Estradiol 3-benzoate

At mid-adolescence (P35, Spear, 2000 (link); Andersen, 2003 (link)), male rats in the socially-stressed group (n = 30, mass at P35 = 222.0 ± 9.04 g; mean ± SE) were exposed to repeated social defeat in the home cage of a larger adult male Sprague-Dawley rat (mass = 397.5 ± 14.5 g; mean ± SE), using a modified version of the resident-intruder paradigm (Miczek, 1979 (link); Koolhaas, Meerlo, De Boer, Strubbe, & Bohus, 1997 (link); Martinez et al., 2002 (link)). Similar to previous social defeat studies (e.g., Covington & Miczek, 2005 (link); Czeh et al., 2007 (link); Vidal et al., 2007 (link)), age-matched male controls (n = 30, mass at P35 = 222.5 ± 7.86 g; mean ± SE) experienced no social defeat or interaction, and were simply placed into a novel empty cage for the duration of each defeat trial to control for handling and novel environment stress. A control treatment group experiencing social contact with a non-aggressive adult male during mid-adolescence was not included, as natural formation of dominant-subordinate hierarchies and variance in intensity of this experience would limit effective use as a control comparison.
To encourage territoriality, each resident male (n = 6) was housed with a sexually receptive female in a large plastic cage (40 × 25 × 17.5 cm) for one week prior to and throughout the course of the social defeat procedure. Females had been anesthetized with ketamine/xylazine (50/10 mg/kg, ip.) and ovariectomized earlier. Female sexual receptivity was induced administration of 17β estradiol benzoate (5 μg/0.1 ml oil, sc., Sigma-Aldrich USA) followed 24 hrs later by progesterone (0.5 mg/0.1 ml oil, sc., Sigma-Aldrich USA; Farmer, Isakson, Coy, & Renner, 1996 (link)). Several different females were used to ensure that each resident was paired with a receptive female every day. Females were removed from resident cages at the start of each defeat session and replaced afterwards. Resident males were assessed for aggressive responses towards an adolescent (P36) male intruder two days before actual experiments began. All residents approached and investigated the intruder thoroughly by sniffing at the anogenital region. The intruder was often held immobile in a crouching position while its neck was groomed roughly by the resident (Miczek & De Boer, 2005 ). Intruders were then attacked and grappled with before being thrown on their backs and displaying submission (Blanchard & Blanchard, 1989 (link); Blanchard, Sakai, McEwen, Weiss, & Blanchard, 1993 (link); Miczek & De Boer, 2005 ), typically within 5 min of introduction. Intruder rats used for these screening trials were not included in any later experiments.
For experimental defeat trials, each adolescent experimental subject was transferred from its home cage to that of a resident, with the interaction video-recorded for later measurement of latency to defeat. Adolescent intruders were considered defeated after exhibiting 3 consecutive submissive postures in response to resident attacks. Adolescents were then confined behind a wire mesh barrier for 35 min, which prevented further physical contact from the resident but allowed transmission of auditory, olfactory and visual cues. Following this, adolescent rats were returned to their home cages. Adolescent subjects were exposed daily to social defeat over a 5 day period, and were confronted with a different resident male each time using a completely randomized design to control for individual variance in defeat intensity. Following social defeat conditioning, subjects and controls were left in their original pairs in their home cages and allowed to mature to early adulthood (P56). There were no differences in mass between defeated subjects and controls at any point during the entire study (data not shown).