Withdrawal from repeated nicotine vapor exposure increases somatic signs of physical dependence, anxiety-like behavior, and brain reward thresholds in adult male rats.

Nicotine vapor consumption via electronic nicotine delivery systems has increased over the last decade. While prior work has shed light on the health effects of nicotine vapor inhalation, its unique effects on the brain and behavior have not been thoroughly explored. In this study we assessed markers of withdrawal following 14 days of nicotine vapor exposure. For Experiment 1, 21 adult male rats were exposed to ambient air or 6, 12, or 24 mg/mL nicotine vapor for 14 consecutive days. Following exposure on day 14, rats were injected with the nicotinic receptor antagonist mecamylamine (3.0 mg/mL) and assessed for somatic withdrawal signs and anxiety-like behavior in the elevated plus maze. For Experiment 2, 12 adult male rats were tested for intracranial self-stimulation (ICSS) immediately following exposure to vehicle vapor (50%/50%, vegetable glycerin/propylene glycol) or 24 mg/mL nicotine vapor, for 14 consecutive days. ICSS behavior was assessed for an additional 14 days, following cessation of repeated vapor exposure. Results reveal that rats with repeated nicotine vapor exposure display an increase in behavioral indicators of withdrawal following injection of mecamylamine (precipitated withdrawal). Additionally, increases in ICSS stimulation thresholds, indicative of reduced brain reward sensitivity, persist following cessation of repeated nicotine vapor exposure (spontaneous withdrawal). These data suggest that repeated e-cigarette use leads to nicotine dependence and withdrawal that affects behavior and brain reward function. Further characterization of the health effects of nicotine vapor is necessary to improve treatment strategies for nicotine use disorder and public health policies related to novel nicotine delivery systems.

Increased Risky Choice and Reduced CHRNB2 Expression in Adult Male Rats Exposed to Nicotine Vapor.

While the cognitive enhancing effects of nicotine use have been well documented, it has also been shown to impair decision making. The goal of this study was to determine if exposure to nicotine vapor increases risky decision making. The study also aims to investigate possible long-term effects of nicotine vapor exposure on the expression of genes coding for cholinergic and dopaminergic receptors in brain. Thirty-two adult male Sprague Dawley rats were exposed to 24 mg/mL nicotine vapor or vehicle control, immediately followed by testing in the probability discounting task for 10 consecutive days. Fifty-four days after the 10-day vapor exposure, animals were sacrificed and expression of genes coding for the α4 and ß2 cholinergic receptor subunits, and dopamine D1 and D2 receptors, were analyzed using RT-PCR. Exposure to nicotine vapor caused an immediate and transient increase in risky choice. Analyses of gene expression identified significant reductions in CHRNB2 and DRD1 in the nucleus accumbens core and CHRNB2 and DRD2 in the medial prefrontal cortex of rats previously exposed to nicotine vapor, relative to vehicle controls. Results provide data on the negative cognitive effects of nicotine vapor exposure and identify cholinergic and dopaminergic mechanisms that may affected with repeated use.

Amino acid systems in the interpeduncular nucleus are altered in a sex-dependent manner during nicotine withdrawal.

Prior work in male rodents established that the medial habenula-interpeduncular nucleus (MHb-IPN) pathway modulates nicotine withdrawal. Specifically, withdrawal severity has been closely associated with inhibitory tone in the IPN via interneurons that release γ-aminobutyric acid (GABA). Inhibitory tone in the IPN is regulated by projections from the MHb that co-release glutamate and acetylcholine. Within the IPN, inhibitory tone is also regulated via corticotropin-releasing factor type 1 (CRF1) receptors that control GABA release from local interneurons. This study extends previous work by comparing sex differences in GABA, glutamate, as well serotonin levels in the IPN during precipitated nicotine withdrawal. Sex differences in withdrawal-induced neurochemical effects were also compared following systemic administration of a CRF1 receptor antagonist. The results revealed that there were no group differences in serotonin levels in the IPN. A major finding was that females displayed a larger withdrawal-induced increases in GABA levels in the IPN than males. Also, withdrawal increased IPN glutamate levels in a similar manner in females and males. Blockade of CRF1 receptors produced a larger suppression of the withdrawal-induced increases in GABA levels in the IPN of females versus males, an effect that was likely related to the robust increase in glutamate following administration of the CRF1 receptor antagonist in females. These data suggest that amino acid systems in the IPN modulate sex differences in the behavioral effects of nicotine withdrawal. Furthermore, our data imply that medications that target stress-induced activation of the IPN may reduce withdrawal severity, particularly in females.

The emergence of insulin resistance following a chronic high-fat diet regimen coincides with an increase in the reinforcing effects of nicotine in a sex-dependent manner.

The present study assessed the sex-dependent effects of insulin resistance on the reinforcing effects of nicotine. Female and male rats received a chronic high-fat diet (HFD) or regular diet (RD) for 8 weeks. A subset of rats then received vehicle or a dose of streptozotocin (STZ; 25 mg/kg) that induces insulin resistance. To assess insulin resistance, glucose levels were measured 15, 30, 60, 120, and 180 min after an insulin injection (0.75 U/kg). Nine days later, the rats were given extended access to intravenous self-administration (IVSA) of nicotine (0.015, 0.03, 0.06 mg/kg) in an operant box where they consumed their respective diet ad libitum and performed responses for water deliveries. Each nicotine dose was delivered for 4 days with 3 intermittent days of abstinence in their home cage. The day after the last IVSA session, physical signs were compared following administration of mecamylamine (3.0 mg/kg) to precipitate nicotine withdrawal. The results revealed that there were no changes in insulin resistance or nicotine intake in HFD alone rats regardless of sex. Insulin resistance was observed in HFD-fed rats that received STZ, and the magnitude of this effect was greater in males versus females. Our major finding was that nicotine intake was greater among HFD + STZ female rats as compared to males. Lastly, the physical signs of withdrawal were similar across all groups. Our results suggest that females diagnosed with disorders that disrupt insulin signaling, such as diabetes may be at risk of greater vulnerability to nicotine use due to enhanced reinforcing effects of this drug.

Estradiol promotes and progesterone reduces anxiety-like behavior produced by nicotine withdrawal in female rats.

This study assessed sex differences and the role of ovarian hormones in nicotine withdrawal. Study 1 compared physical signs, anxiety-like behavior, and corticosterone levels in male, intact female, and ovariectomized (OVX) female rats during nicotine withdrawal. Estradiol (E2) and progesterone levels were also assessed in intact females that were tested during different phases of the 4-day estrous cycle. Study 2 assessed the role of ovarian hormones in withdrawal by comparing the same measures in OVX rats that received vehicle, E2, or E2+progesterone prior to testing. Briefly, rats received a sham surgery or an ovariectomy procedure. Fifteen days later, rats were prepared with a pump that delivered nicotine for 14 days. On the test day, rats received saline or the nicotinic receptor antagonist, mecamylamine to precipitate withdrawal. Physical signs and anxiety-like behavior were assessed on the elevated plus maze (EPM) and light-dark transfer (LDT) tests. During withdrawal, intact females displayed greater anxiety-like behavior and increases in corticosterone levels as compared to male and OVX rats. Females tested in the estrus phase (when E2 is relatively low) displayed less anxiety-like behavior and had lower corticosterone levels versus all other phases. Anxiety-like behavior and corticosterone levels were positively correlated with E2 and negatively correlated with progesterone levels. Intact females displaying high E2/low progesterone showed greater anxiety-like behavior and corticosterone levels as compared to females displaying low E2/high progesterone. Lastly, OVX-E2 rats displayed greater anxiety-like behavior than OVX-E2+progesterone rats. These data suggest that E2 promotes and progesterone reduces anxiety-like behavior produced by nicotine withdrawal.

Overexpression of corticotropin-releasing factor in the nucleus accumbens enhances the reinforcing effects of nicotine in intact female versus male and ovariectomized female rats.

This study assessed the role of stress systems in the nucleus accumbens (NAc) in promoting sex differences in the reinforcing effects of nicotine. Intravenous self-administration (IVSA) of various doses of nicotine was compared following overexpression of corticotropin-releasing factor (CRF) in the NAc of female and male rats. Ovariectomized (OVX) females were also included to assess the role of ovarian hormones in promoting nicotine reinforcement. Rats received intra-NAc administration of an adeno-associated vector that overexpressed CRF (AAV2/5-CRF) or green fluorescent protein (AAV2/5-GFP). All rats were then given extended access (23 h/day) to an inactive and an active lever that delivered nicotine. Separate groups of rats received intra-NAc AAV2/5-CRF and saline IVSA. Rats were also allowed to nose-poke for food and water during IVSA testing. At the end of the study, the NAc was dissected and rt-qPCR methods were used to estimate CRF overexpression and changes in CRF receptors (CRFr1, CRFr2) and the CRF receptor internalizing protein, ß-arrestin2 (Arrb2). Overexpression of CRF in the NAc increased nicotine IVSA to a larger extent in intact female versus male and OVX females. Food intake was increased to a larger extent in intact and OVX females as compared to males. The increase in CRF gene expression was similar across all groups; however, in females, overexpression of CRF resulted in a larger increase in CRFr1 and CRFr2 relative to males. In males, overexpression of CRF produced a larger increase in Arrb2 than females, suggesting greater CRF receptor internalization. Our results suggest that stress systems in the NAc promote the reinforcing effectiveness of nicotine in female rats in an ovarian hormone-dependent manner.

Insulin modulates the strong reinforcing effects of nicotine and changes in insulin biomarkers in a rodent model of diabetes.

This study examined whether the strong reinforcing effects of nicotine and changes in insulin biomarkers observed in diabetic rats are modulated via insulin. A model of diabetes was employed involving administration of streptozotocin (STZ), which produces hypoinsulinemia in rats. The present study included vehicle- or STZ-treated rats that received sham surgery or insulin pellets. Two weeks later, the rats were given extended access to intravenous self-administration (IVSA) of saline or nicotine. Concomitant changes in food intake, water responses, and body weight were assessed during 12 days of IVSA. After the last session, plasma levels of insulin, leptin, amylin, and glucagon-like peptide-1 (GLP-1) were assessed using Luminex® technology. In a separate cohort, phosphorylated insulin receptor substrate-2 (pIRS-2) and insulin growth factor-1 receptor ß (IGF-1Rß) were assessed in the nucleus accumbens (NAc) and ventral tegmental area (VTA) of vehicle- or STZ-treated rats that received sham surgery or an insulin pellet. STZ-treated rats displayed an increase in glucose levels, a decrease in body weight, and an increase in nicotine, food, and water intake relative to controls. STZ-treated rats also displayed a decrease in plasma insulin and leptin levels and an increase in amylin and GLP-1 levels relative to controls. Importantly, all of the STZ-induced changes in behavior and insulin biomarkers were prevented by insulin supplementation. STZ-treated rats also displayed a decrease in pIRS-2 and IGF-1Rß in the NAc (but not VTA), an effect that was also prevented by insulin. These data suggest that insulin systems in the NAc modulate the strong reinforcing effects of nicotine in male diabetic rats.

Sex differences in nicotine intravenous self-administration: A meta-analytic review.

OBJECTIVE: This report reflects a meta-analysis that systematically reviewed the literature on intravenous self-administration (IVSA) of nicotine in female and male rats. The goal was to determine if sex differences in nicotine IVSA exist, estimate the magnitude of the effect, and identify potential moderators of the relationship between sex differences and nicotine consumption. METHODS: Extensive search procedures identified 20 studies that met the inclusion criteria of employing both female and male rats in nicotine IVSA procedures. The meta-analysis was conducted on effect size values that were calculated from mean total intake or nicotine deliveries using the Hedges' unbiased gu statistic. RESULTS: A random effects analysis revealed that overall females self-administered more nicotine than males (weighted gu=0.18, 95% CI [0.003, 0.34]). Subsequent moderator variable analyses revealed that certain procedural conditions influenced the magnitude of sex differences in nicotine IVSA. Specifically, higher reinforcement requirements (>FR1) and extended-access sessions (23h) were associated with greater nicotine IVSA in females versus males. Females also displayed higher nicotine intake than males when the experiment included a light cue that signaled nicotine delivery. Sex differences were not influenced by the diurnal phase of testing, dose of nicotine, or prior operant training. CONCLUSION: Overall, the results revealed that female rats display higher levels of nicotine IVSA than males, suggesting that the strong reinforcing effects of nicotine promote tobacco use in women.

Estradiol promotes the rewarding effects of nicotine in female rats.

It is presently unclear whether ovarian hormones, such as estradiol (E2), promote the rewarding effects of nicotine in females. Thus, we compared extended access to nicotine intravenous self-administration (IVSA) in intact male, intact female, and OVX female rats (Study 1) as well as OVX females that received vehicle or E2 supplementation (Study 2). The E2 supplementation procedure involved a 4-day injection regimen involving 2 days of vehicle and 2 days of E2 administration. Two doses of E2 (25 or 250µg) were assessed in separate groups of OVX females in order to examine the dose-dependent effects of this hormone on the rewarding effects of nicotine. The rats were given 23-hour access to nicotine IVSA using an escalating dose regimen (0.015, 0.03, and 0.06mg/kg/0.1mL). Each dose was self-administered for 4 days with 3 intervening days of nicotine abstinence. The results revealed that intact females displayed higher levels of nicotine intake as compared to males. Also, intact females displayed higher levels of nicotine intake versus OVX females. Lastly, our results revealed that OVX rats that received E2 supplementation displayed a dose-dependent increase in nicotine intake as compared to OVX rats that received vehicle. Together, our results suggest that the rewarding effects of nicotine are enhanced in female rats via the presence of the ovarian hormone, E2.