Pharmacokinetic, behavioral, and brain activity effects of Δ9-tetrahydrocannabinol in adolescent male and female rats

Abstract

Δ⁹-tetrahydrocannabinol (THC) is the intoxicating constituent of cannabis and is responsible for the drug's reinforcing effects. Retrospective human studies suggest that cannabis use during adolescence is linked to long-term negative psychological outcomes, but in such studies it is difficult to distinguish the effects of THC from those of coexisting factors. Therefore, translationally relevant animal models are required to properly investigate THC effects in adolescents. However, though the relevance of these studies depends upon human-relevant dosing, surprisingly little is known about THC pharmacology and its effects on behavior and brain activity in adolescent rodents-especially in females. Here, we conducted a systematic investigation of THC pharmacokinetics, metabolism and distribution in blood and brain, and of THC effects upon behavior and neural activity in adolescent Long Evans rats of both sexes. We administered THC during an early-middle adolescent window (postnatal days 27-45) in which the brain may be particularly sensitive to developmental perturbation by THC. We determined the pharmacokinetic profile of THC and its main first-pass metabolites (11-hydroxy-THC and 11-nor-9-carboxy-THC) in blood and brain following acute injection (0.5 or 5 mg/kg, intraperitoneal). We also evaluated THC effects on behavioral assays of anxiety, locomotion, and place conditioning, as well as c-Fos expression in 14 brain regions. Confirming previous work, we find marked sex differences in THC metabolism, including a female-specific elevation in the bioactive metabolite 11-hydroxy-THC. Furthermore, we find dose-dependent and sex-dependent effects on behavior, neural activity, and functional connectivity across multiple nodes of brain stress and reward networks. Our findings are relevant for interpreting results of rat adolescent THC exposure studies, and may lend new insights into how THC impacts the brain in a sex-dependent manner.

Fig. 1. Plasma concentrations of THC and its first-pass metabolites.

Concentration profile of THC, 11-OH-THC, and 11-COOH-THC in plasma after IP injection of THC, 0.5 mg/kg (left panel: a–e) or 5 mg/kg (right panel: g–l), in female (black) or male (gray) adolescent rats. Lines represent the mean ± SEM, n = 4/sex and dose group at each timepoint (15, 30, 60, 120, 240, 480 min post THC). Adjacent bar graphs represent total exposure (area under the curve, AUC, pmol/min/mL) to THC, 11-OH-THC and 11-COOH-THC, respectively, in females or males after 0.5 mg/kg (b, d, f) or 5 mg/kg THC (h, j, l). Bars represent the mean ± SEM, *p < 0.05, **p < 0.01.

Fig. 2. Brain concentrations of THC and its first-pass metabolites.

Concentration profile of THC, 11-OH-THC, and 11-COOH-THC in brain tissue after IP injection of THC, 0.5 mg/kg (left panel, a–e) or 5 mg/kg (right panel, g–l), in female (black) or male (gray) adolescent rats, n = 4/group. Adjacent bar graphs represent total exposure (area under the curve, AUC, pmol/min/mL) to THC, 11-OH-THC and 11-COOH-THC, respectively, for females or males after 0.5 mg/kg (b, d, f) or 5 mg/kg THC (h, j, l). *p < 0.05, **p < 0.01.

Fig. 3. THC effects on adolescent male and female anxiety, activity, and reward/aversion-related behaviors.

Effects of vehicle (Veh; White bars/dashed lines) or THC (0.5 mg/kg: gray bars and lines; 5 mg/kg: black bars and lines) on behaviors are shown. In adolescent female rats (a–h), (a) THC did not affect the percentage of time spent on the open arms of the elevated plus maze (conducted 30–35 min post-THC), but b) in the open field test (30–40 min post-THC), 5 mg/kg THC decreased time spent in the center of the novel environment, which is considered to be an anxiety-like phenotype. c Locomotor activity was also suppressed over the 60 min session by 5 mg/kg THC, and (d) this effect was especially prominent from 30–45 min after i.p. injection (the first 15 min of the session). e Rearing onto the hind legs was also strongly suppressed by 5 mg/kg THC, (f) again most prominently in the first 15 min of the test. g When injected 10 min prior to 30 min training sessions, neither THC dose induced either a conditioned place preference or aversion in females, (h) nor did THC affect time spent in the unpaired neutral zone in the 3-chamber apparatus. In adolescent male rats (i–p), neither THC dose affected (i) percent time on the open arms of the plus maze, (j) percent time in the center of the open field, or (k) locomotor activity in the whole session, or (l) at any timepoint. As in females, (m) rearing was suppressed by 5 mg/kg THC, (n) especially in the first 15 min of the test. o 0.5 mg/kg THC induced a place preference, while 5 mg/kg THC induced a place aversion. p Neither dose impacted time in the central neutral zone. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 4. THC effects on c-Fos expression.

In females (a–c; g–i), THC dose-dependently induced Fos immunoreactivity in several structures, as indicated by *symbol under bars that significantly differ from vehicle in that structure. Vehicle-treated rats = white bars, 0.5 mg/kg THC rats = gray bars, 5 mg/kg THC rats = black. Individual rat data is represented with dots. In males (d–f; j–l), data are represented using the same scheme. *p < 0.05, **p < 0.01, ***p < 0.001 as indicated, or adjacent to structure name in Dose main effect without significant post-hoc. BLA basolateral amygdala, CeA central amygdala, BNST bed nucleus of the stria terminalis, PLC prelimbic medial prefrontal cortex, ILC infralimbic mPFC, CA1-3 cornus ammoni regions 1-3, DG dentate gyrus, NAcC/NAcSh nucleus accumbens core/shell, RVP/CVP rostral/caudal ventral pallidum, MHb/LHb medial/lateral habenula, tVTA tail of the ventral tegmental area.

Fig. 5. THC effects on functional connectivity within reward and stress networks.

Effects of 0.5 mg/kg THC on functional coupling (left; blue lines representing strength of Fos co-activation, relative to vehicle-injected rats), or functional de-coupling (right; magenta lines representing strength of Fos de-coupling, relative to vehicle rats) is shown in females (a), and males (b). Effects of 5 mg/kg THC, relative to vehicle, are represented using the same logic in females (e), and males (f). Effects of THC doses on global network connectivity (THC-induced change in overall regional Fos cross-correlation) are shown in females (c) and males (d). *p = 0.01, ***p < 0.0001.