Epigenetic mechanisms of nicotine dependence.

Smoking continues to be a leading cause of preventable disease and death worldwide. Nicotine dependence generates a lifelong propensity towards cravings and relapse, presenting an ongoing challenge for the development of treatments. Accumulating evidence supports a role for epigenetics in the development and maintenance of addiction to many drugs of abuse, however, the involvement of epigenetics in nicotine dependence is less clear. Here we review evidence that nicotine interacts with epigenetic mechanisms to enable the maintenance of nicotine-seeking across time. Research across species suggests that nicotine increases permissive histone acetylation, decreases repressive histone methylation, and modulates levels of DNA methylation and noncoding RNA expression throughout the brain. These changes are linked to the promoter regions of genes critical for learning and memory, reward processing and addiction. Pharmacological manipulation of enzymes that catalyze core epigenetic modifications regulate nicotine reward and associative learning, demonstrating a functional role of epigenetic modifications in nicotine dependence. These findings are consistent with nicotine promoting an overall permissive chromatin state at genes important for learning, memory and reward. By exploring these links through next-generation sequencing technologies, epigenetics provides a promising avenue for future interventions to treat nicotine dependence.

Deficiency in the neural cell adhesion molecule 2 (NCAM2) reduces axonal levels of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), affects axonal organization in the hippocampus, and leads to behavioral deficits.

The neural cell adhesion molecule 2 (NCAM2) regulates axonal organization in the central nervous system via mechanisms that have remained poorly understood. We now show that NCAM2 increases axonal levels of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), a protease that regulates axonal guidance. In brains of NCAM2-deficient mice, BACE1 levels are reduced in hippocampal mossy fiber projections, and the infrapyramidal bundle of these projections is shortened. This abnormal axonal organization correlates with impaired short-term spatial memory and cognitive flexibility in NCAM2-deficient male and female mice. Self-grooming, rearing, digging and olfactory acuity are increased in NCAM2-deficient male mice, when compared with littermate wild-type mice of the same sex. NCAM2-deficient female mice also show increased self-grooming, but are reduced in rearing, and do not differ from female wild-type mice in olfactory acuity and digging behavior. Our results indicate that errors in axonal guidance and organization caused by impaired BACE1 function can underlie the manifestation of neurodevelopmental disorders, including autism as found in humans with deletions of the NCAM2 gene.

The neural substrates of higher-order conditioning: A review.

Sensory preconditioned and second-order conditioned responding are each well-documented. The former occurs in subjects (typically rats) exposed to pairings of two relatively neutral stimuli, S2 and S1, and then to pairings of S1 and a motivationally significant event [an unconditioned stimulus (US)]; the latter occurs when the order of these experiences is reversed with rats being exposed to S1-US pairings and then to S2-S1 pairings. In both cases, rats respond when tested with S2 in a manner appropriate to the affective nature of the US, e.g., approach when the US is appetitive and withdrawal when it is aversive. This paper reviews the neural substrates of sensory preconditioning and second-order conditioning. It identifies commonalities and differences in the substrates of these so-called higher-order conditioning protocols and discusses these commonalities/differences in relation to what is learned. In so doing, the review highlights ways in which these types of conditioning enhance our understanding of how the brain encodes and retrieves different types of information to generate appropriate behavior.

Chronic exposure to cafeteria-style diet in rats alters sweet taste preference and reduces motivation for, but not 'liking' of sucrose.

Obesity is associated with changes to taste perception and brain reward circuitry. It is important to understand how these effects alter the preference for palatable foods and drinks, given that these are widely consumed, and leading risk factors for obesity. This study examined the effects of diet-induced obesity on sweet taste preference by analysing the microstructure of licking for sugar solutions and assessing pERK expression in the nucleus accumbens shell and insula. Adult male Sprague-Dawley rats were fed standard chow (Control; n = 16) or a varied, palatable cafeteria diet (Caf; n = 16) for 12 weeks. Two-choice preference tests between 2%, 8% and 32% sucrose solutions were conducted at baseline and in weeks 11-12 of the diet. Rats in the Caf group trebled energy intake and doubled weight gain relative to controls. In tests held under water restriction after 11 weeks of diet, the Control group reliably preferred higher sucrose concentrations (i.e., 32% > 8% > 2%). Relative to controls, the Caf group showed a stronger preference for 32% vs. 2% sucrose, lower preference for 32% vs. 8% sucrose, and were indifferent to 8% vs. 2% sucrose. Testing without water restriction increased preference for higher sucrose concentrations in both groups. Chronic Caf diet increased the latency to lick, decreased total licks and reduced alternations between spouts, but did not alter lick cluster size, a measure of hedonic appraisal, on any test. Following a final exposure to a novel sucrose concentration, neuronal activity (pERK) in the insula and nucleus accumbens shell was significantly reduced in the Caf group. Results indicate that differences in 'liking' do not underlie obesity-induced changes to sweet taste preference.

Social isolation enhances cued-reinstatement of sucrose and nicotine seeking, but this is reversed by a return to social housing.

Physical or perceived (i.e. loneliness) social isolation is increasing in Western cultures. Unfortunately, social isolation is associated with a range of negative physical and mental health outcomes, including increased incidence of obesity and smoking. Here we monitored the impact of social isolation on a range of physical measures, and then tested whether social isolation in adult rats changes how reward-related stimuli motivate sucrose- or nicotine-seeking. Socially isolated rats showed elevated baseline CORT, gained significantly less weight across the study, were more active in response to a novel or familiar environment. Isolated rats also acquired nose-poking for a food pellet more rapidly, and showed increased susceptibility to cue-, but not reward-induced reinstatement. Notably, these effects are partially mitigated by a return to group housing, suggesting that they are not necessarily permanent, and that a return to a social setting can quickly reverse any deficits or changes associated with social isolation. This study advances our understanding of altered reward-processing in socially isolated individuals and reiterates the importance of socialisation in the treatment of disorders such as overeating and addiction.

Pre-quit nicotine decreases nicotine self-administration and attenuates cue- and drug-induced reinstatement.

BACKGROUND: Administration of smoking cessation medications in anticipation of a nominated quit date can promote abstinence. How this occurs is not widely understood, but may be due to the disruption of contingencies between smoking behaviour and acute drug effects. AIMS: The aim of this study was to explore this relationship, we examined the effect of pre-quit nicotine replacement therapy on susceptibility to relapse in an animal model of nicotine dependence. METHODS: Rats were trained to intravenously self-administer nicotine across 20 days. Continuous low-dose nicotine was administered via a mini-osmotic pump either across the last 7 days of self-administration and across 6 days of extinction, or across extinction only. Cue- and drug-induced reinstatements of responding were then measured with mini-pumps retained, the day after mini-pump removal or one week later. RESULTS: Pre-quit nicotine administration markedly reduced self-administration across the last days of training as the response, and its associated cues, no longer reliably predicted an acute drug effect. Pre-quit, but not post-quit, nicotine administration significantly attenuated cue-induced reinstatement once mini-pumps were removed, indicating that the contingency disruption across training reduced the conditioned reinforcing properties of the cue at test. Both pre-quit and post-quit nicotine attenuated nicotine-primed reinstatement. CONCLUSIONS: Together these results suggest that administration of a nicotine replacement prior to a nominated quit date may enhance resistance to relapse via disruption of the contingency between a response, its associated cues, and a rewarding nicotine effect.

Rats choose high doses of nicotine in order to compensate for changes in its price and availability.

Restricting when and where smoking can occur is a major focus of public health policies in Western countries. In conjunction with increased taxation, these approaches have contributed to a reduction in smoking uptake among adolescents, yet the consequences for established smokers are less clear. In order to further explore this relationship, we developed a novel animal model of restricted nicotine self-administration. Rats were trained to choose between three doses of nicotine (15, 30 and 60 µg/kg/infusion) under conditions where nicotine was (1) freely available at a low cost (20-second post-infusion time-out, fixed-ratio 1 [FR1]), (2) available under restricted access at a low cost (300-second post-infusion time-out, FR1), or (3) freely available at a high cost (20-second post-infusion time-out, FR5). We demonstrate that as access to nicotine is restricted or when cost increases, rats compensate for these changes by increasing their intake of the highest dose of nicotine available. This preference was impervious to treatment with the smoking cessation medication varenicline, but was reduced when the cost of the highest dose only was increased, or when nicotine was again made freely available at a low cost. These results provide the first evidence in rats that nicotine availability and cost influence nicotine choice independently of variations in nicotine and context exposure. They imply that established smokers may compensate for changes in the availability and cost of tobacco by increasing their rate of smoking when they are free to do so.

Open-field PET: Simultaneous brain functional imaging and behavioural response measurements in freely moving small animals.

A comprehensive understanding of how the brain responds to a changing environment requires techniques capable of recording functional outputs at the whole-brain level in response to external stimuli. Positron emission tomography (PET) is an exquisitely sensitive technique for imaging brain function but the need for anaesthesia to avoid motion artefacts precludes concurrent behavioural response studies. Here, we report a technique that combines motion-compensated PET with a robotically-controlled animal enclosure to enable simultaneous brain imaging and behavioural recordings in unrestrained small animals. The technique was used to measure in vivo displacement of [11C]raclopride from dopamine D2 receptors (D2R) concurrently with changes in the behaviour of awake, freely moving rats following administration of unlabelled raclopride or amphetamine. The timing and magnitude of [11C]raclopride displacement from D2R were reliably estimated and, in the case of amphetamine, these changes coincided with a marked increase in stereotyped behaviours and hyper-locomotion. The technique, therefore, allows simultaneous measurement of changes in brain function and behavioural responses to external stimuli in conscious unrestrained animals, giving rise to important applications in behavioural neuroscience.

An extended history of drug self-administration results in multiple sources of control over drug seeking behavior.

It is widely recognized that across the development of drug addiction, cues associated with drug use come to exert increasing control over drug seeking and taking behaviors. However, there remain gaps in our knowledge regarding how the different types of drug related cues affect drug seeking and taking behaviors, and how the emergence of cue control over these behaviors relates to the onset of drug seeking compulsions. This paper reviews the literature on drug self-administration in animals to address these gaps. It first identifies the different types of cues that acquire control over reward seeking behavior generally, and examines whether the same types of cues acquire control over drug seeking behavior specifically. It then examines how the role of drug related cues in motivating and reinforcing drug seeking behavior changes across an extended drug-taking history, with a particular focus on the case of nicotine. The evidence reviewed shows that, after an extended history of drug taking, drug seeking behaviors are controlled by contextual cues associated with the development of drug seeking habits, response contingent cues that accompany delivery of the drug, as well as internal states that correlate with levels of drug intake. These multiple sources of control over drug seeking are discussed in relation to the generation of an addicted phenotype in animal models and the hypothesized progression from internal control over drug use to compulsive drug seeking.

Palatable food self-administration and reinstatement are not affected by dual orexin receptor antagonism.

The orexins are widely regarded potential therapeutic targets for a range of disorders of appetitive motivation, including obesity. The motivational activator theory, the first coherent account of the orexin system's role in appetitive motivation, predicts that orexin release motivates appetitive behaviour when the reinforcer is highly salient, available under a high unit-cost or when reward seeking is cue-driven. The present study tested the effect of intracerebroventricular (i.c.v.) administration of the highly potent and commercially available dual orexin receptor antagonist, TCS 1102, on self-administration and reinstatement of palatable food seeking in hungry and sated rats. TCS 1102 was also tested on FR1, FR5, FR10 and PR schedules. Orexin neuron activation was measured by c-Fos/orexin-A immunohistochemistry after cue-induced reinstatement, an extinction test, or a home-cage control. No effect of i.c.v. TCS 1102 was observed on self-administration at any fixed or progressive ratio schedule of reinforcement or reinstatement in hungry or sated rats. Although there was robust recruitment of orexin neurons during behavioural testing conditions, there was no specific activation of these neurons during cue-induced reinstatement when compared to extinction testing conditions. These results suggest that orexin antagonism may not be a useful therapeutic target for obesity as it does not appear to regulate food-seeking, and that the conditions determining orexin involvement as a motivational activator may be less clear than currently understood.

The dual orexin receptor antagonist TCS1102 does not affect reinstatement of nicotine-seeking.

The orexin/hypocretin system is important for appetitive motivation towards multiple drugs of abuse, including nicotine. Both OX1 and OX2 receptors individually have been shown to influence nicotine self-administration and reinstatement. Due to the increasing clinical use of dual orexin receptor antagonists in the treatment of disorders such as insomnia, we examined whether a dual orexin receptor antagonist may also be effective in reducing nicotine seeking. We tested the effect of intracerebroventricular (i.c.v.) administration of the potent and selective dual orexin receptor antagonist TCS1102 on orexin-A-induced food self-administration, nicotine self-administration and reinstatement of nicotine-seeking in rats. Our results show that 30 µg of TCS1102 i.c.v. abolishes orexin-A-induced increases in food self-administration but does not reduce nicotine self-administration. Neither i.c.v. 10 µg nor 30 µg of TCS1102 reduced compound reinstatement after short-term (15 days) self-administration nicotine, but 30 µg transiently reduced cue/nicotine compound reinstatement after chronic self-administration (29 days). These results indicate that TCS1102 has no substantial effect on motivation for nicotine seeking following chronic self-administration and no effect after shorter periods of intake. Orexin receptor antagonists may therefore have little clinical utility against nicotine addiction.

Rats quit nicotine for a sweet reward following an extensive history of nicotine use.

Drug use may be exacerbated in environments which lack alternative means of engaging in rewarding behaviour. When alternative rewards are available, drug use may decrease-an effect that can be harnessed for therapeutic benefit. This idea is particularly well-supported by recent preclinical evidence demonstrating that a majority of rats will readily choose a potent non-drug reward over cocaine or heroin. Here we examine whether the same holds true for nicotine, a drug considered to have one of the highest addiction liabilities amongst drugs of abuse. Rats were trained to nose-poke separately for saccharin or nicotine on alternate days. Using a discrete-trial, forced-choice procedure, rats were then allowed to choose between nicotine and saccharin. This was followed by choice testing after a decrease in saccharin concentration (0.2-0%), omission of the fluid reward, an increase in nicotine concentration and following an extended nicotine self-administration history. All rats demonstrated a clear and immediate preference for saccharin at all times. This was despite variations in reward concentrations, or after an extensive nicotine history. Notably, rats preferred to nose-poke for water over nicotine and would omit responses when no fluid was delivered, rather than resume responding for nicotine. Overall, this study confirms and extends to nicotine previous research on other drugs of abuse, including cocaine and heroin. The ease with which rats quit nicotine in the present study contrasts with the well-known difficulty of humans to quit tobacco smoking. Possible factors that could explain this apparent discrepancy are discussed.

Extended nicotine self-administration increases sensitivity to nicotine, motivation to seek nicotine and the reinforcing properties of nicotine-paired cues.

An array of pharmacological and environmental factors influence the development and maintenance of tobacco addiction. The nature of these influences likely changes across the course of an extended smoking history, during which time drug seeking can become involuntary and uncontrolled. The present study used an animal model to examine the factors that drive nicotine-seeking behavior after either brief (10 days) or extended (40 days) self-administration training. In Experiment 1, extended training increased rats' sensitivity to nicotine, indicated by a leftward shift in the dose-response curve, and their motivation to work for nicotine, indicated by an increase in the break point achieved under a progressive ratio schedule. In Experiment 2, extended training imbued the nicotine-paired cue with the capacity to maintain responding to the same high level as nicotine itself. However, Experiment 3 showed that the mechanisms involved in responding for nicotine or a nicotine-paired cue are dissociable, as treatment with the partial nicotine receptor agonist, varenicline, suppressed responding for nicotine but potentiated responding for the nicotine-paired cue. Hence, across extended nicotine self-administration, pharmacological and environmental influences over nicotine seeking increase such that nicotine seeking is controlled by multiple sources, and therefore highly resistant to change.

Varenicline impairs extinction and enhances reinstatement across repeated cycles of nicotine self-administration in rats.

Varenicline is a partial nicotine receptor agonist widely prescribed as a smoking cessation medication. Repeated (or long-term) use of varenicline has been proposed as a treatment option for tobacco addiction. However the effect of repeated varenicline use on motivation for nicotine is unknown. Here the intravenous nicotine self-administration paradigm in rats was used to model the consequences of varenicline treatment across repeated cycles of administration, extinction and reinstatement. Rats acquired nicotine self-administration across 20 days before undergoing 6 days of extinction, where each extinction session was preceded by a single injection of varenicline or saline. This was followed by a single varenicline-free nicotine-primed reinstatement test. All rats then reacquired nicotine self-administration for 10 days followed by a second cycle of extinction. Across this period, rats either received a second cycle of varenicline (VAR-VAR) or saline (SAL-SAL), or the alternative treatment (SAL-VAR, VAR-SAL), followed by a final reinstatement test. Treatment with varenicline increased responding across the first cycle of extinction, but did not affect responding in the reinstatement test. Across the second cycle, varenicline again increased responding across extinction, and critically, rats treated with varenicline across cycle 1 and saline across cycle 2 (Group VAR-SAL) exhibited more reinstatement than rats in any other group. The effect of VAR on nicotine seeking was not due to its effects on locomotor activity. Instead, the results suggest that a history of VAR can increase vulnerability to reinstatement/relapse when its treatment is discontinued. The possible mechanisms of this increased vulnerability are discussed.

Inhibition of histone deacetylases facilitates extinction and attenuates reinstatement of nicotine self-administration in rats.

Chromatin remodelling is integral to the formation of long-term memories. Recent evidence suggests that histone modification may play a role in the persistence of memories associated with drug use. The present series of experiments aimed to examine the effect of histone deacetylase (HDAC) inhibition on the extinction and reinstatement of nicotine self-administration. Rats were trained to intravenously self-administer nicotine for 12 days on a fixed-ratio 1 schedule. In Experiment 1, responding was then extinguished through removal of nicotine and response-contingent cues. After each extinction session, the HDAC inhibitor, sodium butyrate (NaB), was administered immediately, or six hours after each session. In Experiment 2, response-contingent cues remained available across extinction to increase rates of responding during this phase, and NaB was administered immediately after the session. Finally, in Experiment 3, the effect of NaB treatment on extinction of responding for sucrose pellets was assessed. Across all experiments reinstatement to the cue and/or the reward itself was then tested. In the first experiment, treatment with NaB significantly attenuated nicotine and nicotine + cue reinstatement when administered immediately, but not six hours after each extinction session. When administered after cue-extinction (Expt. 2), NaB treatment specifically facilitated the rate of extinction across sessions, indicating that HDAC inhibition enhanced consolidation of the extinction memory. In contrast, there was no effect of NaB on the extinction and reinstatement of sucrose-seeking (Expt. 3), indicating that the observed effects are specific to a drug context. These results provide the first demonstration that HDAC inhibition facilitates the extinction of responding for an intravenously self-administered drug of abuse and further highlight the potential of HDAC inhibitors in the treatment of drug addiction.

Behavioral and neural substrates of habit formation in rats intravenously self-administering nicotine.

Tobacco addiction involves a transition from occasional, voluntary smoking towards habitual behavior that becomes increasingly resistant to quitting. The development of smoking habits may reflect a loss of behavioral control that can be modeled in rats. This study investigated the behavioral and neural substrates of habit formation in rats exposed to either brief (10 days) or extended (47 days) intravenous (IV) nicotine self-administration training. Following training, the first cohort of rats were exposed to a nicotine devaluation treatment, which involved repeated pairings of IV nicotine with lithium injection. They were then tested for sensitivity of responding to nicotine devaluation under extinction and reinstatement conditions. The second cohort of rats received equivalent self-administration training followed by processing of brain tissue for c-Fos immunohistochemistry. After brief training, devaluation suppressed nicotine-seeking during tests of extinction and reinstatement, confirming that responding is initially sensitive to current nicotine value, and therefore, goal directed. In contrast, after extended training, devaluation had no effect on extinction or reinstatement of responding, indicating that responding had become habitual. Complementary neuroanalysis revealed that extended nicotine self-administration was associated with increased c-Fos expression in brain regions implicated in habitual control of reward seeking, including activation of the dorsolateral striatum and substantia nigra pars compacta. These findings provide evidence of direct devaluation of an IV drug reward, that nicotine self-administration is initially goal-directed but becomes habitual with extended training, and that this behavioral transition involves activation of brain areas associated with the nigrostriatal system.

High levels of intravenous mephedrone (4-methylmethcathinone) self-administration in rats: neural consequences and comparison with methamphetamine.

Mephedrone (MMC) is a relatively new recreational drug that has rapidly increased in popularity in recent years. This study explored the characteristics of intravenous MMC self-administration in the rat, with methamphetamine (METH) used as a comparator drug. Male Sprague-Dawley rats were trained to nose poke for intravenous MMC or METH in daily 2 h sessions over a 10 d acquisition period. Dose-response functions were then established under fixed- and progressive-ratio (FR and PR) schedules over three subsequent weeks of testing. Brains were analyzed ex vivo for striatal serotonin (5-HT) and dopamine (DA) levels and metabolites, while autoradiography assessed changes in the regional density of 5-HT and serotonin transporter (SERT) and DA transporter (DAT) and induction of the inflammation marker translocator protein (TSPO). Results showed that MMC was readily and vigorously self-administered via the intravenous route. Under a FR1 schedule, peak responding for MMC was obtained at 0.1 mg/kg/infusion, versus 0.01 mg/kg/infusion for METH. Break points under a PR schedule peaked at 1 mg/kg/infusion MMC versus 0.3 mg/kg/infusion for METH. Final intakes of MMC were 31.3 mg/kg/d compared to 4 mg/kg/d for METH. Rats self-administering MMC, but not METH, gained weight at a slower rate than control rats. METH, but not MMC, self-administration elevated TSPO receptor density in the nucleus accumbens and hippocampus, while MMC, but not METH, self-administration decreased striatal 5-hydroxyindolacetic acid (5-HIAA) concentrations. In summary, MMC supported high levels of self-administration, matching or exceeding those previously reported with other drugs of abuse.

Long-term effects of chronic oral Ritalin administration on cognitive and neural development in adolescent wistar kyoto rats.

The diagnosis of Attention Deficit Hyperactivity Disorder (ADHD) often results in chronic treatment with psychostimulants such as methylphenidate (MPH, Ritalin®). With increases in misdiagnosis of ADHD, children may be inappropriately exposed to chronic psychostimulant treatment during development. The aim of this study was to assess the effect of chronic Ritalin treatment on cognitive and neural development in misdiagnosed "normal" (Wistar Kyoto, WKY) rats and in Spontaneously Hypertensive Rats (SHR), a model of ADHD. Adolescent male animals were treated for four weeks with oral Ritalin® (2 × 2 mg/kg/day) or distilled water (dH2O). The effect of chronic treatment on delayed reinforcement tasks (DRT) and tyrosine hydroxylase immunoreactivity (TH-ir) in the prefrontal cortex was assessed. Two weeks following chronic treatment, WKY rats previously exposed to MPH chose the delayed reinforcer significantly less than the dH2O treated controls in both the DRT and extinction task. MPH treatment did not significantly alter cognitive performance in the SHR. TH-ir in the infralimbic cortex was significantly altered by age and behavioural experience in WKY and SHR, however this effect was not evident in WKY rats treated with MPH. These results suggest that chronic treatment with MPH throughout adolescence in "normal" WKY rats increased impulsive choice and altered catecholamine development when compared to vehicle controls.