Surgical Protocol for Ovariectomized and/or Adrenalectomized Rats

The rats underwent surgery using xylazine (12 mg/kg) and ketamine (70 mg/kg) anesthesia. Ovariectomy was performed on some rats (n = 12) as previously described (Frye et al., 2008b). Briefly, a ~3-cm incision was made in the dorsal region of the flank, just anterior to the kidney. The ovary was isolated from the surrounding adipose tissue, ligated with surgical silk (4–0 USP, 1.5 m), and extirpated. The muscle wall and skin were closed with 2–3 silk sutures, and the procedure was repeated for the ovary on the alternate side. Some rats also underwent ADX (n = 6) at the time of OVX as per prior methods (Rhodes et al., 2004). For ADX, the adrenal gland was located via proximity to the kidney; the gland was isolated with a forceps and extirpated. One OVX/ADX rat died prior to surgical completion. Following surgery and prior to testing, the animals were monitored for changes in weight, righting response, flank stimulation response, and/or muscle tone (Marshall and Teitelbaum, 1974). No rats failed these assessments in the present study. Given that ADX rats are rendered sodium deficient, they were provided continuous access to a bottle of sodium chloride (0.9%) in addition to drinking water. All rats that had surgery were allowed 10 days for recovery and hormonal washout prior to testing.

Paced mating was conducted as per previous methods (Erskine, 1985). In brief, the paced mating apparatus (37.5 × 75 × 30 cm) was equally divided by a Plexiglass partition, which contained a small (5 cm in diameter) hole in the bottom center, allowing the female (but not the stimulus male) free access to both sides of the apparatus. The frequency of mating contacts (intromissions) was recorded, and a lordosis quotient was calculated [(frequency of female dorsiflexion during a sexual contact/total sexual contacts by a male) * 100] during a 15-min test. The frequency of proceptive/solicitation behavior (ear wiggling, hopping, and darting) prior to intromission was recorded and calculated as a proceptivity quotient [(frequency of proceptive behaviors/total sexual contacts by a male) * 100]. The percentage of defensive aggressive behaviors (vocalizing and attack) that females displayed in response to male intromission was calculated as a defensive aggression quotient [(frequency of defensive aggression/total sexual contacts by a male) * 100]. Following intromission, the percentage of times the experimental female left the chamber containing the male (% exits) following sexual contacts was recorded. The effects of ovarian and/or adrenal gland extirpation to reduce the sexual response of female rodents to male mounting have been established (Davidson et al., 1968; Komisaruk and Diakow, 1973; Feder et al., 1974), and the present behavioral data is confirmatory in this regard.