Materials and reagents

Biological materials

1. Juvenile (4–5 weeks old) stimulus mice matched to the sex and strain of experimental mice (see General Note 1). Testing can be conducted with up to five mice per observer, per round of observation.

a. Juvenile mice can be reused in subsequent rounds of observation, so the amount required is one that is sufficient to provide one round of experimental mice with sex- and strain-matched juveniles. For example, 5 juvenile mice could be used for 20 experimental mice. This would result in four rounds of observation.

b. If juvenile mice are of the same coat color as the experimental mouse, mark the tail of the juveniles with a bright-colored marker for ease of observation and to differentiate between juveniles themselves.

Reagents

1. Tap water

2. 1:10,000 pure vanilla extract (McCormick & Co., Inc., Hunt Valley, MD) diluted in tap water

3. Pooled intact male & female mouse urine purchased from a commercial vendor (Biochemed Pharmacologicals, catalog number: NC1569592), ~2.2 mL/20 mice as a social odor (see also General Notes 1 and 2)

4. 70% EtOH spray

Laboratory supplies

1. Small static cages (186 mm × 298 mm × 128 mm) without bedding, nesting material, food, or water bottle

2. Small static cages (186 mm × 298 mm × 128 mm) with a layer of bedding but without nesting material, food, or water bottle

3. Large static cages (257 mm × 483 mm × 152 mm) with bedding but without nesting material, food, water bottle, or metal wire lids

4. Wire mesh cups (e.g., Amazon, Amazon Basics wire mesh pen cup, reference: B07VVB7DSK)

5. Bottles filled with tap water (e.g., EMD Milipore Corporation, Milipore Express^® PLUS 0.22 mm PES 150 mL bottle unit) or any other available object to serve as a weight on top of the wire mesh cups to prevent the mouse from tipping them while exploring

6. Novel objects (e.g., Simport Scientific Inc., catalog number: C576, SecurTainer^TM II tamper-evident container, 40 mL, reference: C576-40MA). These may be changed based on available resources but must fit in the wire mesh cups and be similar in size to a juvenile mouse

7. Pressed cotton batting nestlets (e.g., Ancare Corp, reference: NES3600)

8. GeneMate 20–200 μL autoclavable single-channel pipettor (ISC BioExpress Inc. or an equivalent model) and pipette tips

9. 15.24 cm cotton swabs (e.g., Tyco Healthcare Group LP, Kendall Curity^TM single tipped applicators, reference: 8884540500)

10. 15 mL polystyrene conical tubes (e.g., Corning, Falcon^® 15 mL polystyrene centrifuge tube, catalog number: 352095)

11. Tube rack (recommended) (e.g., Heathrow Scientific LLC, 5.39 cm wd, 9.52 cm ht, 17.46 cm lg, polypropylene, Grainger, catalog number: 55PT41)

12. 31.5 × 33.5 cm Whatman 3 MM CHR chromatography blotting paper (Cytiva, catalog number: 3030-335)

13. One stopwatch per experimenter + one additional

14. One clipboard per experimenter

15. Coding sheets for instantaneous sampling

16. Optional: sticky notes to keep track of mouse IDs

17. If needed: cages to temporarily store cage mates or house overnight for nest building

Equipment

1. UV transilluminator (e.g., Ultra-Violet Products, Inc., model: chromato-vue transilluminator 0–62)

2. High-resolution camera (high-resolution phone camera is OK; you must ensure that either the same camera is used across assessments or that megapixel count is identical between cameras used)

Software and datasets

1. Microsoft Excel or any other spreadsheet-based software

2. ImageJ (Version 1.53m [46])

3. Statistical software of your choosing to produce data in a reproducible manner, adequate for the comparisons you are making, based on your experimental design

Procedure