Materials and Reagents

1. Identical plastic containers (Figures 1A and 1B): Dimensions 6.5 x 4.5 cm, assembled weight ~100 g (Julia Pet Jar 130 ml) (Princeware, catalog number: 9462 )
   
2. Bedding material made from non-uniform corn cobb granules of 3-4 mm average diameter (Spar Cobb, Sagar Industries, Bangalore, India)
   
3. Food vessel: A lid for smaller plastic container was used as food vessel (Figure 1) (Julia Pet Jar 60 ml) (Princeware, catalog number: 9461 )
   Note: It is filled with ground flavored food and placed above the bedding material inside the plastic container. The lid of plastic container is closed such that the centers for food vessel and hole in the lid are aligned vertically.
   
4. C57/B6 mice (males, 8-12 weeks of age) housed in pairs after weaning until the beginning of habituation sessions. In order to clearly identify observer mice from demonstrators during interaction sessions in our experiments, demonstrator mice are marked with one hole in each ear pinna under general anesthesia after one week of weaning. Animals are separated for individual housing just before first habituation session.12-16 mice per group provide acceptable power during statistical analyses. The mice are reared in 12 h light/dark cycle and all the experiments are conducted during the light-on phase (06:30 AM-18:30 PM). All the procedures involving animals were performed with approval from institute animal ethics committee, IISc Bangalore
   Note: One important consideration is co-housing of observer mouse with demonstrator mouse after weaning. In our method, we do not use a wire mesh/screen during social interaction to separate ObMice and demoMice, in order for the mice interaction to resemble their natural setting. To avoid excessive fighting during social interaction, we randomly select two just-weaned mice and co-house them in the same cage for one month. At the age of ~2 months, these mice are separated to be housed individually for further steps of the protocol and one of them is assigned to become a demonstrator for its cage-mate. Social interaction between two familiar male mice may also help in making it more effective for STFP in comparison to interaction with a stranger male. So, it is crucial to collect a large number of mice in comparable age group at the same time. Either observer or demonstrator mouse can be marked by making a hole in their ear pinna in order to identify them correctly after social interaction session.
   
5. Mouse food pellets (Nutrilab Rodent Feed, Provimi)
   
6. Powdered condiments as flavoring agents (Cocoa, Cinnamon, Thyme, Basil; SNAPIN herbs and spices, Lotus household product, India)
   Note: Source of all the condiments must be consistent from the beginning to the end of the experiment.
   
7. 70% ethanol to clean all the components of food apparatus
   Note: Ethanol cleaning is carried out a day before the experiment session. All clean components are dried in warm air flow overnight to remove any odor trace from ethanol.
   
8. Sodium hypochlorite solution (4% NaOCl solution) (Fisher Scientific, catalog number: SS290-1 )
   Note: It is further diluted to make cleaning solution (see Recipes) for removing prevailing odor from the components of food apparatus and animal cages. After each experiment session, all the components of food apparatus are submerged in a cleaning solution for 15-20 min followed by thorough rinsing in tap water. Cleaned components are then air dried and stored in hygienic conditions.
   
9. X% ‘Condiment’ flavored food (see Recipes)
   
10. Cleaning solution (see Recipes)
    

Equipment

1. Plier for making metal trays
   
2. Paint for metal trays: white paint if black coat mice are used and vice versa
   
3. Transparent Perspex sheets with holes drilled along short central axis (Figures 1E)
   
4. Aluminum/Tin metal sheet (sized to fit in the test cage): 1-2 mm thickness for making spill-proof trays to hold food containers. The vertical walls of these trays are 2 cm high. Length and breadth of these trays can be adjusted for achieving best fit within the test cage. For our setup, the dimensions were 16 x 13 cm
   
5. Weighing balance: with sensitivity up to 10 mg
   
6. Electronic grinder (Morphy Richards, model: Icon Essentials ) for making powdered food
   
7. Full HD webcam (Logitech, model: C920 ) for multiplexed video monitoring
   
8. Polarizer filter (RG610, RG series color glass filters, Optica, Optics India; it is optional) to avoid reflections from transparent Perspex sheets covering the test-cages
   
9. Webcam mounting assembly: We used a long wooden stick (300 x 10 x 5 cm³) with a hole at the center along its length and an M6 screw to fix the webcam above test-cage assembly
   
10. Individually ventilated cages (IVCs)/polycarbonate cages for individual housing of mice (IVC; 36 long x 14 wide x 12.5 cm high) (Citizen Industries, catalog number: 11 )
    
11. Portable electronic drill (Robert Bosch, model: Bosch GSB 10 RE Professional ) with drill bits to make holes of 1 cm diameter in the lids of food container
    

Software

1. Free, open source media player such as VLC. Any software for position tracking may also be implemented for estimating time spent near food containers
   
2. Origin software
   
3. Microsoft Excel
   
4. ImageJ plugins (such as Analyze particle)
   

Procedure