Data were analyzed by a t-test, one-way or two-way ANOVA with Prism 9 software , as appropriate. 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 two-tailed.In these studies, we sought to examine how adolescent exposure to nicotine, THC, or co-exposure may alter later reward- and relapse-related behaviors. For translational relevance to youth, THC was administered orally as related to edible consumption, and nicotine was administered via e-cigarette aerosol exposure. However, for the nicotine treatment, we aimed to compare to our prior findings with subcutaneous injections, so this additional group was included. Given the different routes of nicotine administration, we desired to first validate the respective level of nicotine’s metabolite, cotinine, for both methods. Moreover, given other findings in our lab suggesting that THC may alter nicotine metabolism [unpublished data], we also examined cotinine levels in the THC and nicotine coexposure groups. Higher blood cotinine levels were found across all nicotine-treated groups compared to the vehicle , thereby validating the measure. When comparing among drug-treated groups,vertical grow rack system co-exposure to nicotine vapor and the higher dose of THC led to lower cotinine levels as compared to nicotine vapor alone or co-exposure to nicotine vapor and the lower dose of THC .
This indicates that the high dose of THC did alter nicotine metabolism. Importantly, males exposed to only nicotine, whether via injections or vapor, did not differ in blood cotinine levels, indicating that both of these administration methods resulted in similar levels of nicotine exposure.Next, since adolescent drug exposure could possibly alter general growth, body weight was examined across the adolescent treatment days and in adulthood. Body weight was measured throughout the treatment period and in adulthood .The post-hoc analysis revealed that males exposed to either the lower or higher dose of THC, as well as those co-exposed to nicotine and the higher dose of THC , gained less weight from PND 38 to 49, as compared to control subjects. The post-hoc analysis revealed that adolescent exposure to the higher dose of THC led to lower body weight than vehicle exposure . Thus, these data indicate that adolescent exposure to a higher dose of THC, but not when co-administered with nicotine, induced persistent changes in body growth into adulthood.To examine whether adolescent drug exposure altered the subjects’ ability to learn an operant task, groups were examined for their ability to press a lever to earn food reward. Post-hoc analysis revealed that co-exposure of nicotine and lower dose of THC in adolescence led to a higher level of active lever pressing than the control group in adulthood, but only in session 3 . In session 7, males exposed to nicotine vape alone exhibited a lower level of active lever presses than the control .
With regard to subjects co-exposed to nicotine and the higher dose of THC, a higher level of active lever pressing was found for sessions 6 , 7 and 8 .Thus, to further investigate if the active lever differences are reflected in the number of food pellets obtained across sessions 6-8, we next compared the mean pellets earned. with the exception of the co-exposure nicotine and higher dose THC group that earned significantly more food pellets compared to the control. Therefore, these findings indicate that higher dose THC and nicotine co-exposure during adolescence in males induces more persistent effects on the drive to obtain food in the operant paradigm in adulthood.Next, to determine whether adolescent nicotine and/or THC exposure alters the reinforcing properties of nicotine in adulthood, mice were assessed for intravenous nicotine self-administration. Specifically, compared to control subjects, significantly more nicotine infusions were earned following adolescent exposure to the lower and higher dose of THC , and co-exposure to nicotine and the lower dose of THC . Given these findings with THC altering later nicotine intake, it is surprising to note that differences were not found with co-exposure to nicotine and the higher dose of THC, even though this group exhibited a greater drive to obtain food reward. Taken together, these results indicate that adolescent use of cannabinoids have a persistent effect on reward consumption, which is dependent on THC dose, nicotine co-exposure, and type of reward.Since drugs of abuse may differentially alter development dependent on sex, we next examined whether adolescent nicotine, THC, or co-exposure in females results in similar physiological and behavioral outcomes in adulthood.As above, all of the nicotine treatment groups resulted in a significant level of detectable cotinine .
When comparing among treatment conditions, injections of nicotine resulted in lower cotinine levels than nicotine vapor and co-exposure of nicotine and low dose THC , although it is important to note that all of these groups exhibited levels of cotinine >50 ng/ml which is in the range of that found with human e-cigarette and tobacco smokers. Interestingly, similar to that observed in males, females exhibited significantly lower cotinine levels with nicotine vapor and the higher dose of THC as compared to nicotine vapor alone or co-exposure to nicotine vapor and the lower dose of THC , suggesting that the high dose of THC interacts with nicotine metabolism. Posthoc analyses revealed that the females gained less weight from PND 38 to 49 if exposed to either the lower or higher dose of THC or coexposed to nicotine and the higher dose of THC , compared to vehicle. The posthoc test revealed that the high dose of THC, either in the absence or presence of nicotine , led to decreased body weight differences that were maintained into adulthood, compared to vehicle. Even so, a trend was noted with higher dose of THC potentially resulting in a lower body weight than vehicle . Together, these data indicate that a higher dose of THC during adolescence may have persistent developmental effects on body growth.We next focused our investigations of operant food training in the female mice. The post-hoc analysis revealed that females exposed to the higher dose of THC exhibited a higher level of active lever pressing for sessions 7 and 8 compared to control. Further, co-exposure to nicotine and the lower dose of THC resulted in greater active lever pressing across sessions 6 and 7 , but no difference on the final session 8 compared to the control. Thus, these findings indicate that regardless of adolescent exposure, females were able to acquire the food training task,grow rack with lights although high dose THC may have led to increased responding to obtain food pellets in later sessions, an effect not found with nicotine co-exposure. We then examined intravenous nicotine self-administration during adulthood in female subjects with a history of drug exposure.The post-hoc analysis revealed that a higher dose of THC during adolescence led to increased nicotine intake in adulthood compared to vehicle . While not statistically significant, we also noted a trend with the co-exposure nicotine and lower dose of THC group having higher intake compared to control . Taken together, these findings reveal that a high dose of THC during adolescence increases the drive to consume both food and nicotine in adulthood, an effect which appears to have been counteracted by the co-exposure of nicotine.Re-exposure to the auditory, visual, and/or olfactory cues associated with drug taking has been shown to enhance relapse-related behaviors. Thus, after intravenous nicotine self-administration acquisition, we examined lever pressing behavior for a visual and auditory cue in the absence of nicotine infusions. Since this procedure has been mainly used in rats, it was important to first demonstrate that control subjects could exhibit a robust incubation of nicotine craving effect, as validation of this protocol in mice. For our analysis, we also included comparisons of active lever pressing that correspond to the nicotine self-administration data presented in Figures 1E and 2D .
These data were important to include to determine whether the mice exhibited an extinction burst on the first day of incubation testing , which could have implications for interpretation of the later incubation effect on Day 24. Therefore, the post-hoc analysis compared incubation day 1 to the other sessions . In the post-hoc analysis, there was a significant increase in active lever pressing comparing incubation Day 1 to Day 24 for both males and females . However, active lever pressing did not differ when comparing responding for nicotine infusions to incubation Day 1 . Thus, these findings demonstrate that incubation of nicotine craving can be readily detected in mice. For the nicotine vapor group, the post-hoc analysis revealed an increase in active lever pressing on Day 24, as compared to Day 1, of incubation , but no differences were found comparing Nicotine to Day 1. This effect was interesting given that these groups did not differ in the level of cotinine, suggesting that the differences in duration of daily adolescent exposure may be relevant. While both of the ANOVAs indicated a statistically significant effect, post-hoc analyses did not reveal significant differences among sessions, although with nicotine vapor exposure a trend was noted between the Nicotine and Day 1 sessions suggesting a potential burst in responding. Given the differences found in body weight and food training with some of the THC exposure groups, we predicted that significant differences would also be found for incubation of craving. These findings suggest that the higher dose of THC during adolescence may have led to overall increased active lever pressing, suggesting either overall increased general activity , or alternatively, a premature incubation effect with higher immediate and persistent drug seeking behavior.Finally, we sought to determine whether nicotine and THC together would have unique effects on relapse-related behaviors. Surprisingly, co-exposure elicited differential outcomes compared to what we previously reported for single drug exposure.For all of the above co-exposure comparisons, statistically significant differences were not found between baseline Nicotine and incubation Day 1. Together, these findings indicate that nicotine and THC can interact to induce a differential effect than either substance alone during development, thereby sustaining a heightened response to drug associated cues to propagate increased nicotine seeking behavior and potential risk of relapse.This study sought to determine whether prior nicotine and/or THC exposure during adolescence would alter operant learning, drug reinforcement, and nicotine seeking behaviors. Importantly, we found that nicotine exposure in adolescence regardless of route of administration resulted in significantly high levels of cotinine in both sexes; but coexposure with the higher dose of THC altered the metabolism of nicotine as evidenced by significantly lower cotinine levels in these subjects than those exposed to nicotine alone. Males that were co-exposed to nicotine and the higher dose of THC in adolescence also exhibited increased food self-administration in adulthood. In contrast, none of the female groups differed in food self-administration. Furthermore, males that were exposed to either dose of THC alone in adolescence or co-exposed to nicotine and the lower dose of THC had increased nicotine intake in adulthood. Whereas females with adolescent exposure to only the higher dose of THC exhibited increased nicotine intake in adulthood. Following nicotine self-administration, both male and female control mice exhibited increased nicotine-seeking behaviors following a 24-day abstinence period. Males exposed to nicotine vapor or either dose of THC alone also demonstrated this increased cue-induced nicotine seeking. However, adolescent exposure to nicotine via injections in either sex or to nicotine vapor in females did not result in this later enhanced nicotine seeking behavior. Interestingly, for both sexes, co-exposure to nicotine and THC at either dose in adolescence does result in this incubation of nicotine craving effect in adulthood, even when single drug exposure does not. Nicotine and cannabis use during adolescence has been shown to have lasting implications on later learning and memory. However, our findings did not reveal any differences in the subjects’ abilities to learn the operant food training task in either sex. All groups were able to sufficiently dissociate between the active and inactive levers during training and further achieved the lever pressing criteria within a similar number of sessions. Rather, differences in lever pressing behavior were only found in later sessions once the learning already occurred. Males co-exposed to the higher dose THC and nicotine during adolescence as well as females exposed to the higher dose of THC alone demonstrated a higher level of responding for this task and maintained a more persistent drive to obtain food, which may be indicative of greater hedonic value of food for these subjects.