Chronic exposure to cigarette smoke extract increases nicotine withdrawal symptoms in adult and adolescent male rats.

The aim of the current study was to determine whether non-nicotine constituents of cigarette smoke contribute to nicotine dependence in adolescent and adult male Sprague Dawley rats. For 10 days animals were given three times daily intravenous injections of nicotine (1.5 mg/kg/day) or cigarette smoke extract (CSE) containing an equivalent dose of nicotine. Both spontaneous and mecamylamine-precipitated withdrawal were then measured. Chronic treatment with CSE induced significantly greater somatic and affective withdrawal signs than nicotine in both adolescents and adults. Mecamylamine-precipitated somatic signs were similar at both ages. In contrast, animals spontaneously withdrawn from chronic drug treatment exhibited significant age differences: whereas adolescents chronically treated with nicotine did not show somatic signs, those treated with CSE showed similar physical withdrawal to those of adults. Mecamylamine did not precipitate anxiety-like behavior at either age. However, both adolescents and adults showed significant anxiety in a light-dark box test 18 h after spontaneous withdrawal. Anxiety-like behavior was still evident in an open field test 1 month after termination of drug treatment, with adolescents showing significantly greater affective symptoms than adults. Our findings indicate that non-nicotine constituents of cigarette smoke do contribute to dependence in both adolescents and adults and emphasize the importance of including smoke constituents with nicotine in animal models of tobacco dependence.

Chronic exposure to cigarette smoke extract upregulates nicotinic receptor binding in adult and adolescent rats.

Heavy smokers display increased radioligand binding of nicotinic acetylcholine receptors (nAChRs). This "upregulation" is thought to be a contributing factor to tobacco dependence. Although cigarette smoke contains thousands of constituents that can contribute to nicotine dependence, it is not well understood whether non-nicotine constituents contribute to nAChR upregulation. In this study, we used an aqueous cigarette smoke extract (CSE), which contains nicotine and soluble constituents of cigarette smoke, to induce nAChR upregulation in adult and adolescent rats. To do this, male rats were exposed to nicotine or CSE (1.5 mg/kg/day nicotine equivalent, intravenously) daily for ten days. This experimental procedure produces equivalent levels of brain and plasma nicotine in nicotine- and CSE-treated animals. We then assessed nAChR upregulation using quantitative autoradiography to measure changes in three nAChR types. Adolescents were found to have consistently greater α4ß2 nAChR binding than adults in many brain regions. Chronic nicotine exposure did not significantly increase nAChR binding in any brain region at either age. Chronic CSE exposure selectively increased α4ß2 nAChR binding in adolescent medial amygdala and α7 binding in adolescent central amygdala and lateral hypothalamus. CSE also increased α3ß4 nAChR binding in the medial habenula and interpeduncular nucleus, and α7 binding in the medial amygdala, independent of age. Overall, this work provides evidence that cigarette smoke constituents influence nAChR upregulation in an age-, nAChR type- and region-dependent manner.

Differences in mechanisms underlying reinstatement of cigarette smoke extract- and nicotine-seeking behavior in rats.

Despite extensive research, current therapies for smoking cessation are largely ineffective at maintaining abstinence for more than a year. Whereas most preclinical studies use nicotine alone, the goal of the present study was to evaluate whether inclusion of non-nicotine tobacco constituents provides better face validity for the development of new pharmacological therapies for smoking cessation. Here, we trained adult male rats to self-administer nicotine alone or cigarette smoke extract (CSE), which contains nicotine and other aqueous constituents of cigarette smoke. After stable self-administration behavior was established, animals underwent extinction training followed by drug and cue primed reinstatement testing. We show that animals that self-administered CSE had significant reinstatement in all drug and drug + cue stimulus conditions whereas animals that self-administered nicotine only showed significant reinstatement in the drug + cue conditions. AT-1001, an α3ß4 nicotinic acetylcholine receptor (nAChR) functional antagonist, attenuated drug + cue-primed reinstatement of both CSE- and nicotine-seeking behavior. However, AT-1001 was less potent in blocking drug-primed reinstatement in animals that had self-administered CSE than in those that had self-administered nicotine alone. This was the case even when nicotine was used to prime reinstatement in animals that had self-administered CSE, suggesting that prior CSE exposure had altered the functional role of α3ß4-containing nAChRs in drug-seeking behavior. These findings confirm the importance of non-nicotine tobacco constituents and α3ß4* nAChRs in cue- and nicotine-primed craving. They also suggest that tests using CSE may be more valid models to study tobacco dependence than use of nicotine alone.

Cigarette Smoke Extract: A Preclinical Model of Tobacco Dependence.

Animal models are used to study many human diseases, one of which is tobacco addiction. Most preclinical models use nicotine alone, although there are >7000 constituents present in tobacco smoke. The clinical literature suggests that cigarettes have a strong addictive potential, which is not paralleled in preclinical studies using nicotine alone. In order to address the gap between clinical and preclinical literature on tobacco dependence, cigarette smoke extracts containing tobacco constituents have been developed. This unit describes a procedure for producing an aqueous cigarette smoke extract (CSE) which animals readily self-administer. In addition, we describe how to make the apparatus for producing CSE and how to analyze the solution for nicotine content. © 2016 by John Wiley & Sons, Inc.

Comparison of the reinforcing properties of nicotine and cigarette smoke extract in rats.

Tobacco dependence is difficult to treat, with the vast majority of those who try to quit relapsing within the first year. Improvements in smoking cessation therapies may be achieved by improving current preclinical research methods. However, most experimental tests in animals use nicotine alone, ignoring the 8000 other constituents found in tobacco smoke. To improve on this model, we have used self-administration to test the reinforcing properties of aqueous cigarette smoke extract (CSE) in rats, made by bubbling cigarette smoke through a saline solution. CSE is more potent than nicotine alone in both the acquisition and maintenance of self-administration, but did not exhibit higher progressive ratio responding. Mecamylamine and varenicline had similar potencies to block nicotine and CSE self-administration, indicating the involvement of nicotinic receptors in CSE reinforcement. Following extinction of responding, reinstatement was triggered by exposing animals to a pharmacological stressor, yohimbine (2.5 mg/kg, i.p.), alone and in combination with cues. Animals that self-administered CSE were significantly more sensitive to stress-induced reinstatement than those that self-administered nicotine. Ligand binding autoradiography studies showed nicotine and CSE to have similar affinities for different nicotinic receptor types. CSE significantly reduced MAO-A and MAO-B activities in vitro, whereas nicotine did not. Although CSE inhibition of MAO-A activity in vitro was found to be partially irreversible, irreversible inhibition was not observed in vivo. These experiments show that CSE is an effective reinforcer acting via nicotinic receptors. Furthermore, it better models MAO inhibition and is more sensitive to stress-induced reinstatement than nicotine alone, which is a potent trigger for relapse in smokers.

Nicotinic receptors in addiction pathways.

Neuronal nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels that consist of pentameric combinations of α and ß subunits. These receptors are widely distributed throughout the brain and are highly expressed in addiction circuitry. The role of nAChRs in regulating neuronal activity and motivated behavior is complex and varies both in and among brain regions. The rich diversity of central nAChRs has hampered the characterization of their structure and function with use of classic pharmacological techniques. However, recent molecular approaches using null mutant mice with specific regional lentiviral re-expression, in combination with neuroanatomical and electrophysiological techniques, have allowed the elucidation of the influence of different nAChR types on neuronal circuit activity and behavior. This review will address the influence of nAChRs on limbic dopamine circuitry and the medial habenula-interpeduncular nucleus complex, which are critical mediators of reinforced behavior. Characterization of the mechanisms underlying regulation of addiction pathways by endogenous cholinergic transmission and by nicotine may lead to the identification of new therapeutic targets for treating tobacco dependence and other addictions.