Hormonal status and age differentially affect tolerance to the disruptive effects of delta-9-tetrahydrocannabinol (Δ(9)-THC) on learning in female rats

Abstract

The effects of hormone status and age on the development of tolerance to Δ(9)-THC were assessed in sham-operated (intact) or ovariectomized (OVX) female rats that received either intraperitoneal saline or 5.6 mg/kg of Δ(9)-THC daily from postnatal day (PD) 75-180 (early adulthood onward) or PD 35-140 (adolescence onward). During this time, the four groups for each age (i.e., intact/saline, intact/THC, OVX/saline, and OVX/THC) were trained in a learning and performance procedure and dose-effect curves were established for Δ(9)-THC (0.56-56 mg/kg) and the cannabinoid type-1 receptor (CB1R) antagonist rimonabant (0.32-10 mg/kg). Despite the persistence of small rate-decreasing and error-increasing effects in intact and OVX females from both ages during chronic Δ(9)-THC, all of the Δ(9)-THC groups developed tolerance. However, the magnitude of tolerance, as well as the effect of hormone status, varied with the age at which chronic Δ(9)-THC was initiated. There was no evidence of dependence in any of the groups. Hippocampal protein expression of CB1R, AHA1 (a co-chaperone of CB1R) and HSP90β (a molecular chaperone modulated by AHA-1) was affected more by OVX than chronic Δ(9)-THC; striatal protein expression was not consistently affected by either manipulation. Hippocampal brain-derived neurotrophic factor expression varied with age, hormone status, and chronic treatment. Thus, hormonal status differentially affects the development of tolerance to the disruptive effects of delta-9-tetrahydrocannabinol (Δ(9)-THC) on learning and performance behavior in adolescent, but not adult, female rats. These factors and their interactions also differentially affect cannabinoid signaling proteins in the hippocampus and striatum, and ultimately, neural plasticity.

Keywords: age of initiation; cannabinoid receptors; chronic Δ9-tetrahydrocannabinol; learning; ovariectomy; rat.

FIGURE 1

Timeline of manipulations for the two age groups of female subjects that were administered chronic Δ⁹-THC after either ovariectomy (OVX) or a sham operation.

FIGURE 2

Bar graphs showing the effects of hormonal status and chronic Δ⁹-THC administration from either early adulthood (n = 6/group) or adolescence (n = 6/group) on baseline response rates and percent errors under the multiple schedule of repeated acquisition and performance of response sequences. These data represent the grand mean for 6–20 occasions in each subject when an injection of saline was substituted for the daily chronic dose of Δ⁹-THC. The first of these occasions occurred after a minimum of 65 days after chronic Δ⁹-THC in each age group (early adult or adolescent). As shown by the horizontal bars and letters, there was a significant main effect of chronic Δ⁹-THC on both response rate and percent errors in each component, but no effect of hormone status and no interaction between chronic Δ⁹-THC and hormone status.

FIGURE 3

Acute effects of Δ⁹-THC in intact and OVX females that received either saline (A, upper four panels) or 5.6 mg/kg of Δ⁹-THC (B, lower four panels) daily from early adulthood to sacrifice and that were responding under an acquisition (A, graph legend) and performance (P, graph legend) procedure. Data points and vertical lines above C in each panel indicate the grand mean and standard error of the mean (SEM) for 9–19 saline (control) injections. The data points and vertical lines in the dose-effect curves represent a grand mean and SEM for that dose, as each dose was administered on 2–4 occasions in each subject. Asterisks alone or in combination with brackets indicate significant differences between particular doses of Δ⁹-THC and acute saline (control) injections. 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 (e.g., instances in which responding was eliminated entirely and the percentage of errors could not be calculated, or simply differences in the potency of Δ⁹-THC across subjects).

FIGURE 4

Acute effects of Δ⁹-THC in intact and OVX females that received either saline (A) or 5.6 mg/kg of Δ⁹-THC (B) daily from adolescence to sacrifice and that were responding under an acquisition and performance procedure. Data points and vertical lines above C in each panel indicate the grand mean and SEM for 6–20 saline (control) injections. Asterisks alone or in combination with brackets indicate significant differences between particular doses of Δ⁹-THC and acute saline (control) injections, whereas a cross indicates a significant difference between hormone groups at a particular dose. For additional details, see legend for Figure 3.

FIGURE 5

Acute effects of rimonabant in intact and OVX females that received either saline (A) or 5.6 mg/kg of Δ⁹-THC (B) daily from adolescence or early adulthood to sacrifice and that were responding under an acquisition and performance procedure. The data for the two age groups were combined as there was no marked difference between these age groups. Data points and vertical lines above C in each panel indicate the grand mean and SEM for 3–10 vehicle (control) injections administered to each subject in each treatment group. In the upper panels (response rate), asterisks alone or in combination with brackets indicate significant differences between particular doses of Δ⁹-THC and acute saline (control) injections, whereas crosses indicate a significant difference from the intact/saline group under control conditions or after particular doses of rimonabant. In the bottom panels (percent errors), there were no significant interactions, only main effects for dose and treatment group. Therefore, the asterisks with brackets indicate significant differences from control injections for all of the groups at every dose of rimonabant, whereas the crosses with brackets indicate the treatment groups that were significantly different from the intact/saline group irrespective of dose.

FIGURE 6

Effects of chronic Δ⁹-THC and hormone status on the endogenous levels of CB1R, AHA1, and HSP90β in the hippocampus. Above each bar graph are western blots of protein levels in hippocampal extracts from the female rats in each of the four chronically treated groups. The extracts were separated by 10% SDS-PAGE, transferred onto PDVF membranes, and subjected to western blotting with specific antibodies for each protein. The data were quantified using a GE ImageQuant (LAS-4000 Plus) and the Image J program (Version 1.54). Levels of each protein were corrected for the β-actin levels in the same samples and presented as a ratio of protein to actin in relative units. Asterisks associated with a bar indicate significant main effects of hormone status, as there were no significant main effects of chronic treatment and no significant interaction between chronic Δ⁹-THC and hormone status in hippocampus.

FIGURE 7

Effects of chronic Δ⁹-THC and hormone status on the endogenous levels of CB1R, AHA1, and HSP90β in the striatum. Above each bar graph are western blots of protein levels in striatal extracts from the female rats in each of the four chronically treated groups. Levels of each protein were corrected for the β-actin levels in the same samples and presented as a ratio of protein to actin in relative units. Asterisks associated with a bar indicate significant main effects of hormone status, whereas letters indicate significant differences among the four treatment groups when there were significant main effects for chronic Δ⁹-THC and hormone status or a significant interaction between the factors (e.g., “a” is different from “b,” but not different from “a,b,c”).

FIGURE 8

Effects of chronic Δ⁹-THC and hormone status on the endogenous levels of BDNF in the hippocampus. Above each bar graph are western blots of protein levels in hippocampal extracts from the female rats in each of the four chronically treated groups. Levels of each protein were corrected for the β-actin levels in the same samples and presented as a ratio of protein to actin in relative units. Asterisks associated with a bar indicate significant main effects of hormone status, whereas letters indicate significant differences among the four treatment groups when there were significant main effects for both chronic Δ⁹-THC and hormone status (e.g., “a” is different from “b,” but not different from “a,c”).