Adolescent substance users have also been found to exhibit abnormalities in brain function, structure, and volume. However, given the nature of human studies, it is difficult to establish a causal link between early life exposure and the development of these conditions, especially as drug co-use is not often considered and may partially explain inconsistent findings noted in prior studies. Nicotine acts in the brain via the neuronal nicotinic acetylcholine receptors, which are ligand-gated ion channels expressed on both presynaptic and postsynaptic membranes. Rodent models have shown that adolescent nicotine exposure alone may lead to behavioral alterations during adulthood. For instance, in male and female rats, adolescent nicotine enhances nicotine reward and intake during adulthood. Nicotine during adolescence has also been shown to increase depression-associated behaviors, decrease exploratory activity, and induce deficits in context conditioning to shock-associated cues in adult rats. However, in these studies, differences were not found with anxiety associated behaviors, extinction of contextual conditioning, or cued fear responses. In mice, sex dependent effects have been noted, with adolescent nicotine consumption leading to decreased anxiety-associated behaviors in adult females, but not males. With regard to cannabinoids, Δ9-tetrahydrocannabinol has been classified as the main psychoactive component in cannabis and exerts its actions on cannabinoid 1 and cannabinoid 2 receptors in the brain and periphery. Differential patterns of expression for these receptors are found across adolescent development and between males and females,drying room and notably CB1 receptors exhibit the highest level of expression during the developmental period of mid-adolescence.
Following THC administration in adolescence, adult female, but not male, rats exhibit depression-associated behaviors, but no changes in anxiety-associated or general locomotor behaviors were observed. Interestingly, the depression-associated behavioral effects found in females were paralleled by significantly reduced CB1 receptor expression and activity in the amygdala, ventral tegmental area and nucleus accumbens, whereas similar changes were not found in the ventral tegmental area and nucleus accumbens of males. Further, administration of WIN 55,212–2, a CB1 and CB2 specific agonist, during adolescence has similarly been shown to increase depressive-like behaviors, as well as palatable food intake, during adulthood in male rats. Together, these prior findings demonstrate that early life exposure to either nicotine or cannabinoid agonists alone can alter later affective and cognitive function, which introduces the possibility of potential synergistic or opposing effects under co-use conditions. In the current studies, we sought to examine whether nicotine and cannabinoid coexposure during mid-adolescence would result in altered affective and reward-seeking behavior during adulthood. While prior studies have examined each drug and/or behavioral measure independently, the current investigations represent the first study of a co-exposure condition, which is commonly found in human subjects, and the resulting effects on multiple cognitive and affective measures. To this end, adolescent mice were exposed to the cannabinoid receptor agonist, WIN55,212–2, and/or nicotine and then assessed for cognitive, anxiety-related and depression-related behaviors during adulthood.
Drug exposure occurred during postnatal day 38–49, which corresponds to mid-adolescence in rodents or ~13–17 years of age in humans. Based on prior evidence of differential responses for males and females with drug-related effects and baseline receptor expression, male and female mice were examined in a within-sex manner. Further, given that significant differences were found in behavioral measures at the moderate dose of the cannabinoid agonist, a second study was then conducted to examine whether these effects would be maintained with a lower dose of the cannabinoid agonist. Together, these studies provide evidence that adolescent drug exposure alters affective and reward-related behaviors during adulthood in a sex- and drug-dependent manner.Male and female wildtype C57BL/6J mice were derived from breeders in our laboratory animal facilities. Mice were maintained in an environmentally controlled vivarium on a 12 h reversed light/dark cycle. Food and water were provided ad libitum until behavioral training commenced. During food training, subjects were mildly food restricted to 85–90% of their free-feeding bodyweight, and water was provided ad libitum. Following food training and the lever reversal task, food and water were again provided ad libitum for at least 5 days prior to subsequent behavioral assessments. All experiments were conducted in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee at the University of California, Irvine.The cannabinoid receptor agonist WIN55,212–2 mesylate was dissolved in vehicle containing 1% DMSO, 1% Tween-80, and 98% saline . The doses of WIN55,212–2 administered were 2 or 0.2mg/kg intraperitoneally .
The moderate dose of WIN was selected based on prior studies demonstrating altered neural function with adolescent exposure in mice and rats, and the low dose of WIN was selected since this amount of drug has been shown to sustain daily reinforcing self-administration behavior in adolescent rats. -Nicotine hydrogen tartrate salt was dissolved in 0.9% sterile saline and adjusted to pH 7.4. Nicotine was administered at a dose of 0.36 mg/kg, subcutaneous ; this dose is within the rewarding range of the dose response function that also elicits a behavioral response in adolescent C57BL/6J mice. Peripheral injections were administered at a volume of 10 mL/kg.On PND 70, subjects were mildly food restricted and trained to press a lever in an operant chamber for food pellets under a fixedratio 5, time out 20 s schedule of reinforcement. Each session was performed using 2 retractable levers . Completion of the response criteria on the active lever resulted in the delivery of a food pellet. Responses on the inactive lever were recorded but had no scheduled consequences. Once stable responding was achieved , the lever assignment was switched to examine cognitive flexibility. In the reversal task, the previous inactive lever became active, in that food pellets were earned in accordance with the established FR5TO20s schedule. In contrast, the previously active lever became inactive, in which responses were recorded but without scheduled consequence. All behavioral responses were automatically recorded by Med Associates software.The elevated plus maze was composed of 4 opaque runways 5 cm wide and 35 cm in length, which were elevated 40 cm from the floor. Two opposing closed runways had opaque walls 15 cm in height, whereas the other two opposing sides did not contain walls . Subjects were placed in the center portion of the elevated plus maze and behavior was recorded for 5 min with a video camera. Behavior was scored by two blinded experimenters with ANY-maze software.Subjects were habituated to sucrose pellet consumption for 2 days prior to sucrose testing, during which time 60 mg of sucrose pellets was provided for each subject in the home cage. On the third day, subjects were individually examined in home cage conditions, but were single housed and provided 200 mg of total sucrose pellets in a dish. All subjects were maintained under ad libitum full food conditions, and thus were not food restricted during testing. Sucrose eaten was recorded at specified intervals by experimenters blinded to the group condition. At the end of each session, experimenters examined the cage for breakage or disintegration of sucrose pellets; this occurred on only a few occasions and in these instances, the remnant amount was calculated and included in the final mg amount of sucrose remaining. Mice were required to consume at least one 20mg sucrose pellet within the first 30-min time period for inclusion in the study.Subjects were examined for their daily intake of mouse chow. Mice were restricted to daily feeding sessions of 6 hr periods. During these sessions, subjects were individually housed and provided full access to consume 6–8 grams of standard chow , and water was provided in the feed cages ad libitum. Food was weighed prior to and after each session. After 3 days of habituation to the feeding protocol, data were collected on the fourth day and analyzed across groups.A cylindrical tank was filled with room temperature water at a level of 15cm from the bottom of the tank. For testing, each subject was held by the tail, and slowly placed in the water. Mice were videotaped for the 5 min swim test duration. Analysis of distance traveled was assessed with AnyMaze software,how to trim cannabis and the quantity of immobility bouts was hand scored by two separate experimenters to ensure accurate assessments.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, one-way or two-way ANOVA with Prism 7 software , as appropriate. Data obtain across sessions was 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.In an initial cohort, we assessed whether drug condition would affect change in body weight during the duration of the drug injections from postnatal day 38 to PND 49 . Change in body weight was also compared to adulthood at PND70, prior to the commencement of behavioral assessments. Groups did not differ in body weight at PND 38 following random group assignment.For both males and females, post-hoc tests revealed significant differences between the number of active and inactive lever presses for all groups from sessions 3–7, but the groups did not differ from one another when comparing responding among drug conditions on each lever. After establishing consistent responding on the active lever, cognitive flexibility was examined in the reversal task. Subjects were required to switch their lever pressing behavior, as the active and inactive lever assignments were reversed. After establishing consistent responding on the active lever, cognitive flexibility was examined in the reversal task. Subjects were required to switch their lever pressing behavior, as the active and inactive lever assignments were reversed. Given the growing incidence of nicotine and cannabis experimentation during adolescence, we sought to examine whether such exposure would lead to altered behavioral effects during adulthood. In these studies, we found that male adolescent exposure to a moderate dose of the cannabinoid receptor agonist, WIN55,212–2 , led to increased cognitive flexibility in a learning reversal task, decreased anxiety-associated behaviors, and increased natural reward consumption, but no differences in general locomotor or depression-related behavior. Interestingly, the co-exposure condition of both nicotine and the moderate dose of WIN led to similar behavioral profiles as WIN alone in these measures, suggesting that a potentiative or additive effect was not present for these behaviors. However, with regard to the number of lane crosses in the elevated plus maze, the nicotine and WIN co-exposure condition appeared to exert a counteractive effect on the WIN-induced increase in exploratory behavior at the moderate dose, suggesting an opposing effect with adolescent exposure to both drugs. With regard to females, the moderate dose of WIN induced a lower body weight during the adolescent period, but co-exposure with nicotine appeared to exert an opposing effect that resulted in no difference from the control condition. However, these effects of WIN on body weight were transitory, as the difference in females did not persist into adulthood. For the behavioral assessments, female subjects were overall more resistant to the long-term effects of adolescent drug exposure. Group differences were only found in the sucrose consumption test, in which the moderate dose WIN females exhibited decreased natural reward consumption compared to the control females. However, differences from the control were not found with the female nicotine and WIN co-exposure condition for sucrose consumption, suggesting that the presence of nicotine ameliorated the actions of WIN on reward circuitry during the adolescent period. In contrast, adolescent exposure to a low dose of WIN had no effect on physiological or behavioral measures, either alone or in the presence of nicotine, for both males and females. Taken together, these findings demonstrate that while adolescent cannabinoid agonist exposure at a moderate dose exerts variable effects on both physiological and behavioral measures in males and females, co-administration of nicotine surprisingly counteracted some of these effects by normalizing to control levels. While prior studies have examined the effects of adolescent exposure of either nicotine or WIN alone on later behaviors, the current findings represent the first examination of the effects of co-exposure during mid-adolescence and subsequent long-term effects on adult behavior. This age range was selected based on the correlation to human adolescence with higher levels of experimentation and more recurrent patterns of drug consumption than that found in younger individuals. With regard to nicotine alone, opposing effects have been found in male Sprague-Dawley rats with increased depression-associated behaviors, but no difference in anxiety-associated behaviors, during adulthood. However, these behavioral differences were only found at higher nicotine doses approximately twice that administered in the current study.