Materials and Reagents

1. Mice 
   Adult (12 weeks of age or older) male mice (C57BL/6J) (Charles River, L’Arbresle, France, Strain Code 027), weighing 22-28 g at the beginning of the experiments. Mice are housed individually, each in their respective cages, in an environment with controlled temperature (around 23 °C) and humidity under a 12-12 h light-dark cycle with free access to food. Young adult mice (> 12 weeks) are used to avoid a bias due to the increase of body weight of the developmental growth curve.
   Note: C57BL/6J mouse is chosen because it is widely used as a model for diet induced obesity since it is prone to develop severe obesity, elevated adiposity, glucose intolerance and moderate insulin resistance. However, other strains and ages of mice may also be used, but taking into account that strains such as SWR/J and A/J mice are more resistant to obesity development. The weight at the beginning of the experiments is just an indication (mean weight of C57BL/6J 12-weeks mouse). Of note, the initial weight of the animals could differ if a different strain is used.
   
2. Diet
   In the diet-induced obesity protocol, animals can be allocated to receive free access of chocolate-mixture (CM) or exclusive access of high-fat diet (HF) for the study of obesity development as shown in Figure 1A. From now on, CM experiment is referred to as condition 1 and HF experiment as condition 2.
   
   
   Figure 1. Schematic representation of the main experimental procedures. A. Two different experiments were carried out each of them with a separate control group, namely condition 1 and condition 2. Control group in both conditions 1 and 2 received standard chow (SC) ad libitum. After the habituation period, in condition 1 case group was provided with a free-choice between a standard chow diet and a chocolate-mixture diet (CM), while in condition 2 case group was offered only a high-fat (HF) hypercaloric food. Sample size is shown in parentheses denoting the number of mice in each group. B. Timeline of the limited access to energy-dense foods test in condition 1 (above) and condition 2 (below). Overweight mice were offered CM (condition 1) or HF (condition 2) pellets a single hour during the light (resting) phase of the light-dark cycle for 3 consecutive days to measure feeding inflexibility. C. Representation of the setup used to evaluate compulsive-intake in diet-induced obese mice of condition 1 (above) and obese mice of condition 2 (below). Mice have restricted the access to food to 2 h per day at the beginning of the dark phase during 6 consecutive days. On Days 1, 3 and 5 control mice received standard chow, while case mice in condition 1 are given standard chow/chocolate mixture and case mice in condition 2 are presented with high-fat diet. On Days 2, 4 and 6 all animals received only standard chow.
   
   
   1. Standard chow (SDS, Witham, UK, catalog number: 801151)
      Standard chow contained 10.76 MJ kg^-1 digestible energy (17.5% coming from protein, 75% from carbohydrate and 7.4% from fat; Table 1). Typically, standard chow carbohydrates are contributed to by 45% starch and approximately 4% simple sugars (monosaccharide plus disaccharides) as a proportion of total carbohydrates by weight. 
   2. Pellets of a mixture of commercial chocolates (CM)
      CM are made from equal amounts of Bounty^®, Snickers^®, Mars^® and Milka^® chocolates (local supermarket). CM pellets contained 20.6 MJ kg^-1 digestible energy (17% coming from protein, 52% from carbohydrate and 24% from fat; carbohydrate is contributed to by 8% starch and approximately 44% simple sugars (monosaccharide plus disaccharides, Table 1).
   3. High-Fat (HF) diet (Test Diet^®, catalog number: 58Y1)
      HF consisted of commercial pellets of purified diet with 60% energy derived from fat. HF pellets contained 22 MJ kg^-1 digestible energy (24% coming from protein, 30% from carbohydrate and 35% from fat; carbohydrate is contributed to by 5.4% starch and approximately 6.4% simple sugars (monosaccharide plus disaccharides, Table 1).
   Notes:
   
   1. Diets listed above describe what has been used successfully using the protocol provided; however, using alternative diets with similar composition could also be successful.
   2. Food was renewed every 3-4 days to ensure the maintenance of the organoleptic properties. We have consistently observed in several of our experiments that around 15% of mice under a high-energy free choice obesogenic diet do not gain weight (obese-resistant). 
   
   Table 1. Nutritional composition and energy content of the experimental diets. The composition of the SC and HF diets was provided by the manufacturers. CM diet was prepared in the lab and its composition was analyzed by Alquimisa laboratories S.L (Salamanca, Spain).
   
   
   
   
   
3. Quinine hydrochloride dehydrated (Sigma-Aldrich, Madrid, Spain, catalog number: Q1125) for the adulteration of food in order to test mice feeding inflexibility.

Equipment

1. Grinder and spoon for chocolate mixture (Bounty^®, Snickers^®, Mars^® and Milka^®) pellet preparation
2. Diet storage refrigerator
3. Personal computer for data recording using RS232/USB port
4. PheCOMP System (Multitake model, Panlab, Barcelona, Spain, catalog number: LE0851B)
   The equipment consists of experimental cages (Mouse polycarbonate home cages; Allentown Caging Equipment, ACE, 197 weight x 306 depth x 212 height mm, see Bura et al., 2010) provided with a grid floor and a filter top. The platform supporting each cage contains 2 infrared frames (16 x 16 beams, 16 mm spaced) allowing simultaneous recordings of horizontal activity and rearing (Panlab LE8827 Rearing IR frame).
   Notes:
   
   1. The cages contain 2 feeder units and 2 drinking units and give intake data resolutions of 0.02 grams per second allowing higher precision of the amount of food (g) or liquid consumed (ml) compared to standard cages; see Figure 2. The quantity of water drunk from a nipple, and any possible dripping, are also accounted by using the same system of transducers with 20-μl precision. The modular structure allows multiple dispenser combinations made them suitable for our free-choice experiments.
   2. One cage can only be used for one mouse.
   3. Before each experiment, the weight transducer system of the cages are calibrated. To do so, the manufacturer (Panlab, Barcelona, Spain) provides a 20 grams precision weight that is used to calibrate both the drinkers and feeders.
   
   
   Figure 2. Representative images for housing cages used during the study. A. Standard individualized cage for mouse housing. B. A PheCOMP cage (Panlab, Spain), the equipment used to automatically record the food/water intake and activity of individualized mice.
   

Software

1. Compulse software (Panlab, Barcelona, Spain) for automated data collection
2. GraphPad PRISM 6.0 software (GraphPad Software. Inc., San Diego, CA, USA), a software for graphing
3. SPSS 17.0 (SPSS Inc, Chicago, IL, USA) statistical analysis software
4. “mtb2int.pl” and “int2combo.pl” Perl scripts for processing Phecomp cages output files into intervals comma-separated values (CSV) files. The scripts are available on github at: https://github.com/cbcrg/phecomp/blob/master/lib/perl/ 
5. Pergola (Espinosa-Carrasco et al., 2018b) (https://github.com/cbcrg/pergola): Pergola is a Python library and command line tool for the conversion of longitudinal behavioral data into genomic file formats for its analysis and visualization
6. Integrative Genomics Viewer (IGV) (Robinson et al., 2011), (http://software.broadinstitute.org/software/igv/), a desktop application for the visualization of genomic data
7. Pergola web-server (Espinosa-Carrasco, 2019) (http://pergola.crg.eu/), a user-friendly web interface to process and visualize longitudinal behavioral data using Pergola
   

Procedure