Sterile physiological saline

The alcohol challenge consisted of 3 (Exp. 1) or 3.5 (Exp. 2, 3) g/kg ethanol (20% v/v). Sterile physiological saline (0.9%, TEKnova, Hollister, CA) was used to make the ethanol solution and vehicle-exposed rats were given equivolume saline only. Both solutions were given using intraperitoneal (i.p.) administration, and cage mates were assigned to the same experimental condition.

The goal of Experiment 2 was to determine whether sensitivity of the pituitary gland would be altered in female rats exposed to footshock during adolescence. To do this, adolescent female rats (N = 32, n = 8/group) were either exposed to footshock in early adolescence (P29-31) or remained in their home cages and subsequently remained undisturbed in the colony until adulthood. Adult (P79-P81) rats were then injected with either 0 or 1.0 μg/kg CRH (i.p.; Catalog # C3042, Sigma-Aldrich, St. Louis, MO) dissolved in sterile physiological saline (0.9%, TEKnova, Hollister, CA). This dose was selected based upon recent findings from our lab demonstrating an intermediate-level ACTH and CORT response when injected peripherally (Hueston & Deak, 2014 (link)). Studies to date have not investigated the differences in magnitude of the response to CRH injection between males and female rats, however in humans a 1.0 μg/kg did not elicit differential responding in males and females (Gallucci et al., 1993). One hour after injection, rats were then rapidly decapitated under stress-free conditions and trunk blood was collected for measurement of ACTH and CORT (Figure 2A).

Since there were no effects of hormone-mediated stimulation of either the pituitary gland (Experiment 2) or the adrenal glands (Experiment 3), we performed one final test of HPA axis regulation to assess negative feedback regulation. Thus, Experiment 4 utilized a dexamethasone suppression test since injection of dexamethasone engages negative feedback mechanisms and suppresses the production and release of CORT (Hueston & Deak, 2014 (link); Riegle & Hess, 1972 (link)). Adolescent female rats (N = 32, n = 8/group) were either exposed to footshock in early adolescence (P29-31) or remained in homecages and subsequently remained undisturbed in the colony until adulthood. Adult (P77-P80) rats were then injected with 50 μg/kg dexamethasone (subcutaneous; catalog # D1756, Sigma-Aldrich, St. Louis, MO) dissolved in 50% propylene glycol (catalog # P4347, Sigma-Aldrich, St. Louis, MO) and 50% sterile physiological saline (0.9%, TEKnova, Hollister, CA) or vehicle, and 90 min later were placed in restraint for 30 min. Rats were then rapidly decapitated under low stress conditions and trunk blood was collected for measurement of ACTH, CORT and progesterone (Figure 4A). This dose and timing was selected in order to maximally suppress the CORT response and was based upon previous findings from our lab (Hueston et al., 2014 (link)).