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.

Sex and diet-dependent gene alterations in human and rat brains with a history of nicotine exposure.

Introduction: Chronic nicotine exposure induces changes in the expression of key regulatory genes associated with metabolic function and neuronal alterations in the brain. Many bioregulatory genes have been associated with exposure to nicotine, but the modulating effects of sex and diet on gene expression in nicotine-exposed brains have been largely unexplored. Both humans and rodents display motivation for nicotine use and the emergence of withdrawal symptoms during abstinence. Research comparing pre-clinical models with human subjects provides an important opportunity to understand common biomarkers of the harmful effects of nicotine as well as information that may help guide the development of more effective interventions for nicotine cessation. Methods: Human postmortem dorsolateral prefrontal cortex (dLPFC) tissue BA9 was collected from female and male subjects, smokers and non-smokers (N = 12 per group). Rat frontal lobes were collected from female and male rats that received a regular diet (RD) or a high-fat diet (HFD) (N = 12 per group) for 14 days following implantation of a osmotic mini-pump (Alzet) that delivered nicotine continuously. Controls (control-s) received a sham surgical procedure. RNA was extracted from tissue from human and rat samples and reversed-transcribed to cDNA. Gene expression of CHRNA10 (Cholinergic receptor nicotinic alpha 10), CERKL (Ceramide Kinase-Like), SMYD1 (SET and MYD Domin Containing 1), and FA2H (Fatty Acid 2-Hydrolase) in humans was compared to rats in each subset of groups and quantified by qPCR methods. Additionally, protein expression of FA2H was analyzed by immunohistochemistry (IHC) in human dLPFC. Results: Humans with a history of smoking displayed decreased CHRNA10 (p = 0.0005), CERKL (p ≤ 0.0001), and SMYD1 (p = 0.0005) expression and increased FA2H (p = 0.0097) expression compared to non-smokers (p < 0.05). Similar patterns of results were observed in nicotine exposed vs. control rats. Interestingly, sex-related differences in gene expression for CERKL and FA2H were observed. In addition, ANCOVA analysis showed a significant effect of nicotine in a sex-different manner, including an increase in CERKL in male and female rats with RD or HFD. In rats exposed to an HFD, FA2H gene expression was lower in nicotine-treated rats compared to RD rats treated with nicotine. Protein expression of FA2H (p = 0.001) by IHC was significantly higher in smokers compared to non-smokers. Conclusion: These results suggest that a history of long-term nicotine exposure in humans alters the expression of sphingolipid metabolism-related (CERKL, SMYD1, and FA2H) and neuronal (CHRNA10) marker genes similarly as compared to rats. Sex- and diet-dependent differences appear in nicotine-exposed rats, critical in regulating sphingolipid metabolism and nicotinic acetylcholine receptors. This research enhances the construct validity of rat models of nicotine usage by showing a similar pattern of changes in gene expression in human subjects with a smoking history.

Alcohol self-administration and nicotine withdrawal alter biomarkers of stress and inflammation and prefrontal cortex changes in Gß subunits.

Background: Although alcohol and nicotine are often used together, the biological consequences of these substances are not well understood. Identifying shared targets will inform cessation pharmacotherapies and provide a deeper understanding of how co-use of alcohol and nicotine impacts health, including biomarkers of stress and inflammation.Objective: We examined the effects of nicotine exposure and withdrawal on alcohol self-administration (SA), stress and inflammatory biomarkers, and a G-protein coupled receptor subunit (Gß) in brain areas associated with drug use.Methods: Male rats were trained to SA alcohol and then received a nicotine pump (n = 7-8 per group). We assessed alcohol intake for 12 days during nicotine exposure and then following pump removal to elicit withdrawal. After the behavioral studies, we assessed plasma leptin, corticosterone, and interleukin-1ß (IL-1ß), and Gß protein expression in the amygdala, nucleus accumbens (NAc), and prefrontal cortex (PFC).Results: Nicotine exposure or withdrawal did not alter alcohol intake (p > .05). Alcohol and nicotine withdrawal elevated corticosterone levels (p = .015) and decreased Gß levels in the PFC (p = .004). In the absence of nicotine, alcohol SA suppressed IL-1ß levels (p = .039). Chronic exposure to nicotine or withdrawal during alcohol SA did not alter leptin levels or Gß expression in the amygdala or NAc (p's > .05).Conclusions: The combination of alcohol SA and nicotine withdrawal produced a persistent increase in stress biomarkers and a suppression in Gß expression in the PFC, providing an important first step toward understanding the common biological mechanisms of alcohol/nicotine misuse.

Effectiveness of pharmacotherapies for diabetes on nicotine, food, and water intake in insulin-resistant rats.

The intersectionality between diabetes medications and nicotine consumption was assessed in female and male rats. Briefly, the rats were fed a high-fat diet (HFD) or regular diet (RD) for 4 weeks. Then separate groups received vehicle or a low dose of streptozotocin (STZ; 25 mg/kg). Three days later, insulin resistance was assessed by measuring plasma glucose levels for 180 min following an injection of insulin (0.75 U/kg). The rats were then prepared with jugular catheters, and they were given 23 h access to nicotine intravenous self-administration (IVSA) in 4 days cycles with 3 days of forced abstinence in their home cages where they consumed their respective diet. During the IVSA sessions, operant responses for food and water and changes in body weight were recorded. Prior to administration of the pharmacotherapies, the rats were given access to two doses of nicotine (0.015 then 0.03 mg/kg for the remainder of the study). Then, daily injections of the pharmacotherapies were given at the onset of dark cycle (6 p.m.) in the following order: 1) dapagliflozin (3.0 then 10.0 mg/kg), 2) insulin (0.75 U/kg twice), and 3) bromocriptine (3.0 then 10.0 mg/kg). The results suggest that our HFD+STZ regiment induced insulin resistance in female and male rats. Also, the HFD-fed rats displayed higher nicotine intake than RD controls, regardless of sex. Administration of insulin, but not dapagliflozin or bromocriptine, normalized nicotine intake in HFD-fed rats to control levels. These results have clinical implications regarding the potential efficacy of insulin to control excessive nicotine intake in persons with diabetes.

Early-life adversity increases anxiety-like behavior and modifies synaptic protein expression in a region-specific manner.

Early-life adversity (ELA) can induce persistent neurological changes and increase the risk for developing affective or substance use disorders. Disruptions to the reward circuitry of the brain and pathways serving motivation and emotion have been implicated in the link between ELA and altered adult behavior. The molecular mechanisms that mediate the long-term effects of ELA, however, are not fully understood. We examined whether ELA in the form of neonatal maternal separation (MatSep) modifies behavior and synaptic protein expression in adults. We hypothesized that MatSep would affect dopaminergic and glutamatergic signaling and enhance sensitivity to methamphetamine (Meth) reward or increase anxiety. Male Wistar rats were subjected to MatSep for 180 min/d on postnatal days (PND) 2-14 and allowed to grow to adulthood (PND 60) with no further manipulation. The hippocampus (Hipp), medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and caudate putamen (CPu) were isolated from one subgroup of animals and subjected to Western blot and protein quantitation for tyrosine hydroxylase (TH), α-synuclein (ALPHA), NMDA receptor (NMDAR), dopamine receptor-1 (D1) and -2 (D2), dopamine transporter (DAT), and postsynaptic density 95 (PSD95). Separate group of animals were tested for anxiety-like behavior and conditioned place preference (CPP) to Meth at 0.0, 0.1, and 1.0 mg/kg doses. MatSep rats displayed an increase in basal levels of anxiety-like behavior compared to control animals. MatSep rats also demonstrated CPP to Meth, but their responses did not differ significantly from controls at any drug dose. Increased NMDAR, D2, and ALPHA expression was observed in the NAc and CPu following MatSep; D2 and ALPHA levels were also elevated in the mPFC, along with DAT. MatSep rats had reduced D1 expression in the mPFC and Hipp, with the Hipp also showing a reduction in D2. Only the CPu showed elevated TH and decreased DAT expression levels. No significant changes were found in PSD95 expression in MatSep rats. In conclusion, ELA is associated with long-lasting and region-specific changes in synaptic protein expression that diminish dopamine neurotransmission and increase anxiety-like behavior in adults. These findings illustrate potential mechanisms through which ELA may increase vulnerability to stress-related illness.

Sex and age differences in approach behavior toward a port that delivers nicotine vapor.

The goal of our laboratory is to study the mechanisms that promote nicotine use, particularly in vulnerable populations. To more closely mimic human use patterns, the present study employed nicotine vapor methods involving passive exposure for 14 days in adolescent and adult female and male rats. Age and sex differences in approach behavior (nosepokes) were assessed in a port that delivered nicotine plumes on Day 1 and 14 of our exposure regimen. Controls received ambient air in exposure chambers. After the final session, rats received a nicotinic receptor antagonist to precipitate withdrawal. Then, physical signs, anxiety-like behavior, and plasma levels of cotinine (a nicotine metabolite) were assessed. Over time, females displayed a larger increase in approach behavior to the nicotine port than males, an effect that was larger in adolescents. Nosepoke responses in adolescent females were correlated with anxiety-like behavior, but not physical signs of withdrawal. Adolescents gained more weight than adults regardless of treatment, and the weight gain was larger in male adolescents. Female adolescents also displayed the highest levels of cotinine than all other groups. These findings suggest that nicotine vapor produces greater motivational effects in adolescent females as compared to their adult and male counterparts.

Exposure to Nicotine Vapor Produced by an Electronic Nicotine Delivery System Causes Short-Term Increases in Impulsive Choice in Adult Male Rats.

INTRODUCTION: Traditional cigarette use influences cost-benefit decision making by promoting impulsive choice. However, the impact of nicotine exposure via electronic nicotine delivery systems on impulsivity remains unclear. Hence, the present study examined the short- and long-term effects of nicotine vapor on impulsive choice. METHODS: Twenty-four adult male rats were trained in the delay discounting task to choose between small immediate food rewards and large delayed food rewards. After 24 days of training in the task rats were exposed to vapor containing either 0, 12, or 24 mg/mL of nicotine for 10 days. To validate inhalation of nicotine vapor serum cotinine levels were analyzed on exposure days 1, 5, and 10 using enzyme-linked immunosorbent assay. Following vapor exposure, rats were retrained in the discounting task until rats displayed stable responding and the effects of nicotine vapor on choice preference were assessed. RESULTS: Rats exposed to 12 and 24 mg/mL nicotine vapor displayed higher serum cotinine levels than control rats exposed to 0 mg/mL vapor. There were no differences in impulsive choice between any vapor exposure groups when tested 15 days after exposure, across 6 days of stable responding, suggesting that nicotine vapor does not have long lasting effects on impulsive choice. Interestingly, a subsequent nicotine vapor challenge revealed short-term increases in impulsive choice immediately following a single exposure to 24 mg/mL nicotine vapor, relative to choice preference immediately following exposure to 0 mg/mL vapor. CONCLUSIONS: These results suggest that exposure to nicotine vapor causes immediate, short-term increases in impulsive choice. IMPLICATIONS: E-cigarette use is increasing at an alarming rate, particularly among adolescents and young adults. This is concerning given the lack of research into the effects of nicotine vapor exposure on the brain and behavior. The present study describes a viable rodent model of human e-cigarette use and suggests that exposure to nicotine vapor produces short-term increases in impulsive choice.

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.

Converging vulnerability factors for compulsive food and drug use.

Highly palatable foods and substance of abuse have intersecting neurobiological, metabolic and behavioral effects relevant for understanding vulnerability to conditions related to food (e.g., obesity, binge eating disorder) and drug (e.g., substance use disorder) misuse. Here, we review data from animal models, clinical populations and epidemiological evidence in behavioral, genetic, pathophysiologic and therapeutic domains. Results suggest that consumption of highly palatable food and drugs of abuse both impact and conversely are regulated by metabolic hormones and metabolic status. Palatable foods high in fat and/or sugar can elicit adaptation in brain reward and withdrawal circuitry akin to substances of abuse. Intake of or withdrawal from palatable food can impact behavioral sensitivity to drugs of abuse and vice versa. A robust literature suggests common substrates and roles for negative reinforcement, negative affect, negative urgency, and impulse control deficits, with both highly palatable foods and substances of abuse. Candidate genetic risk loci shared by obesity and alcohol use disorders have been identified in molecules classically associated with both metabolic and motivational functions. Finally, certain drugs may have overlapping therapeutic potential to treat obesity, diabetes, binge-related eating disorders and substance use disorders. Taken together, data are consistent with the hypotheses that compulsive food and substance use share overlapping, interacting substrates at neurobiological and metabolic levels and that motivated behavior associated with feeding or substance use might constitute vulnerability factors for one another. This article is part of the special issue on 'Vulnerabilities to Substance Abuse'.

Female rats display greater nicotine withdrawal-induced cellular activation of a central portion of the interpeduncular nucleus versus males: A study of Fos immunoreactivity within provisionally assigned interpeduncular subnuclei.

BACKGROUND: The interpeduncular nucleus (>1840) (IPN) has been shown to modulate the behavioral effects of nicotine withdrawal in male rodents. To date, the contribution of this brain structure to sex differences in withdrawal is largely unexplored. METHODS: This study compared neuronal activation, as reported by observable Fos expression in the IPN of nicotine-dependent female and male rats experiencing withdrawal. We provisionally localized the Fos-expressing cells to certain IPN subnuclei within Swanson's standardized brain atlas (2018). Adult female and male rats were prepared with a pump that delivered nicotine (3.2 mg/kg/day; base) continuously. Controls received a sham surgery. Fourteen days later, the rats received administration of saline or the nicotinic receptor antagonist, mecamylamine (3.0 mg/kg; salt), and physical signs and anxiety-like behavior were assessed. The rats were then euthanized and brain sections containing the IPN were processed for Fos immunofluorescence to infer the possible IPN subnuclei displaying differential activation between sexes. RESULTS: Both female and male rats displayed withdrawal-induced Fos expression within the IPN. Compared to males, female rats displayed greater numbers of withdrawal-induced Fos-positive cells within a circumscribed portion of the IPN that may fall within the cytoarchitectural boundaries of the central subnucleus (>1840) (IPNc). The withdrawal-induced activation of the IPN was correlated with negative affective states in females, but not males. CONCLUSION: These data suggest that a centrally located group of IPN cells, presumably situated partly or completely within the IPNc, play a role in modulating sex differences in negative affective states produced by withdrawal.

Insulin restores the neurochemical effects of nicotine in the mesolimbic pathway of diabetic rats.

This study examined whether insulin modulates the neurochemical effects of nicotine in the mesolimbic pathway of diabetic rats. The rats received vehicle or streptozotocin (STZ) to induce hypoinsulinemia. A subset of STZ-treated rats was implanted with insulin pellets that rapidly normalized glucose levels. Two-weeks later, dialysis probes were implanted into the nucleus accumbens (NAc) and ipsilateral ventral tegmental area (VTA). The next day, dialysate samples were collected during baseline and then following systemic administration of nicotine. Samples were also collected following intra-VTA administration of the gamma-aminobutyric acid (GABA)A receptor antagonist, bicuculline. Dopamine, GABA, glutamate, and acetylcholine (ACh) levels were assessed using liquid chromatography/mass spectrometry (LC/MS). The results revealed that vehicle-treated rats displayed a nicotine-induced increase in NAc dopamine levels. In contrast, STZ-treated rats did not display any changes in NAc dopamine following nicotine administration, an effect that was likely related to a concomitant increase in GABA and decrease in glutamate levels in both the NAc and VTA. Intra-VTA administration of bicuculline increased NAc dopamine in vehicle-treated rats, and this effect was absent in STZ-treated rats. Vehicle-treated rats displayed a nicotine-induced increase in ACh levels in the NAc (but not VTA), an effect that was lower in the NAc of STZ-treated rats. Insulin supplementation normalized the neurochemical effects of nicotine in the NAc and VTA of STZ-treated rats, suggesting that insulin modulates the neurochemical effects of nicotine in the mesolimbic pathway of diabetic rats.

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.

Examination of nicotine and saccharin reward in the Goto-Kakizaki diabetic rat model.

Morbidity and mortality attributed to type 2 diabetes have exponentially increased in the US. At exceptionally high risk is a subpopulation of persons with type 2 diabetes who smoke, which are shown to have decreased success rates of smoking cessation than euglycemic smokers. Preclinical research in our laboratory has shown that the rewarding effects of nicotine are enhanced in the streptozotocin and high-fat diet rodent model of diabetes. It is presently unclear whether this enhancement of nicotine reward can be demonstrated in other insulin resistant rat models. This study aimed to determine if a similar increase in nicotine reward is found in Goto-Kakizaki (GK) rats, a model of the spontaneous formation of insulin resistance in an inbred sub-strain of Wistar rat. Nicotine conditioned place preference (CPP) was examined in Sprague-Dawley (SD), Wistar, and GK rats. A robust nicotine CPP was found in SD and Wistar rats, but nicotine CPP was not detected in GK rats. Locomotor activity was also evaluated in all three strains, and GK rats demonstrated significantly less activity as compared to SD and Wistar rats. To further assess reward behavior in GK rats, consumption of saccharin solution was measured over a 48 -h period. GK rats showed a significant increase in saccharin intake compared to SD rats. These findings suggest that GK rats experience an enhanced hedonic processing as compared to SD rats. The lack of nicotine CPP in GK rats may be due to deficits in learning and memory, thus hindering their ability to acquire or express a place preference.

Relationship Between Nicotine Intake and Reward Function in Rats With Intermittent Short Versus Long Access to Nicotine.

INTRODUCTION: Tobacco use improves mood states and smoking cessation leads to anhedonia, which contributes to relapse. Animal studies have shown that noncontingent nicotine administration enhances brain reward function and leads to dependence. However, little is known about the effects of nicotine self-administration on the state of the reward system. METHODS: To investigate the relationship between nicotine self-administration and reward function, rats were prepared with intracranial self-stimulation electrodes and intravenous catheters. The rats were trained on the intracranial self-stimulation procedure and allowed to self-administer 0.03 mg/kg/infusion of nicotine. All rats self-administered nicotine daily for 10 days (1 hour/day) and were then switched to an intermittent short access (ShA, 1 hour/day) or long access (LgA, 23 hour/day) schedule (2 days/week, 5 weeks). RESULTS: During the first 10 daily, 1-hour sessions, nicotine self-administration decreased the reward thresholds, which indicates that nicotine potentiates reward function. After switching to the intermittent LgA or ShA schedule, nicotine intake was lower in the ShA rats than the LgA rats. The LgA rats increased their nicotine intake over time and they gradually consumed a higher percentage of their nicotine during the light phase. The nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine induced a larger increase in reward thresholds (ie, anhedonia) in the LgA rats than the ShA rats. In the LgA rats, nAChR blockade with mecamylamine decreased nicotine intake for 2 hours and this was followed by a rebound increase in nicotine intake. CONCLUSIONS: A brief period of nicotine self-administration enhances reward function and a high level of nicotine intake leads to dependence. IMPLICATIONS: These animal studies indicate that there is a strong relationship between the level of nicotine intake and brain reward function. A high level of nicotine intake was more rewarding than a low level of nicotine intake and nicotine dependence was observed after long, but not short, access to nicotine. This powerful combination of nicotine reward and withdrawal makes it difficult to quit smoking. Blockade of nAChRs temporarily decreased nicotine intake, but this was followed by a large rebound increase in nicotine intake. Therefore, nAChR blockade might not decrease the use of combustible cigarettes or electronic cigarettes.

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.

Sex differences in cholinergic systems in the interpeduncular nucleus following nicotine exposure and withdrawal.

The medial habenula-interpeduncular nucleus (MHb-IPN) pathway modulates negative affective states produced by nicotine withdrawal. Sex differences in the contribution of acetylcholine (ACh) systems in this pathway have not been explored. Thus, this study assessed ACh levels and gene expression of α- and ß-containing nicotinic acetylcholine receptor (nAChR) subunits in the IPN of female and male rats following nicotine treatment and withdrawal. Rats were prepared with a pump that delivered nicotine for 14 days, and naïve controls received a sham surgery. In Study 1, rats were prepared with a probe in the IPN, and ACh levels were measured following saline and then mecamylamine administration. In Study 2, separate groups of naïve control or nicotine-treated rats received saline or mecamylamine and physical signs and anxiety-like behavior were assessed using elevated plus maze (EPM) procedures. The IPN was then dissected and mRNA levels were assessed using RT-qPCR methods. Nicotine treatment increased ACh levels to a larger extent in females than males. Nicotine withdrawal produced a similar increase in physical signs; however, females displayed greater anxiety-like behavior than males. In females, gene expression of α5 increased following nicotine treatment and withdrawal. In males, α7 increased following nicotine treatment and α2 and α3 increased during nicotine withdrawal. Both females and males displayed an increase in ß3 and ß4 during nicotine withdrawal. In females, anxiety-like behavior was correlated with α4, α5, and ß2 gene expression in the IPN. These results suggest that sex differences in withdrawal are modulated via cholinergic systems in the IPN.

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.