Materials and Reagents

1. Male and female juvenile (4-5-week-old) Sprague-Dawley rats
   Notes:
   1. We breed and maintain our own colony at the University of Maryland School of Medicine. This facilitates early-life treatments with pharmacological agents predicted to affect masculinization and avoids stress due to animal shipping or relocation. Animals are kept on a 12:12 h reverse light:dark cycle.
   2. Rats are tested as sex-, treatment- and age-matched non-sibling pairs. These “play pairs” will remain constant across multiple days of testing such that each animal is playing with the same play partner every day. To achieve this, we plan experiments to include rats from at least two separate litters. Depending on the number of experimental conditions, we will test at least two cohorts of play pairs (2 sets of paired litters, or 4 litters total) (see Figure 1A for experimental design and timeline).
   3. At birth, litter size is culled to 12-14 pups per dam to avoid confounds of over/under nutrition and maternal care. Litters are kept as sex- and treatment-balanced as possible, and litters with an initial sex ratio of > 2:1 males:females (or vice versa) are not used in our experiments due to potential in utero effects of prenatal testosterone.
2. Pharmacological agents of interest
   Note: Depending on the hypothesis under study and the experimental timing of treatments, animals may need to be treated much earlier in life (prenatal exposure, postnatal masculinization, etc.; see VanRyzin et al., 2019 for examples).
   
   
   Figure 1. Representative experimental timeline and behavior illustrations. A. On the day of birth, pups from two litters are sexed, culled and treated (if necessary), and assigned to sex- and treatment-matched pairs with the opposite litter. The pups are allowed to grow undisturbed until the juvenile age. Beginning at P26, play behavior can be assessed daily throughout the juvenile period. B. Illustrations depicting rats engaging in the three main rough-and-tumble play behaviors–pouncing, pinning, and boxing.
   

Equipment

1. Custom plexiglass testing arena 49 cm x 37 cm, 24 cm high (see Figure 2A)
2. Behavioral testing room equipped with red light
3. One to two lamp stands with red light bulbs for illumination of the testing arenas
4. Video recording device and tripod stand
   Note: We typically use a smart phone with a tripod adapter to record behavioral tests.
5. TEK-Fresh bedding (Envigo, catalog number: 7099)
6. Fur-marking pen or permanent marker (Stoelting, catalog number: 50440-1)
7. Stopwatch or timer
8. Cleaning solution
   Note: Several different cleaning solutions may be used to sanitize the arenas after testing. We typically use MB-10 (Quip Laboratories).
   

Procedure