Catheters consisted of a 6 cm length of silastic tubing fitted to guide cannula bent at a curved right angle and encased in dental acrylic. The catheter tubing was passed subcutaneously from the animal’s back to the right jugular vein, and a 1 cm length of the catheter tip was inserted into the vein and tied with surgical silk suture. Following the surgical procedure, animals were allowed ≥48 hours to recover from surgery, then provided access to again respond for food reward. Mice were then permitted to acquire intravenous nicotine self-administration during 1 hour daily sessions, 6–7 days per week, at the standard training dose of nicotine . Each session was performed using two retractable levers . Completion of the response criteria on the active lever resulted in the delivery of an intravenous nicotine infusion . Responses on the inactive lever were recorded but had no scheduled consequences. Catheters were flushed daily with physiological sterile saline solution containing heparin . Catheter integrity was tested with the short-acting barbiturate anesthetic Brevital . Subjects and their data were removed from the study due to death or if the catheter integrity was compromised as determined by visual leakage or Brevital assessment. Behavioral responses were automatically recorded by Med Associates software.The experimental design is outlined in Figure 2.1. Following adolescent injections, mice remained drug-free until adulthood . Thereafter, they were examined for differences in cognitive behavior as reported previously. For these investigations, to ascertain the dose–response function,flood drain tray mice were tested according to the established mouse intravenous self-administration protocol.
Following an acquisition period of at least 7 days on the training dose , the animals were presented with a different dose of nicotine for at least 5 days, and the mean intake for the last two sessions was used for statistical analyses. In between each dose, subjects were returned to the 0.1 mg/kg/infusion dose for 2 days or until intake returned to baseline levels. The dose– response function occurred over a total ~35 sessions with testing sessions occurring 6 days per week. Thereafter, mice were stabilized on the moderate 0.1 mg/kg/infusion dose across three baseline sessions after successfully passing the Brevital catheter patency test. Then, subjects were challenged with an injection of the low dose WIN or vehicle control, 20 minutes prior to the nicotine self-administration session. Injections of vehicle or low dose WIN were administered in a random, counterbalanced design both within and across groups, and subjects were permitted at least 2 baseline days in between WIN/vehicle administration to return to baseline levels of nicotine intake. After the crossover experiment with the single, acute dose of WIN, mice were chronically pretreated with the same low WIN dose prior to each session across seven consecutive sessions, and nicotine intake on the seventh session was used to determine the effects of chronic coexposure during adulthood for all groups. Since the control groups for the moderate and low dose WIN cohorts exhibited similar effects with pretreatment, data were compiled into one graph for each sex. Finally, mice were again returned to self-administer the 0.1 mg/kg/infusion dose, and after achieving baseline levels of responding, they were then transitioned to respond for saline infusions . Eleven mice were required to be excluded due to death/cannibalization by cagemates , and six were excluded due to compromised catheter integrity .
Given that these studies sought to investigate the effects of drug exposure relative to the control condition within each sex, statistical comparisons were performed separately for males and females based on this a priori hypothesis. Data were analyzed by a t test, oneway or two-way analysis of variance with Prism 7 software , as appropriate. Data obtained across sessions were analyzed with a repeated measures two-way ANOVA. Significant main or interaction effects were followed by Bonferroni post hoc comparison with correction for multiple comparisons. The criterion for significance was set at α = 0.05.Adolescent exposure groups were examined for differences in nicotine intake during adulthood across low, moderate, and high self-administration doses . This approach allows for the assessment of the dose–response function, which provides a measure of responding across nicotine doses with increasing value of reinforcement and doses inducing greater aversion and/or satiation. Specifically, the WIN and nicotine/WIN adolescent exposure groups exhibited a significantly increased number of nicotine infusions compared with the control and nicotine adolescent exposure groups . Further, the groups did not differ in their saline level of responding, indicating that these differences were not due to a general increase in lever pressing behavior. Since both the WIN and nicotine/WIN exposure conditions involved a moderate dose of the cannabinoid agonist , we next addressed the possibility that this WIN dose could have masked the effects of nicotine in an interactive effect. Thus, we examined a separate cohort of mice exposed to a low dose of WIN , either in the presence or absence of nicotine. The post hoc analysis revealed an upward shift in the dose–response function for the nicotine exposure group, as compared with both the WIN and coexposure nicotine and WIN groups.
Specifically, at the 0.03 mg/kg/infusion dose, the adolescent nicotine group exhibited a significantly greater number of nicotine infusions than the adolescent WIN and nicotine/WIN coexposure groups. At the moderate 0.1 mg/kg/infusion dose, the nicotine group also demonstrated a statistically significant increase from the WIN group and nicotine/WIN coexposure group , and the nicotine/WIN coexposure group was also significantly decreased compared with the control group . No other groups significantly differed from the control, or at the saline and high dose of nicotine . Thereafter, a second set of female mice were examined for differences with the lower dose of WIN. To examine whether further exposure in adulthood to a cannabinoid agonist subsequently alters nicotine intake, mice were pretreated with the low dose of the cannabinoid agonist or vehicle prior to a nicotine self-administration session; thereafter, the mice were then repeatedly administered the low dose of the cannabinoid agonist prior to seven consecutive nicotine self-administration sessions . Interestingly, when we examined the adolescent-exposed nicotine and WIN groups, a stark contrast in responding was evidenced.Specifically, in post hoc analyses, the vehicle condition exhibited a higher level of nicotine infusions compared with pretreatment with the cannabinoid after one session and after seven consecutive sessions . Further, chronic administration of the cannabinoid agonist significantly reduced nicotine intake to a greater extent than the acute condition .In these studies, we found that adolescent cannabinoid and/or nicotine exposure exert a lasting impact on susceptibility to drug reinforcement, which is evidenced in adulthood. However, these effects were dependent on the substance of abuse , dose of the cannabinoid, and sex. Specifically, adult males exhibited increased nicotine self-administration at the lower rewarding nicotine dose following adolescent cannabinoid agonist exposure at the moderate dose , either alone or with nicotine coadministration. In contrast, adult females demonstrated an opposing effect following adolescent cannabinoid exposure at the moderate dose, in which such exposure resulted in decreased nicotine intake compared with nicotine exposure alone. However, differences were not induced within either sex with adolescent exposure to the lower dose of the cannabinoid agonist . Furthermore, after maintaining nicotine self administration, pretreatment with the low dose of the cannabinoid agonist attenuated nicotine intake in both male and female control mice,flood and drain tray and this lowering effect was evidenced both acutely and after chronic pairings. Surprisingly, the cannabinoid agonist was ineffective in altering nicotine intake in mice previously exposed to nicotine, the cannabinoid agonist, or both during adolescence; an effect that was present at both the lower and moderate doses of the cannabinoid agonist.Nicotine self-administration produces an inverted U-shaped dose–response curve, which represents the competing positive and negative properties of nicotine. The increased responding for nicotine over the ascending limb of the curve reflects the increasing reinforcing effects of nicotine as the unit dose increases. In contrast, the decreased responding over the descending limb of the curve reflects the increasing aversive properties of nicotine or satiation. Mesolimbic dopamine neurons have been primarily implicated in modulating the rewarding and reinforcing aspects of the drug, whereas the aversive signaling of nicotine appears to involve the habenulo-interpeduncular pathway.
Our findings suggest that adolescent cannabinoid exposure most likely alters the function of the mesolimbic pathway, as differences were found primarily on the ascending limb of the dose– response function. In support of this notion, adolescent cannabinoid or nicotine exposure has previously been shown to alter monoaminergic signaling.However, in our study, nicotine alone was ineffective in altering later drug-taking behaviors in males, either in combination with the cannabinoid or alone. Since studies have shown that of those adolescents age 12–17 who smoke, the majority smoke one or less than one cigarette per day , the current studies focused on a rewarding dose with once daily exposure of a rewarding dose. Thus, the current results have particular relevance to experimental patterns of drug consumption found in youth. Differential patterns of expression of the cannabinoid receptors are found across adolescent development and between males and females, and CB1Rs exhibit highest level of expression during the developmental period of mid-adolescence. Thus, the potential for exogenous cannabinoids to alter synaptic and neural circuit function may be considered greatest during this time period. Indeed, prior studies have revealed an effect of CB1R activation on adolescent brain development and indicate a correlation between adolescent exposure and later cognition and reward-related function. For instance, we found that adult males exposed during adolescence to the moderate 2 mg/kg dose of WIN exhibited increased cognitive flexibility in a learning reversal task, decreased anxiety associated behaviors, and increased natural reward consumption with the same exposure paradigm. The coexposure condition of both nicotine and the moderate dose of WIN also led to similar behavioral profiles as WIN alone in these measures, suggesting that a potentiative or additive effect was not present similar to that found in the current studies with nicotine intake. With regard to females, they were found to be overall more resistant to the long-term effects of adolescent drug exposure, in which the moderate dose WIN females exhibited decreased natural reward consumption compared with the control females. Interestingly, CB1R knockout mice are resistant to nicotine-mediated locomotion and conditioned place preference, but do not differ in nicotine self-administration, as compared with wild-type mice, which suggests that the lack of CB1Rs during adulthood may affect generalized locomotor behavior and drug-conditioned memory function, but perhaps not the motivation to consume nicotine. However, given the constitutive knockout of the gene in these mice, it is possible that compensatory mechanisms occurred during development, resulting in altered expression of other receptors, potentially including cannabinoid 2 receptors and/or nAChRs.Both single and co-use of nicotine and cannabinoid products are prevalent during adolescence and adulthood. Thus, we further examined coexposure during adulthood, under both control conditions and following adolescent drug exposure. In the control group, we found that the low dose of the cannabinoid agonist reduced nicotine intake in adulthood. This represents the first demonstration of the effects of a cannabinoid agonist on intravenous nicotine self-administration in mice. These results were surprising since the CB1 receptor antagonists rimonabant and taranabant have also been shown to reduce nicotine consumption. However, when one considers that additive effects may be induced on brain reward circuitries, such as that found with reduced brain stimulation thresholds in the presence of rewarding doses of nicotine, it is likely that the presence of the cannabinoid agonist augmented the activity of the reward circuits in the brain, leading to a reduction in nicotine intake while maintaining similar circuit activation to support drug reinforcement. However, this stipulation will need to be more directly tested in future studies. We further found that the effectiveness of the cannabinoid agonist in reducing nicotine self-administration is dependent on prior drug exposure during adolescence, as all of the adolescent nicotine and/or WIN exposure groups did not differ in nicotine intake with WIN pretreatment in adulthood. Of further note, we found that this lack of responsiveness to the dampening effects of the cannabinoid agonist on nicotine intake also occurred in adolescent groups exposed to the low dose of the cannabinoid agonist. It is important to note that this level of exposure did not induce any other detectable behavioral effects during adulthood, either in this study or in our prior analysis of cognitive, anxiety-, and depression associated behaviors.