BL/6J mice using this approach.

Presented here is a protocol for the 2BC/CIE model of alcohol dependence in mice to study alcohol use disorder.

Alcohol use disorder (AUD) is a chronic alcohol-related disorder that typically presents as uncontrolled drinking and preoccupation with alcohol. A key ...

Intermittent-Access Two-Bottle Choice Drinking with Lick Detection in Rats

Modeling Alcohol Consumption in Rodents Using Two-Bottle Choice Home Cage Drinking and Microstructural Analysis

Two-bottle choice home cage drinking is one of the most widely used paradigms to study ethanol consumption in rodents. In its simplest form, animals are provided with access to two drinking bottles, one of which contains regular tap water and the other ethanol, with daily intake measured by change in bottle weight over the 24 h period. Consequently, this approach requires no specialized laboratory equipment. While such ease of implementation is likely the greatest contributor to its widespread adoption by preclinical alcohol researchers, the resolution of drinking data acquired using this approach is limited by the number of times the researcher measures bottle weight (e.g., once daily). However, the desire to examine drinking patterns in the context of overall intake, pharmacological interventions, and neuronal manipulations has prompted the development of home cage lick detection systems that can acquire data at the level of individual licks. Although a number of these systems have been developed recently, the open-source system, LIQ HD (Lick Instance Quantifier Home cage Device), has garnered significant attention in the field for its affordability and user friendliness. Although exciting, this system was designed for use in mice. Here, we review appropriate procedures for standard and lickometer-equipped two-bottle choice home cage drinking. We also introduce methods for adapting the LIQ HD system to rats including hardware modifications to accommodate larger cage size and a redesigned 3D printed bottle holder compatible with standard off-the-shelf drinking bottles. Using this approach, researchers can examine daily drinking patterns in addition to levels of intake in many rats in parallel thereby increasing the resolution of acquired data with minimal investment in additional resources. These methods provide researchers with the flexibility to use either standard bottles or a lickometer-equipped apparatus to interrogate the neurobiological mechanisms underlying alcohol drinking depending on their precise experimental needs.

This protocol describes a standard intermittent-access two-bottle choice home cage drinking paradigm to model alcohol consumption in rats. In addition, it provides step-by-step instructions to augment the standard protocol with a DIY lick detection system that enables microstructural analysis of drinking behavior.

Two-bottle choice home cage drinking is one of the most widely used paradigms to study ethanol consumption in rodents. In its simplest form, animals are ...

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

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 ...