To test irritability-like behavior after WIN exposure, we used the bottle-brush test, based on the experimental method that was designed previously for mice and slightly modified to better monitor rat behavior. Currently, this model is increasingly used by both our laboratory and others as a measure of negative emotional states in animal models of addiction. This method has advantages over other behavioral paradigms that measure aggressive/ defensive behaviors, such as the social dominance/subordination paradigms and resident/intruder confrontation paradigm, because the experimenter has greater control over the mechanical stimulus and thus better precision in ensuring uniform provocation. Furthermore, the “social” factor in eliciting agonistic behavior and the risk of physical injury during an agonistic encounter are both circumvented in the bottle-brush test. The mechanical stimulus of the moving bottle-brush has also been found to be more effective in provoking these behaviors compared with either deceased or stuffed animals. In the present study, the animals were randomized, and three trained observers scored the rats’ behaviors in real-time as described below. Te observers were blinded to treatment of the animals. Testing consisted of ten 10-s trials with 10-s intertrial intervals in plastic cages with clean bedding. A bottle-brush was rotated rapidly toward the rat’s whiskers. Both aggressive responses and defensive responses were recorded. The behavioral responses were chosen based on Riittinen et al. and Lagerspetz and Portin. Total aggressive and defensive scores were calculated for each animal based on the average score of the observers. Both aggressive and defensive behaviors were summed to calculate the total irritability score. Irritability-like behavior reflects a composite measure of aggressive vs. defensive responses. Irritability-like behavior was assessed 6 days afer the last injection of WIN/vehicle in adolescence and again in adulthood 18h into withdrawal afer the escalation of cocaine self-administration .

The rats were anesthetized by isofurane inhalation, plant growing stand and intravenous catheters were aseptically inserted in the right jugular vein using a modified version of a procedure that was described previously. The right jugular vein was punctured with a 22-gauge needle, and the tubing was inserted and secured inside the vein by tying the vein with suture thread. The catheter assembly consisted of an 18 cm length of MicroRenathane tubing that was attached to a guide cannula . The guide cannula was bent at a near right angle, embedded in dental acrylic, and anchored with a mesh . The catheter exited through a small incision on the back, and the base was sealed with a small plastic cap and metal cover cap. The catheters were flushed daily with heparinized saline in 0.9% bacteriostatic sodium chloride that contained 20mg/0.2ml of the antibiotic Cefazolin .Self-administration in adulthood was performed in operant conditioning chambers that were enclosed in lit, sound-attenuating, ventilated environmental cubicles. The front door and back wall of the chambers were constructed of transparent plastic, and the other walls were opaque metal. Each chamber was equipped with two retractable levers that were located on the front panel. Cocaine was delivered through plastic catheter tubing that was connected to an infusion pump, which was activated by responses on the right lever. Responses on the left lever were recorded but did not have any scheduled consequences. Activation of the pump resulted in the delivery of 0.1 ml of cocaine . A computer controlled fluid delivery and behavioral data recording. The rats were first trained to self-administer cocaine under a fixed-ratio 1 schedule of reinforcement in daily 1-h sessions. Each active lever press resulted in the delivery of one cocaine dose. A 20-s timeout period followed each cocaine infusion. During the TO period, responses on the active lever did not have scheduled consequences. This TO period occurred concurrently with illumination of a cue light that was located above the active lever to signal delivery of the positive reinforcement. The rats were trained to self-administer cocaine in 14 sessions until a stable baseline of reinforcement was achieved .

The criterion for the acquisition of cocaine self-administration was defined as the intake of at least 2.5 mg/kg cocaine in the 1-h self-administration session, requiring at least five lever presses. This criterion was adapted from previous publications. After the 14-session acquisition period, the rats were subjected to fourteen 6-h cocaine self-administration sessions to allow them to escalate their cocaine intake. To study the motivation to seek cocaine, a progressive-ratio schedule of reinforcement was used, in which the response requirement began at one lever press/infusion and increased exponentially according to the following equation: lever presses/infusion=[5×e] − 5. The session duration was limited to 6h or ended when a rat failed to achieve the response requirement within 1h. The PR sessions were conducted after the training/acquisition phase and again after a stable level of escalation was achieved. In the course of the experiment, which lasted for more than 3 months, some rats were excluded at different stages of the experiment. Two rats were excluded during the acquisition phase because of the failure of catheter patency. In the course of the escalation phase, three rats were excluded from the study because of the failure of catheter patency at the end of the study, and one rat in the vehicle group died unexpectedly during the day of from cocaine self-administration before the study was completed, thus leaving n=6 rats/group for the final analysis. The present study found that adolescent WIN exposure increased irritability-like behavior in adolescence, which persisted into adulthood, induced cross-sensitization to the locomotor-stimulating effect of cocaine in adolescence, which did not persist into adulthood, decreased the speed of acquisition but not the rate of cocaine self-administration in adulthood, and had no effect on the escalation of cocaine self-administration in adulthood.

Overall, these results demonstrate that although cannabinoid exposure in adolescence induces irritability-like behavior and cross-sensitization to the psychostimulant effect of cocaine during adolescence, it does not promote cocaine self-administration once the animals reach adulthood. However, the effect of adolescent WIN exposure on cocaine self-administration in adolescence was not investigated in the present study because the animals reached adulthood by the time they had recovered from the surgeries that were required for self-administration. Reductions of both body weight and food intake were observed during WIN treatment. Although the activation of cannabinoid receptors typically produces an increase in food intake in adulthood accumulating evidence suggests that adolescent exposure to THC or WIN in rats decreases food intake and body weight. The increase in water intake during WIN exposure in the present study confirms the role of cannabinoid receptors in homeostatic responses that regulate not only energy homeostasis but also fluid balance. Irritability, anxiety, and dysphoria are key negative emotional states that characterize the withdrawal syndrome in humans, which arises when access to the drug is prevented and contributes to drug relapse. Irritability has also been reported to be greater in adolescents at higher risk for substance use. Irritability-like behavior has also been shown to increase during withdrawal from alcohol and nicotine in rodents. However, to our knowledge, whether early exposure to cannabinoids affects irritability-like behavior has not been studied in animal models. In the present study, we found that WIN exposure induced irritability-like behavior in adolescence and adulthood, suggesting that cannabinoid exposure in adolescence induces long-lasting neurobehavioral adaptations that can persist months after WIN exposure. However, further studies are needed to investigate whether this finding has translational relevance. An alternative explanation is that, despite blind randomization of the subjects to the two groups, the increase in irritability-like behavior that was observed in WIN-treated rats may be attributable to preexisting differences in irritability-like behavior. Further studies are needed to investigate whether this fnding has translational relevance. Numerous human studies demonstrate that early cannabis use is associated with greater vulnerability to the later development of drug addiction and psychiatric illness. A recent study reported a pivotal role for cannabinoid receptors as molecular mediators of adolescent behavior and suggested that cannabinoid receptors may be important in adolescent-onset mental health disorders. Chronic adolescent exposure to WIN has also been shown to induce anxiety-like behavior in rats. However, plant grow table contradictory findings have also been published, with either no change or even a decrease in anxiety-like behavior after cannabinoid exposure in adolescence. Rats that were exposed to cannabis smoke were also reported to exhibit a decrease in anxiety-like behavior. Interestingly, a previous study also demonstrated that long-term cognitive and behavioral dysfunction that was induced by adolescent THC exposure could be prevented by concurrent cannabidiol treatment. Importantly, WIN acts as a full cannabinoid receptor agonist, in contrast to THC, which only acts as a partial agonist. Moreover, cannabis is known to consist of dozens of additional phytocannabinoids apart from THC.

Furthermore, different strains of cannabis differ in their THC content, and THC levels in cannabis have increased year after year because of consumer demand, thus making direct comparisons of human data across time and across studies difficult. Nevertheless, we chose this model of early cannabinoid exposure and followed it precisely because it has been shown to induce cocaine cross-sensitization, thus supporting the gateway hypothesis. Further studies are needed to investigate whether the long-term irritability-like behavior that was observed in the present study can be prevented by concurrent cannabidiol treatment or whether adolescent exposure to cannabis smoke induces long-lasting irritability-like behavior in rats. Epidemiological data consistently document that cannabis exposure precedes the use of other illicit drugs. However, epidemiological data cannot provide causal evidence of this sequence. Animal models are particularly useful for studying effects that are related to cross-sensitization because they allow sequential administrations of the studied drugs while controlling for confounding variables. Several studies have reported behavioral cross-sensitization between cannabinoids and stimulants in rodents. WIN treatment during adolescence in rats induces long-lasting cross-tolerance to morphine, cocaine, and amphetamine, potentiates amphetamine-induced psychomotor sensitization, and induces cocaine-induced psychomotor sensitization in adolescence. WIN exposure also leads to increases in methylenedioxymethamphetamine-induced and cocaine-induced conditioned place preference. In the present study, WIN exposure in adolescence induced cross-sensitization to the stimulatory effect of cocaine in adolescence. However, this effect was no longer present in adulthood when the rats had self-administered cocaine for several weeks, suggesting that cannabinoid exposure in adolescence may increase the psychomotor effects of cocaine during the first exposure to cocaine, but this effect is not necessarily long-lasting. Cannabinoid exposure increased irritability-like behavior and the psychomotor effects of cocaine, but it did not promote the acquisition or escalation of cocaine self-administration. Indeed, we observed the slower acquisition of cocaine self-administration with 1-h short-access to cocaine in male rats with prior exposure to WIN compared with controls. In contrast, a previous study reported a trend toward an increase in cocaine self-administration during the short acquisition phase in female rats with prior exposure to the cannabinoid receptor agonist CP55,940 but not in male rats. However, this study did not discriminate between inactive and active levers, and no diference in cocaine self-administration was observed during the 14-day maintenance phase in either sex. A recent study showed that adolescent WIN exposure caused impairments in an attentional set-shifing task, a measure of cognitive fexibility, in adulthood. An alternative hypothesis is that the slower acquisition of cocaine self-administration in adulthood that was observed in the present study may be attributable to cognitive impairment that slows the acquisition of operant responding. In humans, several studies have indicated that the adolescent use of cannabis can lead to long-term cognitive deficits, including problems with attention and memory. During escalation, no differences were observed between the rats that were exposed to vehicle in adolescence and the rats that were exposed to WIN in adolescence. This suggests that if cognitive impairments affected the initial acquisition of self-administration, then they did not produce long-term deficits. Te model of long-access to cocaine self-administration is one of the most validated animal models of cocaine use disorder and drug addiction in general. This model has been shown to result in all seven of the diagnostic criteria of the Diagnostic and Statistical Manual of Mental Disorders, 4th edition , and seven of the 11 DSM-5 criteria, including most of the criteria that are required for severe use disorder: tolerance, withdrawal, substance taken in larger amount than intended, unsuccessful efforts to quit, considerable time spent to obtain the drug, important social, work, or recreational activities given up because of use, and continued use despite adverse consequences.