Ovarian hormones and chronic administration during adolescence modify the discriminative stimulus effects of delta-9-tetrahydrocannabinol (Δ⁹-THC) in adult female rats

Abstract

Marijuana abuse during adolescence may alter its abuse liability during adulthood by modifying the interoceptive (discriminative) stimuli produced, especially in females due to an interaction with ovarian hormones. To examine this possibility, either gonadally intact or ovariectomized (OVX) female rats received 40 intraperitoneal injections of saline or 5.6 mg/kg of Δ⁹-THC daily during adolescence, yielding 4 experimental groups (intact/saline, intact/Δ⁹-THC, OVX/saline, and OVX/Δ⁹-THC). These groups were then trained to discriminate Δ⁹-THC (0.32-3.2 mg/kg) from saline under a fixed-ratio (FR) 20 schedule of food presentation. After a training dose was established for the subjects in each group, varying doses of Δ⁹-THC were substituted for the training dose to obtain dose-effect (generalization) curves for drug-lever responding and response rate. The results showed that: 1) the OVX/saline group had a substantially higher mean response rate under control conditions than the other three groups, 2) both OVX groups had higher percentages of THC-lever responding than the intact groups at doses of Δ⁹-THC lower than the training dose, and 3) the OVX/Δ⁹-THC group was significantly less sensitive to the rate-decreasing effects of Δ⁹-THC compared to other groups. Furthermore, at sacrifice, western blot analyses indicated that chronic Δ⁹-THC in OVX and intact females decreased cannabinoid type-1 receptor (CB1R) levels in the striatum, and decreased phosphorylation of cyclic adenosine monophosphate response element binding protein (p-CREB) in the hippocampus. In contrast to the hippocampus, chronic Δ⁹-THC selectively increased p-CREB in the OVX/saline group in the striatum. Extracellular signal-regulated kinase (ERK) was not significantly affected by either hormone status or chronic Δ⁹-THC. In summary, these data in female rats suggest that cannabinoid abuse by adolescent human females could alter their subsequent responsiveness to cannabinoids as adults and have serious consequences for brain development.

Fig. 1

Timeline of manipulations for the subjects in each experimental group.

Fig. 2

Effects of Δ⁹-THC on the percentage of Δ⁹-THC-lever responding (upper panels) and overall response rate (lower panels) in adult female rats that received chronic Δ⁹-THC (intact/Δ⁹-THC), ovariectomy (OVX/saline), or both (OVX/Δ⁹-THC) during adolescence. The control group received a sham surgery and chronic saline during adolescence (intact/saline). Subjects in each group discriminated different training doses while responding under a FR-20 schedule of food reinforcement (see Table 1). Doses of Δ⁹-THC were administered i.p. 20 min before the start of the session, which began with a 10-min timeout. Data points and vertical lines above “C” in each panel represent the grand mean and SEM for each group, which was calculated from the means for 4 to 11 injections of saline or vehicle for each subject in that group. The data points and vertical lines in the dose-effect curves for each group in each panel also represent a grand mean and SEM. The grand mean and SEM for each dose was calculated from the means for 1 to 5 determinations of that dose in each subject. Numerical values in parentheses and adjacent to a data point indicate the number of subjects represented by that point when it differed from the total number of subjects for that group. Asterisks along with a bracket indicate doses of Δ⁹-THC that were significantly different from saline administration (control) for both chronically-treated groups as there was neither a main effect of chronic treatment nor an interaction. Asterisks without a bracket indicate doses of Δ⁹-THC that were significantly different from saline administration (control) for a particular group. A cross with a bracket indicates the only dependent measure for which there was a significant main effect of chronic treatment.

Fig. 3

Panel A shows CB1R protein expression in the hippocampus and striatum of adult female rats that received chronic Δ⁹-THC (intact/Δ⁹-THC), ovariectomy (OVX/saline), or both (OVX/Δ⁹-THC) during adolescence. The control group received a sham surgery and chronic saline during adolescence (intact/saline). The bar graphs in panel B show the quantification of CB1R levels when they are normalized to B-actin in the hippocampus and striatum. The letters in the bar graph for striatum represent the significant (p<0.05) main effect of Δ⁹-THC as indicated by a two-way ANOVA and Holm-Sidak post hoc tests (i.e., a’s are significantly different from b’s, but not from each other).

Fig. 4

Panel A shows the levels of p-CREB and t-CREB in the hippocampus and striatum of adult female rats that received chronic Δ⁹-THC (intact/Δ⁹-THC), ovariectomy (OVX/saline), or both (OVX/Δ⁹-THC) during adolescence. The control group received a sham surgery and chronic saline during adolescence (intact/saline). The bar graphs in panel B show the quantification of p-CREB in the hippocampus and striatum as a ratio of p-CREB to t-CREB. Letters in the left-hand graph for the hippocampus represent the significant (p<0.05) main effect of Δ⁹-THC as indicated by a two-way ANOVA and Holm-Sidak post hoc tests. The asterisk in the right-hand graph for the striatum represents the significant difference from control (intact/Saline), which was established by a post hoc test conducted after a significant interaction was obtained with a two-way ANOVA.

Fig. 5

Panel A shows the levels of p-ERK 1/2 and t-ERK 1/2 in the hippocampus and striatum of adult female rats that received chronic Δ⁹-THC (intact/Δ⁹-THC), ovariectomy (OVX/saline), or both (OVX/Δ⁹-THC) during adolescence. The control group received a sham surgery and chronic saline during adolescence (intact/saline). The bar graphs in panel B show the quantification of p-ERK in the hippocampus and striatum as a ratio of p-ERK to t-ERK. A two-way ANOVA indicated there were no significant main effects of OVX or chronic Δ⁹-THC, and no significant interaction between these two factors.