Given these findings, it is possible that patients may differentially respond to pharmacotherapeutics based on developmental drug exposure, representing a potential underlying factor mitigating individual differences in cessation outcomes. Indeed, given that we found differences in nicotine intake during adulthood with developmental drug exposure, and currently available pharmacotherapeutics such as varenicline also target nAChRs, similar signaling mechanisms may be involved in mitigating the behavioral responses to these drug compounds. Finally, in these studies, we examined the effects of an injected cannabinoid agonist during adolescent development on nicotine self-administration in adulthood. Importantly, these results have direct implications for the use of “spice” synthetic cannabinoids, of which the majority belong to the aminoalkylindole class, including WIN55,212-2. In addition, these findings likely have further implications for cannabis exposure. Δ9- Tetrahydrocannabinol has been characterized as a partial agonist of the CB1R, and therefore, it is possible that the low dose of the WIN agonist could have resulted in the occupation of a fewer number of receptors,hydroponics flood tray thereby inducing an effect more similar to a higher dose of a partial agonist on downstream cellular signaling.
However, this will need to be more systematically addressed in future studies. Moreover, while it is possible that volitional intake during adolescence may differentially alter drug reinforcement, rather than experimenter administered injections, there are some caveats to such an experimental design. First, it is not yet feasible to implant intravenous catheters in adolescent mice. Second, the dose that each animal receives cannot be discretely controlled with self administration studies. This point is further compounded by the fact that coexposure conditions result in different intake amounts of each drug, as compared with single use conditions. Furthermore, both THC and WIN self-administration in rodent models have been difficult to establish in many labs, although some have been successful due to specific doses and reinforcement testing paradigms. In particular, for THC in rats, the combined presence of cannabidiol sustained self-administration behavior in both intravenous and vapor paradigms. This is interesting given that many THC e-cigarette vapes on the market do not contain cannabidiol, at least as indicated on commercial packaging. Given these considerations and with the foundational findings derived from the current studies, it will nevertheless be important in future studies to develop models for volitional adolescent nicotine and cannabinoid self-administration, perhaps via vapor exposure, and then to determine whether the variable, self-titrated levels of each drug differentially impacts nicotine and/or cannabinoid self-administration in adulthood.Nicotine dependence remains the leading cause of preventable death worldwide with over 1.3 billion current users. Nicotine can be consumed in many forms including tobacco cigarettes and in recent years, electronic nicotine delivery systems .
ENDS involve the heating of nicotine-containing liquid to produce an aerosol that users inhale similarly to tobacco smoke. The use of ENDS, also known as e-cigarettes or nicotine vape pens, has drastically increased over the past decade, especially among youth, whereas tobacco cigarette use has declined. Of further concern, adolescent nicotine exposure increases the risk of developing nicotine dependence and other substance use disorders, including cannabis use disorder. Cannabis is the most abused illicit drug with over 200 million people using it around the world. The main psychoactive component in cannabis is Δ9-tetrahydrocannabinol . THC is consumed by humans in many forms including orally in THC-containing foods or drinks known as edibles. Edible THC is appealing to many users because it can be consumed without the smoke of traditional cannabis use and the lipids in food make drug absorption easier. When consumed orally, it has been shown to take at least 30 minutes to see a significant rise in blood THC levels and the gradual return to baseline lasts six hours post-ingestion in humans. Because edibles require a longer time before users begin to feel the effects and the concentrations of THC in these products can be much higher, it is easy for inexperienced users, particularly youth, to consume more THC than intended. Beyond single drug use, co-use of multiple drugs like both cannabis and nicotine containing products is frequent. Around 60% of cigarette smokers report ever using cannabis, and 90% of cannabis users report ever smoking cigarettes in their lifetime.
Youth and young adults who co-use both report consuming more cannabis and nicotine annually. People suffering from these individual and co-occurring substance use disorders may try to abstain from taking the drugs, but relapse is a major concern as most people begin smoking again within the first week. Drug relapse and craving during abstinence has been shown to be triggered by certain cues associated with drug-taking. In humans, nicotine craving in response to nicotine-associated cues increases over time during abstinence. These cues can include the physical environment, people with whom the drug taking typically occurs, as well as any associated visual, auditory, and olfactory sensory perceptions. This behavior is depicted in animal models of research as the incubation of craving paradigm in which cue-induced drug-seeking behavior increases over time during abstinence following drug self administration. In rats, this has been demonstrated for several drugs of abuse, including heroin, alcohol, and nicotine. Although these studies have established important findings for single drug use, it remains to be determined whether polydrug use alters the incubation response and whether similar effects are found for the incubation of nicotine craving in mice. The studies outlined below examined how this incubation of nicotine craving behavior, as well as operant learning and later drug intake, are altered dependent on prior drug exposure. Specifically, adolescent male and female mice were exposed to nicotine via injections or vapor, a lower or higher dose of THC, or both, and assessed in adulthood for differences in operant learning, nicotine intake, and relapse-related drug seeking behaviors. The two routes of administration for nicotine are used because injections have been one of the most common methods of drug exposure in adolescent mice, which allows for comparisons to other studies, whereas the nicotine vapor exposure is highly translational to the current use of ENDS products. Moreover, two doses of THC are administered to account for dose-dependent effects of oral consumption. We proposed that adolescent single and poly-drug exposure alters later nicotine intake and susceptibility to cue-induced relapse in adulthood. The findings from this study can inform how previous drug history can impact the effectiveness of relapse interventions for those in the early stages of abstinence.Male and female wild type C57BL/6J mice were derived from breeders in our laboratory animal facilities. In total 194 mice were assessed in these studies. Mice were maintained in an environmentally controlled vivarium on a 12 h reversed light/dark cycle. Food and water were provided ad libitum until postnatal day 70, at which time subjects were mildly food restricted to 85–90% of their free-feeding body weight for behavioral assessments, while water was continued to be provided ad libitum. All experiments were conducted in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at the University of California, Irvine.For the nicotine injections, -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 considered to be 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. For the aerosolized vape nicotine, -Nicotine hydrogen tartrate salt was dissolved in 50% propylene glycol and 50% glycerin for the 7.5mg/mL concentration and adjusted to pH 7.4 . Our prior study has shown that this concentration elicits significant levels of the nicotine metabolite, cotinine,hydro flood table in the blood of rodents exposed to nicotine vapor. Nicotine or vehicle vapor was administered using LJARI vapor chambers which were programmed to administer 12 puffs of vapor across a one-hour session.
Each vapor administration was programmed for five seconds and air flow was regulated at 1 L/min. THC in ethanol was obtained from the NIH NIDA Drug Distribution Program. Ethanol was first evaporated out under nitrogen and diluted in sesame oil vehicle for oral gavage administration at a volume of ~0.1 ml, adjusted based on body weight. The oral 5 and 10 mg/kg doses are considered moderate and moderately high doses, with other reports in the literature examining up to 20 mg/kg without significant adverse effects [PMID 34652500]. The oral 5 and 10 mg/kg doses have been found to be behaviorally effective in reducing locomotion to 20-50% of baseline levels, and mice will readily consume THC orally at these doses. For these studies, the THC was diluted at 5 mg/kg or 10 mg/kg in sesame oil and was administered via oral gavage at a concentration of 1 mg/ml and 2 mg/ml respectively. The experimental paradigm is outlined in Figure 3.1A. Beginning on postnatal day 38, male and female mice were randomly subdivided into seven experimental groups: Control: saline injections or oral sesame oil and vehicle vape, NIC SC: nicotine injections , NIC Vapor: oral sesame oil and aerosolized nicotine , THC: oral THC and vehicle vape, hTHC: oral THC and vehicle vape, THC/NIC: oral THC and nicotine vapor , and hTHC/NIC: oral THC and nicotine vapor . Mice received treatments across 12 consecutive days from PND 38 to PND 49, and oral vehicle or THC was administered 30 min prior to the 1hr vape sessions. Body weight was recorded prior to each drug exposure. On PND 49, for cotinine analysis, blood was collected randomly from a subset of subjects from each nicotine-exposed experimental group twenty minutes after the last nicotine injection or 1 hr vapor session. Subjects were all tested in multiple smaller cohorts with randomly assigned drug exposure conditions to enhance rigor and reproducibility of the findings. Once stable responding was achieved during operant food training, subjects were put back on full food for a minimum of three days before being surgically catheterized as previously described. Briefly, mice were anesthetized with an isoflurane /oxygen vapor mixture and prepared with intravenous catheters. 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, subjects 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 one hour daily sessions, six days per week, at the standard training dose of nicotine in the same operant chambers as used in food training. Completion of the response criteria on the active lever resulted in the delivery of an intravenous nicotine infusion and a cue light indicating reward delivery. Responses on the inactive lever were recorded but had no scheduled consequences. Mice are permitted to self-administer nicotine for eight days on the lower 0.03 mg/kg/infusion nicotine dose and five days on the moderate 0.1 mg/kg/infusion nicotine dose, which has previously been shown to permit consistent titration of nicotine responding in mice. Behavioral responses were automatically recorded by Med Associates software. Catheters were flushed daily with physiological sterile saline solution containing heparin . To assess nicotine seeking behavior during a state of abstinence, mice began the incubation of nicotine craving paradigm following the final day of intravenous nicotine self administration at the 0.1 mg/kg/infusion dose. One day and 24 days after the last nicotine self-administration session, mice were placed back into the operant chamber and allowed to lever press while experiencing the same sensory cues as during nicotine self-adminstration. However, only the cue light and sound of the nicotine pump were earned after five active lever presses, in the absence of any nicotine infusions. Following the Day 1 incubation of craving assessment, catheter integrity was tested with the short-acting barbiturate anesthetic Brevital to ensure catheters maintained functionality throughout the self-administration period and mice were properly receiving nicotine infusions.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.