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 separately8,20,21,22. Lastly, we standardized a binge-eating model in C57BLC/6 mice, as Corwin had only standardized the model in rats.
Intermittent HPF presentation may resemble unpredictable HPF exposure, leading to increased HPF consumption, as previously reported11. Notably, no habituation to HPF was observed throughout the binge sessions, suggesting that intermittent exposure could serve as a robust model for investigating persistent binge eating behaviors. It has been reported that binge-eating behaviors in rodents should be observed as close to the dark phase of their cycle as possible, as this is when they tend to eat the most23. Corwin recommends offering palatable food 2.5 h before the lights go off24. In our study, we followed this timing for presenting HPF, and we successfully established binge-eating behavior.
Surprisingly, animals consuming HPF continuously or intermittently did not show significant differences in body weight compared to those fed standard chow. One plausible explanation for this observation is the presence of homeostatic regulation processes that govern food intake and body weight. Another factor to consider is that the animals were housed individually, and the room temperature was maintained at approximately 21.5 &#176;C. While some studies have reported no changes in feeding patterns or body weight at temperatures around 21 &#176;C25,26,27, more recent research has examined hypothalamic neural pathways at 21-22 &#176;C, highlighting their activation and the effects on energy homeostasis28. Moving forward, we will consider these findings when evaluating potential changes in food consumption and body weight under more thermoneutral temperature conditions.
Finally, this binge-eating model in mice (Figure 5) is crucial for exploring the physiological foundations of reward and motivation, including the nucleus accumbens (NAc). The NAc is associated with the hedonic value of reinforcing stimuli29 and the response to reinforcing stimuli such as chocolate30. It has been shown that exposure to palatable foods, whether intermittent or continuous, is sufficient to modify the behavior of mice31, resulting in increased anxiety-like behaviors. These behaviors have been characterized in mice exposed to high-fat and high-sugar foods32. We observed that the IG and CoG groups exhibited similar anxiety behaviors despite the different food intake between these groups.
The anxiety behavior induction has been observed with different food regimens in rodent models. The long-term alternate-day fasting caused anxiety in mice33. Mice, when fed a high-fat diet, displayed anxiety-like behavior measured by the open-field test34. Similar results have been described by binge-like sucrose intake35 and limited access to a high-fat diet36 in mice. However, some other studies on mice with different food intake types did not observe changes in anxiety-like behavior37,38. Thus, the neural control of behavioral and food intake is a key to understanding the relationship between both behaviors. Chen et al., in 2020, performed optogenetic activation of GABAergic neurons in the ventral tegmental area and found that this induced anxiety-like behaviors and promoted the intake of palatable foods39. Although intermittent exposure shows significant differences compared to continuous exposure, intermittent access to substances/foods has been observed to produce more robust addiction-like behavioral manifestations40.
However, this study did not evaluate behavioral tests to measure anhedonia and, consequently, depressive-like symptoms. It is recommended that a more comprehensive battery of behavioral tests be included, as similar models have shown increases in anhedonia, such as increased sucrose or saccharin consumption and increased immobility time in the forced swim test. Future studies should incorporate these additional assessments to provide a more thorough understanding of the behavioral effects of palatable food exposure in murine models.

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.

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			<td>ANY-maze Video Tracking Software</td>
			<td>Stoelting Co.</td>
			<td>4.73 version</td>
			<td></td>
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			<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>
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			<td>Gloves Ambiderm (black)</td>
			<td>Ambiderm</td>
			<td>7.50222E+12</td>
			<td></td>
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			<td>GraphPad Prism 9</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
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			<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&#38;Ms 43.8g (Brown packaging)</td>
			<td>M&#38;M&#180;s&#160;</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:&#160;sugar, cocoa mass, skimmed&#160;milk&#160;powder, cocoa butter,&#160;lactose, starch,&#160;milk&#160;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&#160;lecithin, E445), salt, flavourings, palm kernel oil, antioxidant (E306). (May contain:&#160;peanut, hazelnut, almond). Milk chocolate contains milk solids 14% minimum.&#160;Milk chocolate contains vegetable fats in addition to cocoa butter.</td>
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		<tr>
			<td>Open Field maze&#160;</td>
			<td></td>
			<td></td>
			<td></td>
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			<td>Polypropylene Mice Cage</td>
			<td>Orchid Scientific</td>
			<td>SMP 01</td>
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			<td>Rodent drinkers</td>
			<td>Sunnypet</td>
			<td>SP-3656</td>
			<td></td>
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			<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>
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		<tr>
			<td>Plastic bottles</td>
			<td>unike.mx</td>
			<td>S-14487</td>
			<td></td>
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		<tr>
			<td>Sanitary Bed, Sawdust for Animals</td>
			<td>BIOINVERT</td>
			<td>No number</td>
			<td>10 kg</td>
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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). No significant differences in total kcal consumption were observed between the CG and CoG groups (Figure 1A, ns, p &#62; 0.5, Kruskal-Wallis test). On test days (22, 24, and 26), the IG group demonstrated greater kcal consumption than the CG and CoG groups (Figure 1B, ### p &#60; 0.0001, *** p &#60; 0.0001, ** p &#60; 0.001, Kruskal-Wallis test); no differences were observed between the CG and CoG groups (Figure 1B, p &#62; 0.5, Kruskal-Wallis test). The increased caloric intake in the IG group was attributed to the consumption of palatable rather than standard chow. No differences were found in standard chow consumption among the three groups, a representative day (Figure 1C, p &#62; 0.5, Kruskal-Wallis test). Conversely, the IG group consumed more HPF than the CoG group (Figure 1D, *** p &#60; 0.0001). We chose day 26 as a representative day to do these comparisons because it was the last day of bingeing. When we did the average of all the binge days, we found statistical differences since week 1 (Supplementary Figure S1). We conclude that high-calorie food is preferred over balanced food during binge-eating episodes. Furthermore, palatable food is reinforcing when provided intermittently but not continuously. It is also important to note that M&#38;M&#39;s were standardized as a highly palatable food due to the combination of sugars and fats.
We also evaluated consumption every 24 h. From day 8 to day 26, the IG had a higher total kcal consumption over 24 h than the CG (Figure 2A, ###p &#60; 0.0001, Kruskal-Wallis test). Additionally, on certain days (8, 12, 19, 22, 24, 26), the IG consumed more than the CoG (Figure 2A, **p &#60; 0.001, Kruskal-Wallis test). Also, after binge sessions, the IG decreased total kcal intake the following day, suggesting homeostatic compensation by the subjects after the binge period. The IG had a higher total kcal consumption over 24 h than the CG and the CoG on day 26 (Figure 2B, ###p &#60; 0.0001, %%% p &#60; 0.0001, Kruskal-Wallis test). Furthermore, the CoG had a higher intake than the CG but a lower intake than the IG (Figure 2B, %%%p &#60; 0.0001, **p &#60; 0.001, Kruskal-Wallis test). Lastly, body weight increased proportionally according to the growth of each mouse. Surprisingly, we did not find significant differences in body weight among the three groups over the 26 sessions (Figure 2C) (p &#62; 0.5, Kruskal-Wallis test). These results could indicate that there are homeostatic regulation processes in intake and body weight regulation. Additionally, binge eating affects the quantity of both palatable and standard chow consumption.
The open field test showed that CG mice spent extended periods in the arena&#39;s central region than CoG and IG mice (Figure 3A, %%% p &#60; 0.0001, ## p &#60; 0.001, ANOVA One-way Test). CG showed a significant decrease in time to remain in the peripheral area compared with the CoG group (Figure 3B, %% p &#60; 0.001, ANOVA One-way Test) and the IG group (Figure 3B, # p &#60; 0.001, ANOVA One-way Test). No statistically significant differences were found between CoG and IG in the time spent in the center of the apparatus (Figure 3A) or its periphery (Figure 3B). Furthermore, CG traveled more in the central region than CoG (Figure 3C, % p&#60; 0.05, ANOVA One-way Test) and IG (Figure 3C, #p&#60; 0.05, ANOVA One-way Test). However, no statistically significant differences were observed in the distance traveled in the peripheral area among all groups (Figure 3D, p &#62; 0.05, ANOVA One-way Test). Within 5 minutes of exploration in the open field test, entries into the central zone in the CG group were significantly more than in CoG (Figure 3E, % p&#60; 0.05, ANOVA One-way Test) and IG group (Figure 3E, #p&#60; 0.05, ANOVA One-way Test). Another parameter that assesses locomotion in the open field test is the number of line crossings. This is the frequency with which the mouse crosses the lines shown in Figure 4B with its four paws. The number of line crossings is shown in Figure 3F. CG mice outcrossed lines significantly more often than CoG mice (Figure 3F, % p&#60; 0.05, ANOVA One-way Test). No significant change was observed in this parameter when comparing CG mice to IG mice, nor between CoG and IG (Figure 3F, p &#62; 0.05, ANOVA One-way Test). Figure 4A shows representative traces of time spent in each group&#39;s peripheral or center zone of one mouse. In Figure 4B, the different zones of the arena can be observed clearly. These findings suggest that the time spent in different regions is not due to locomotion issues.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67560/67560fig01.jpg" /> 
Figure 1: Binge-type intake and consumption for 2 h. (A) Kilocaloric consumption on day 26 for 2 h of standard chow and M&#38;M&#39;s. No differences were observed in kcal consumption between the CG and CoG (p &#62; 0.5, Kruskal-Wallis test). On the days when palatable food was presented to the IG (days 8, 10, 12, 15, 17, 19, 22, 24, and 26), the IG had a higher total kcal intake than the CoG and CG (***p &#60; 0.0001, ###p &#60; 0.0001, Kruskal-Wallis test). (B) The IG had a higher total kcal intake than the CG and CoG (test days: 22, 24, and 26) (**p &#60; 0.001, ***p &#60; 0.0001, Kruskal-Wallis test). (C) A representative day (day 26) of standard chow intake is shown; there were no differences in standard chow intake among the three groups (ns p &#62; 0.5, Kruskal-Wallis test). (D) A representative day (day 26) of the consumption of the HPF between the CoG and IG is shown. The IG had a higher intake of the HPF than the CoG (*** p &#62; 0.0001, Mann-Whitney test). Abbreviations: ns= no significance, TI= Total intake, SC= Standard chow. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67560/67560fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67560/67560fig02.jpg" /> 
Figure 2: Intake and body weight (24 h). (A) Caloric intake was recorded every 24 h for 26 days of standard chow and M&#38;M&#39;s. We found significant differences between the CG and the IG from day 8 to day 26 (### p &#60; 0.0001, Kruskal-Wallis test). On some days, the IG had a higher intake than the CoG (** p &#60; 0.001, Kruskal-Wallis test). Finally, in some sessions, the CoG consumed more than the CG (%% p &#60; 0.001, Kruskal-Wallis test). (B) On day 26, a representative day, it can be observed that both the IG and the CoG consumed more kcal than the CG and that the IG consumed more kcal than the CG (### p &#60; 0.0001, %%% p &#60; 0.0001, ** p&#60;0.001 Kruskal-Wallis test). (C) No differences were found in the body weight of the three groups (p &#62; 0.5, Kruskal-Wallis test). Abbreviations: CG = Chow group; CoG = Continuous group; IG = Intermittent group; HPF = highly palatable food. Abbreviations: ns = no significance. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67560/67560fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67560/67560fig03.jpg" /> 
Figure 3: Open field test. (A) Time spent in the central zone. Significant differences were found between the CG and the CoG (%%% p &#60; 0.0001, ANOVA One-way Test) and the IG (## p &#60; 0.001, ANOVA One-way Test). (B) Time spent in the peripheral zone. Significant differences between the CG and the CoG (%% p &#60; 0.001, ANOVA One-way Test) and the IG (# p &#60; 0.001, ANOVA One-way Test). (C) Distance traveled in the central zone. There was a significant difference between CG and the CoG (% p&#60; 0.05 ANOVA One-way Test), CG and the IG (#p&#60; 0.05, ANOVA One-way Test). (D) Distance traveled in the peripheral zone. There was no difference among the groups in the distance traveled in the peripheral zone (ns, p &#62; 0.05, ANOVA One-way Test). (E) Number of entries into the central zone. Significant differences were found between the CG group and the CoG group (% p&#60; 0.05), while the IG group showed differences with the CG group but not with the CoG group (# p&#60; 0.05, One-Way ANOVA). (F) Number of line crossings. There was a significant difference between CG and CoG (% p&#60; 0.05); however, no significant differences were found between CG and IG. (ns p &#62; 0.05, ANOVA One-way Test). Abbreviations: ns= no significance. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67560/67560fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67560/67560fig04.jpg" /> 
Figure 4: Test chamber zones and track paths of the mice. (A) Comparison of track paths for representative mice of the CG, CoG, and IG. (B) Delimitations and measurements of the test chamber zones. A 15 x 15 cm zone series was identified and used to evaluate subject tracks. Abbreviations: CG= Chow group; CoG= Continuous group; IG= Intermittent group. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67560/67560fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67560/67560fig05.jpg" /> 
Figure 5. Timeline of the binge-like eating model. This figure illustrates the treatment of the three experimental groups: CG provided with ad libitum access to chow (yellow) and water. CoG given ad libitum access to both chow (yellow) and M&#38;M&#39;s (HPF, brown), along with water. IG granted ad libitum access to chow (yellow) and water but restricted to the HPF (brown) access for only 2 h per day, 3 days per week. On day 22, an open field test was conducted for all three groups before the binge test (red circle). The figure displays the 4-week model duration, with the initial 21 days (green) representing the learning period and the testing period (pink) indicating the phase where binge-like behavior is established. Abbreviations: CG= Chow group; CoG= Continuous group; IG= Intermittent group; HPF= highly palatable food. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67560/67560fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure S1. Weekly average of total intake in 2 h. This figure presents the intake (standard chow and M&#38;M&#39;s, according to each group&#39;s assigned diet) for the three groups: CG (orange), CoG (blue), and IG (purple). Statistical analyses revealed significant differences in intake between the IG vs. CG and CoG groups from week 1 through week 4 (#### p &#60; 0.00001, **** p &#60; 0.00001, Kruskal-Wallis test, Turkey&#180;s multiple comparisons test). No statistically significant differences were observed between the CG and CoG groups across the 4 weeks. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67560/Supplementary Figure 1.zip" target="_blank">Please click here to download this File.</a>

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 ...

Nanyang Technological University

Research

JoVE Journal

Behavior

589 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 ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

Testing for Odor Discrimination and Habituation in Mice

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the mouse can discriminate between non-social odors and social odors, whether the mouse habituates to a repeatedly presented odor, and whether the mouse demonstrates dishabituation when presented with a novel odor. Since many social behavior tests measure the experimental animal&#8217;s response to a familiar or novel mouse, false positives can be avoided by establishing that the animals can detect and discriminate between social odors. There are similar considerations in learning tests such as fear conditioning that use odor to create a novel environment or olfactory cues as an associative stimulus. Deficits in the olfactory system would impair the ability to distinguish between contexts and to form an association with an olfactory cue during fear conditioning. 
In the odor habitation/dishabituation test, the mouse is repeatedly presented with several odors. Each odor is presented three times for two minutes. The investigator records the sniffing time directed towards the odor as the measurement of olfactory responsiveness. A typical mouse shows a decrease in response to the odor over repeated presentations (habituation). The experimenter then presents a novel odor that elicits increased sniffing towards the new odor (dishabituation). After repeated presentation of the novel odor the animal again shows habituation. This protocol involves the presentation of water, two or more non-social odors, and two social odors. In addition to reducing experimental confounds, this test can provide information on the function of the olfactory systems of new knockout, knock-in, and conditional knockout mouse lines.

This manuscript describes a protocol to examine the olfactory system of rodents. The olfactory habituation/dishabituation test will allow investigators to determine whether a mouse habituates to a repeatedly presented odor and whether the mouse demonstrates dishabituation when presented a novel odor.

This video demonstrates a technique to establish the presence of a normally functioning olfactory system in a mouse. The test helps determine whether the ...

Quantifying Social Motivation in Mice Using Operant Conditioning

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered shuttle boxes were equipped with two operant levers (left and right) and a food receptacle in one chamber, which was then divided from the second chamber by an automated guillotine door covered by a wire grid. Different stimulus mice, rotated across testing days, served as a social stimulus behind the wire grid, and were only visible following the opening of the guillotine door. Test mice were trained to lever press in order to open the door and gain access to the stimulus partner for 15 sec. The number of lever presses required to obtain the social reward progressively increased on a fixed schedule of 3. Testing sessions ended after test mice stopped lever pressing for 5 consecutive minutes. The last reinforced ratio or breakpoint can be used as a quantitative measure of social motivation. For the second paradigm, test mice were trained to discriminate between left and right lever presses in order to obtain either a food reward or the social reward. Mice were rewarded for every 3 presses of each respective lever. The number of food and social rewards can be compared as a measurement of the value placed upon each reward. The ratio of each reward type can also be compared between mouse strains and the change in this ratio can be monitored within testing sessions to measure satiation with a given reward type. Both of these operant conditioning paradigms are highly useful for the quantification of social motivation in mouse models of autism and other disorders of social behavior.

This article describes a new protocol for the quantitative measurement of social motivation in mice using operant conditioning for a social reward. The protocol is useful for the measurement of social motivation in mouse models of autism and other disorders of social behavior.

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered ...

Whisker-signaled Eyeblink Classical Conditioning in Head-fixed Mice

Eyeblink conditioning is a common paradigm for investigating the neural mechanisms underlying learning and memory. To better utilize the extensive repertoire of scientific techniques available to study learning and memory at the cellular level, it is ideal to have a stable cranial platform. Because mice do not readily tolerate restraint, they are usually trained while moving about freely in a chamber. Conditioned stimulus (CS) and unconditioned stimulus (US) information are delivered and eyeblink responses recorded via a tether connected to the mouse's head. In the head-fixed apparatus presented here, mice are allowed to run as they desire while their heads are secured to facilitate experimentation. Reliable conditioning of the eyeblink response is obtained with this training apparatus, which allows for the delivery of whisker stimulation as the CS, a periorbital electrical shock as the US, and analysis of electromyographic (EMG) activity from the eyelid to detect blink responses.

The preparation presented here for whisker-signaled eyeblink conditioning in head-fixed mice precisely stimulates specific whiskers while allowing mice to ambulate on a cylindrical treadmill. A whisker stimulation conditioned stimulus (CS) paired with a periorbital shock unconditioned stimulus (US) results in reliable associative learning on this apparatus.

Eyeblink conditioning is a common paradigm for investigating the neural mechanisms underlying learning and memory. To better utilize the extensive ...

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 ...

Measuring Active and Passive Tameness Separately in Mice

Domesticated animals such as dogs and laboratory mice show a high level of tameness, which is important for humans to handle them easily. Tameness has two behavioral components: a reluctance to avoid humans (passive tameness) and a motivation to approach humans (active tameness). To quantify these components in mice, we previously developed behavioral tests for active tameness, passive tameness, and the willingness to stay on a human hand, each designed to be completed within 3 min. The data obtained were used for selective breeding, with a large number of mice analyzed per generation. The active tameness test measures the movement of the mouse toward a human hand and the contact it engages in. The passive tameness test measures the duration of time that a mouse tolerates human touch. In the stay-on-hand test, a mouse is placed on a human hand and touched slowly using the thumb of that hand; the duration of time that the animal remains on the hand is measured. Here, we describe the test set-up and apparatus, explain the procedures, and discuss the appropriate data analysis. Finally, we explain how to interpret the results.

Tameness in animals includes a reduction of avoidance responses to humans (passive tameness) and an increase in active approaches to humans (active tameness). Here, we describe detailed protocols for three behavioral tests (active tameness, passive tameness, and stay-on-hand tests) to separately measure the active and passive tameness of mice.

Domesticated animals such as dogs and laboratory mice show a high level of tameness, which is important for humans to handle them easily. Tameness has two ...

Evaluating Skilled Prehension in Mice Using an Auto-Trainer

We describe a method to introduce na&#239;ve mice to a novel prehension (reach-to-grasp) task. Mice are housed singly in cages with a frontal slot that permits the mouse to reach out of its cage and retrieve food pellets. Minimal food restriction is employed to encourage the mice to perform the food retrieval from the slot. As the mice begin to associate coming to the slot for food, the pellets are manually pulled away to stimulate extension and pronation of their paw to grasp and retrieve the pellet through the frontal slot. When the mice begin to reach for the pellets as they arrive at the slot, the behavioral assay can be performed by measuring the rate at which they successfully grasp and retrieve the desired pellet. They are then introduced to an auto-trainer that automates both the process of providing food pellets for the mouse to grasp, and the recording of successful and failed reaching and grasping attempts. This allows for the collection of reaching data for multiple mice with minimal effort, to be used in experimental analysis as appropriate.

Method to assess the impact of training on motor skills is a useful tool. Unfortunately, most behavioral assessments can be labor intensive and/or expensive.We describe here a robotic method of assessing prehension (reach-to-grasp) skill in mice.

We describe a method to introduce na&#239;ve mice to a novel prehension (reach-to-grasp) task. Mice are housed singly in cages with a frontal slot that ...

Assessment of Memory Function in Pilocarpine-induced Epileptic Mice

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal model of epilepsy, we generated pilocarpine-treated chronic epileptic mice. We present a protocol for three different behavioral tests using these epileptic mice: novel object location (NL), novel object recognition (NO), and pattern separation (PS) tests to evaluate learning and memory for places, objects, and contexts, respectively. We explain how to set the behavioral apparatus and provide experimental procedures for the NL, NO, and PS tests following an open field test that measures the animals&#8217; basal locomotor activities. We also describe the technical advantages of the NL, NO, and PS tests with respect to other behavioral tests for assessing memory function in epileptic mice. Finally, we discuss possible causes and solutions for epileptic mice failing to make 30 s of good contact with the objects during the familiarization sessions, which is a critical step for successful memory tests. Thus, this protocol provides detailed information about how to assess epilepsy-associated memory impairments using mice. The NL, NO, and PS tests are simple, efficient assays that are appropriate for the evaluation of different kinds of memory in epileptic mice.

This article presents experimental procedures for assessing memory impairments in pilocarpine-induced epileptic mice. This protocol can be used to study the pathophysiologic mechanisms of epilepsy-associated cognitive decline, which is one of the most common comorbidities in epilepsy.

Cognitive impairment is one of the most common comorbidities in temporal lobe epilepsy. To recapitulate epilepsy-associated cognitive decline in an animal ...

Chronic Stress Shifts Effort-Related Choice Behavior in a Y-Maze Barrier Task in Mice

Mood disorders, including major depressive disorder, can be precipitated by chronic stress. The Y-maze barrier task is an effort-related choice test that measures motivation to expend effort and obtain reward. In mice, chronic stress exposure significantly impacts motivation to work for a higher value reward when a lesser value reward is freely available compared to unstressed mice. Here we describe the chronic corticosterone administration paradigm, which produces a shift in effortful responding in the Y-maze barrier task. In the Y-maze task, one arm contains 4 food pellets, while the other arm contains only 2 pellets. After mice learn to select the high reward arm, barriers with progressively increasing height are then introduced into the high reward arm over multiple test sessions. Unfortunately, most chronic stress paradigms (including corticosterone and social defeat) were developed in male mice and are less effective in female mice. Therefore, we also discuss chronic non-discriminatory social defeat stress (CNSDS), a stress paradigm we developed that is effective in both male and female mice. Repeating results with multiple distinct chronic stressors in male and female mice combined with increased usage of translationally relevant behavior tasks will help to advance the understanding of how chronic stress can precipitate mood disorders.

The Y-maze barrier task is a behavior test that examines motivation to expend effort for reward. Here, we discuss testing multiple well-validated chronic stressors including chronic corticosterone and social defeat stress with this behavior, as well as the novel chronic non-discriminatory social defeat stress (CNSDS), which is effective in females.

Mood disorders, including major depressive disorder, can be precipitated by chronic stress. The Y-maze barrier task is an effort-related choice test that ...