Psychopharmacology (2002) 162:102–118 DOI 10.1007/s00213-002-1096-0
REVIEW
A. M. Mathieu-Kia · S. H. Kellogg · E. R. Butelman M. J. Kreek
Nicotine addiction: insights from recent animal studies
Received: 13 August 2001 / Accepted: 7 March 2002 / Published online: 15 May 2002 © Springer-Verlag 2002
Abstract Rationale: Recent preclinical behavioral and neurobiological research has characterized important behavioral features and has identified neurobiological substrates that may underlie nicotine reinforcement and addiction. Objective: To examine recent advances on nicotine exposure in preclinical models, from three perspectives: (a) the chronopharmacokinetics of nicotine, (b) behavioral studies on nicotine reinforcement, withdrawal, and reinstatement/relapse, and (c) effects of nicotine on neurobiological substrates after repeated exposure. Results: Preclinical studies can be used to operationally model selected aspects of nicotine reinforcement, withdrawal, and reinstatement or relapse. These may be used to investigate the functional in vivo consequences of acute and long-term changes in neuronal acetylcholine receptor populations that follow nicotine exposure. Behavioral studies focusing on distinct stages of nicotine exposure (e.g., active reinforcement vs. cessation or reinstatement) may also be used in parallel with studies on dopaminergic function, a proposed substrate for the reinforcing effects of nicotine, and of opioid receptor function, a possible site of neuroadaptations secondary to nicotine exposure. Conclusions: While no single current animal model may capture the experience of human smoking or nicotine addiction, increasingly, separate animal models are capturing the full spectrum of behavioral and neurobiological dimensions of this complex condition. Keywords Preclinical model · Drug-seeking behavior · Nicotinic · Dopamine · Opioid
A.M. Mathieu-Kia · S.H. Kellogg (✉) · E.R. Butelman M.J. Kreek Rockefeller University, Laboratory on the Biology of Addictive Diseases, Box 171, 1230 York Avenue, New York, NY 10021-6399, USA e-mail:
[email protected] Tel.: +1-212-3278247, Fax: +1-212-3278574
Introduction Over the past 20 years preclinical research studies have been accumulating on the neurobiology of nicotine addiction. This review emphasizes the recent literature on the pharmacokinetics of nicotine, on animal models to explore the distinct phases of nicotine addiction, and on nicotinic neuropharmacology.
Chronopharmacokinetic characteristics of nicotine Inasmuch as nicotine is thought to be the primary compound in tobacco smoke that establishes and maintains tobacco dependence, knowledge of its pharmacokinetic characteristics is important for understanding the daily smoking cycle. Smoking with frequent or deep inhalation is the route of administration that allows for the most rapid delivery of nicotine to the brain. Nicotine, mainly through pulmonary absorption and via arterial blood, reaches the brain within a few seconds. This pattern is consistent with drugs that have a high abuse potential (Quinn et al. 1997). As it is distributed to other body tissues, circulating nicotine is rapidly oxidized into cotinine, a major metabolite eliminated in urine. In human smokers, as was also shown in recent data using the stable isotopic nicotine technique (Benowitz et al. 1999), the initial distribution half-life (t1/2α) of nicotine in the blood is estimated to be 4–10 min, and the elimination half-life (t1/2β) is approximately 120 min (Benowitz 1988; Lunell et al. 2000; Perez-Stable et al. 1998). Arterial nicotine concentrations peak approximately 20 s after each puff, yielding increases in nicotine concentrations in the order of 10 ng/ml (Rose et al. 1999). This rapid arterial nicotine increase following each puff may be especially relevant to the comparative reinforcing properties of cigarette smoking vs. other forms of nicotine delivery, such as gum, transdermal patch, and nasal spray (e.g., Gourlay and Benowitz 1997; Perkins et al. 1994; Schneider et al. 1996). After termination of each cigarette, the blood level of nicotine rapidly peaks and
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Fig. 1 Proposed circadian profile of circulating nicotine in regular smokers. This theoretical schematic drawing models the intermittent peaking-falling pattern during daylight hours and the circadian fluctuations in nicotine concentration in the blood of human tobacco smokers. Superimposed on these two different patterns of oscillation, the overall level of nicotine gradually increases across the morning, reaches a plateau during the afternoon to the late evening, and finally decreases during the night to achieve low concentrations by the next morning. Ideally, in animal models of nicotine addiction, we should be able to reproduce a similar profile with respect to the analogous circadian rhythm. White horizontal bars Daytime hours; black horizontal bars nighttime hours. This drawing is arithmetically constructed and is based on the short and long metabolic half-lives of nicotine using a model of 8 cigarette smoking episodes a day. This graph is inspired by, and very close to, the experimental data obtained in human smokers (Benowitz et al. 1982)
falls. In addition to this intermittent spike pattern, the overall level of nicotine across the daylight period gradually increases in the morning, reaches maximal level during the afternoon and late evening, and finally falls during the night to achieve low but potentially active concentrations by the next morning (Benowitz et al. 1982). This pattern repeats itself daily (Fig. 1). The potential of nicotine to desensitize nAChRs should also be considered in this context, since receptor desensitization may occur in the presence of relatively low nicotine concentrations (such as may be encountered during “trough” periods; e.g., Marks et al. 1996; see also “Primary molecular site of action: the neuronal nicotinic acetylcholine receptors,” below). In rodent models nicotine is also quickly absorbed and its blood concentration rapidly declines in a biexponential fashion. In rats after ten puffs of cigarette smoke, the t1/2α and t1/2β values of nicotine are 5 and 86 min, respectively (Rotenberg et al. 1980). Similarly, after an acute intra-arterial or intravenous administration of nicotine mean t1/2α and t1/2β of nicotine are approximately 10 and 90 min, respectively (Adir et al. 1976; Kyerematen et al. 1988; Miller et al. 1977; Plowchalk et al. 1992; Sastry et al. 1995). As for acute administration, calculations of the pharmacokinetic parameters in blood from rats chronically treated with nicotine yield a global t1/2 of 44–49 min. This is a consistent value regardless of whether rats receive the subcutaneous nicotine injections two, four, or eight times a day over a 10-day period (Rowell and Li 1997). In the brain the global t1/2 of nico-
tine is slightly longer than in the blood (Plowchalk et al. 1992; Rowell and Li 1997; Sastry et al. 1995). As the number of injections increases, the brain levels of nicotine, which are on average two or three times higher than those in the blood (Donny et al. 2000; Rowell and Li 1997), increase and reach a plateau which is difficult to extrapolate from the blood values (Ghosheh et al. 2001). Additional work is needed to establish the pharmacokinetic profile of nicotine in specific brain regions and in studies of behavioral models of addiction. Furthermore, recent experiments have contributed to the development of possible novel agonist pharmacotherapies for breaking the cycle of nicotine addiction in humans. For example, recent studies have determined that nornicotine (N-demethylated nicotine; an alkaloid present in tobacco, and also a minor nicotine metabolite), was weakly selfadministered by rats but could antagonize intravenous nicotine self-administration (SA; Bardo et al. 1999; Green et al. 2000).
Acquisition and maintenance of nicotine-seeking behavior Numerous experiments have shown that nicotine serves as a positive reinforcer, and this has been extensively documented in diverse animal species (almost exclusively male) including primates, dogs, and rodents (for reviews see Goldberg et al. 1983; Henningfield and Goldberg 1983; Perkins et al. 1999; Stolerman 1999). Although the conditioned place preference assay (Calcagnetti and Schecter 1994; Clark and Fibiger 1987; Jorenby et al. 1990; Risinger and Oakes 1995) has been used in some studies, the intravenous SA assay has been favored as a more direct way of demonstrating that nicotine initiates and sustains drug-seeking behavior (self-administration by other routes has also been studied, e.g., Glick et al. 1996). However, to perfectly reproduce the route, frequency, chronology, and intake pattern of smoking the ideal approach would be to have an animal actually smoke cigarettes or self-inhale smoke on a smoking machine. In fact, rhesus monkeys and baboons do smoke after this behavior has been established in an operant situation. In some extreme cases they would smoke up to 40 cigarettes/day, for periods lasting up to 3 years (Ando
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and Yanagita 1981; Rogers et al. 1985, 1988). Despite the clear analogy to human behavior, studies using smoking protocols with nonhuman primates have been rare in the last decade, probably due to material and technical demands. As is commonly observed in intravenous SA assays, a crucial experimental parameter that governs the acquisition of SA behavior is the unit dose of nicotine (all doses in text are presented as nicotine base, unless otherwise stated). The optimal acquisition dose is thought to be around 0.01–0.03 mg/kg per intravenous injection in many animal species (Corrigall and Coen 1989; Cox et al. 1984; Donny et al. 1995, 1998; Goldberg et al. 1981; Henningfield and Goldberg 1983; Risner and Goldberg 1983; Shoaib and Stolerman 1999; Shoaib et al. 1997; Suto et al. 2001; Tessari et al. 1995). The maintenance of nicotine-seeking behavior in animal models is also dose dependent, and an inverted “U” function is usually found. However, the overall function may be shallower for nicotine than for other reinforcing drugs, and may suggest comparatively less dose regulation for nicotine vs. other reinforcing drugs (Lynch and Carroll 1999; Rose and Corrigall 1997). The inverted “U” shape doseeffect curve has also been observed in conditioned place preference assays with mice (e.g., Risinger and Oakes 1995) and rats (Fudala and Iwamoto 1986). Nicotine-seeking behavior has been examined in a variety of procedural conditions, both analogous to and different from the human smoking cycle. The issue of restricted versus unlimited access to nicotine is central because of concerns about how to best mimic human smoking behavior with respect to both the frequency of cigarette use and the sleep-wake cycle. Restricted access to nicotine has been a common model, which may not capture the experience of human smokers who have access to tobacco at any time. In light of recent legislative interventions in the United States, access to tobacco is becoming more restricted, and increasing numbers of smokers may actually be following a fixed or variable interval pattern of SA (i.e., possibly similar to a “binge” use pattern). As investigations go forward, it will be increasingly useful to systematically compare restricted access, unlimited access, and “binge” models. In an example of research utilizing schedules of repetitive nicotine exposure, nicotine was administered ad libitum to mice in their sole source of drinking water. Mice, as nocturnal animals, drink primarily during their active phase, and as a result their plasma levels of nicotine follow a circadian high-to-low profile similar to that reported in human smokers (for reviews see Pietila and Ahtee 2000). This oral intake approach leads to a relatively slow nicotine absorption pattern that may not fully capture common cigarette use patterns. Valentine et al. (1997), in an attempt to more closely approximate human nicotine use, developed a model in which rats were allowed unlimited access to nicotine using an intravenous SA assay while respecting the circadian/meal cycles. The four doses used (0.00375–0.03 mg/kg) were low compared to the commonly used doses in restricted
access; nevertheless, the rats rapidly initiated SA behavior. However, there was a relatively low rate of SA behavior, and there was not a general distinction between responding on the active and inactive levers. In these conditions of unlimited drug access the maximal nicotine intake was approximately 0.2–1.4 mg/kg per day, vs. 0.4–0.9 mg/kg per hour in the restricted access approach (Corrigall and Coen 1989; Cox et al. 1984; Donny et al. 1995). Other important factors (both similar to and different from the human smoking cycle) that could influence the nicotine-seeking behavior also include previous experimental administration or SA of various compounds such as cocaine (Epping-Jordan et al. 1999; Tessari et al. 1995), caffeine (Tanda and Goldberg 2000) or nicotine itself. Thus, noncontingent exposure to nicotine can facilitate the later acquisition of nicotine SA (Shoaib et al. 1997), the development of conditioned place preference (Shoaib et al. 1994), and the increased intake of nicotine as shown in a two-bottle choice study (Maehler et al. 2000). Although the direct parallel between these findings and human smoking behavior is unclear, these findings may reflect the process by which a relatively small number of cigarette exposures puts an individual at high risk for developing nicotine addiction. Within the SA literature, there has been a growing interest in the difference between the fixed-ratio and the progressive-ratio schedules. Markou et al. (1993) have proposed that the fixed-ratio schedules are a measure of the “hedonic” value of the drug while the progressiveratio schedules are analogous to the experience of motivational incentive or “drug craving.” The “breaking point” is seen as a potential measure of the “cost” of the drug. Donny et al. (2000) used this model to evaluate sex-based, motivational-incentive differences in rats that could potentially parallel the greater difficulty that women have with the cessation of smoking (Perkins et al. 1999). It has been recently proposed that stimuli associated with smoking or with nicotine SA procedures (e.g., stimulus lights) acquire conditioned reinforcer status, and that this contributes to the maintenance of tobacco or nicotine SA (Balfour et al. 2000; Di Chiara 2000; Donny et al. 1999). This may be analogous to the sensory stimuli (e.g., in the respiratory tract) that may be temporally associated with rapid arterial nicotine increases during cigarette smoking in humans (e.g., Rose et al. 1999).
Extinction of nicotine-seeking behavior and withdrawal Smoking cessation or periods of forced nicotine abstinence have been examined in animals undergoing extinction of nicotine-seeking behavior or cessation after chronic experimenter-delivered administration. In rodent SA experiments studying extinction, when saline is substituted for nicotine, an overall decline in drug-seeking behavior is usually observed. During the first substitu-
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tion session some rats begin pressing the bar less frequently while others demonstrated a typical “extinction burst” – a rapid but short-lived increase in bar pressing levels (Corrigall and Coen 1989; Donny et al. 1995; Shoaib et al. 1999). Interestingly, the drug-seeking behavior remains above control levels for 4–13 sessions before being completely extinguished (Chiamulera et al. 1996; Donny et al. 1995; Tessari et al. 1995); this resistance to extinction further supports the view of nicotine as a powerfully addictive compound (e.g., DiChiara 2000). Early and long-lasting behavioral signs of extinction were also produced by preadministration with a nicotinic antagonist (Goldberg and Spealman 1982; Shoaib et al. 1997). Nicotine withdrawal in animals consists of a syndrome of spontaneous or precipitated physical signs – similar to those found during opiate withdrawal (including teeth-chattering/chews, yawns, abdominal writhes/gasps, ptosis, wet shakes/tremors, rearing, and jumping). Increases in body weight, decreases in spontaneous activity, hyperphagia, and hyperdipsia are also used to evaluate the withdrawal intensity (Hildebrand et al. 1997; Levin et al. 1987; Malin et al. 1992). In rats the discontinuation of subcutaneous nicotine infusions (3.2–3.6 mg/kg per day) produces a constellation of spontaneous behavioral signs during the 2 days following cessation (Hildebrand et al. 1997; Malin et al. 1992, 1994; Watkins et al. 2000a). Nicotinic antagonists are also able to precipitate all of these abstinence symptoms, and a subsequent nicotine injection (e.g., 0.15–0.4 mg/kg subcutaneously) attenuates their intensity (Hildebrand et al. 1997; Malin et al. 1994, 1998). In a study using daily intermittent nicotine injections in mice, mild somatic withdrawal signs were reported between 24 and 48 h after the final bolus, and some of them persisted for 3–4 days (Isola et al. 1999). When these studies are contrasted, continuous and intermittent exposures share similar withdrawal symptom while following different temporal courses. It is to be noted that none of the studies to date on withdrawal signs have used a SA model (to our knowledge), and this approach would more closely mimic the behavior of smokers. The intracranial self-stimulation (ICSS) model has also been able to measure the reinforcing properties of nicotine exposure and the state change (or “presumed dysphoria”) that follows its termination. In the ICSS model the effect of nicotine is thought to mimic the power and rewarding effect of each electrical stimulation (for a review see Wise 1996). As a result the ICSS threshold is lower for the nicotine-treated animals than for those receiving vehicle. This stimulus substitution is reached as early as the second day of administration and lasts as long as nicotine is repeatedly injected and not blocked with nicotinic antagonists (Bauco and Wise 1994; Bespalov et al. 1999; Bozarth et al. 1998a, 1998b; Huston-Lyons et al. 1993; Ivanová and Greenshaw 1997). In a contrasting effect, animals maintained on a continuous infusion of nicotine by osmotic pump do not
show this same change in the ICSS (Epping-Jordan et al. 1998). Nonetheless, once the continuous nicotine infusion is terminated, the spontaneous withdrawal condition elevates ICSS thresholds. The rat may be postulated to be in a “dysphoric” state, a condition that may parallel the “negative mood state” seen in smokers, which is typified by irritability, lack of concentration, anxiety, and craving (Benowitz 1988; see also below). Similarly, conditioned place aversion induced by precipitated nicotine withdrawal may provide a sensitive measure of internal states (e.g., discriminative or hedonic) produced by nicotine withdrawal in animals (Ise et al. 2000; Watkins et al. 2000a). Other studies have also attempted to examine the “subjective” or interoceptive state of animals when they experience nicotine withdrawal. Spontaneous withdrawal in rats has been marked by an increased “sensorimotor reactivity” response to acoustic startle (Acri et al. 1991; Harris et al. 1986; Helton et al. 1993, 1997; Rasmussen et al. 1997). The withdrawal state after chronic, repeated nicotine administration is also notable for its significant disruption of operant behaviors not directly linked to the drugseeking behavior. When nicotine access is terminated, the reinforcement potential of food or sweetened solution is suppressed, and this interference is immediately reversed when nicotine is again available (Carroll et al. 1989; Corrigall and Coen 1989). Similarly, the abrupt cessation of nicotine administration in rats trained on a shock-avoidance schedule is also associated with a reduction in lever-pressing responses and with an increase in the number of shocks received (for a review see Balfour 1991). These behavioral alterations are seen as acute signs of withdrawal and could be compared to the loss of the “psychological benefits” of smoking commonly described by smokers during abstinence.
Reinstatement model of nicotine-seeking behavior and relapse Experimental relapse from nicotine addiction has been examined through the reinstatement of previously extinguished nicotine-seeking behaviors, using spontaneous recovery conditions, nicotine priming doses, or stressinducing stimuli. Animals removed from their operant chamber or in saline substitution protocols spontaneously recover drug-seeking behavior at levels comparable to those observed during the preextinction period within one session (Donny et al. 1995; Goldberg et al. 1981; Valentine et al. 1997). Similarly, after 6–20 sessions of saline substitution, the availability of nicotine progressively reinitiates SA to preextinction levels (Goldberg et al. 1981; Tessari et al. 1995). Lastly, after a 3-week period of nicotine withdrawal, rats reexposed to the drug-taking environment spontaneously recover their previous behavior (Shaham et al. 1997). Whatever the experimental condition, spontaneous recovery of SA occurs after either a short or long cessation period, and this phenomenon
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illuminates the connection between environmental factors and relapse. Another approach to study relapse is to use an acute priming nicotine injection to reinstate nicotine SA. An acute priming nicotine dose (either intravenous or subcutaneous) reinstates nicotine SA after 4–13 days of saline substitution (Chiamulera et al. 1996; Shaham et al. 1997). Interestingly, higher levels of reinstatement of nicotine SA are observed with a lower priming dose of nicotine (Chiamulera et al. 1996). Thus, in addition to potentially conditioned environmental stimuli, nicotineseeking behavior can also be reinitiated through noncontingent nicotine reexposure. Intermittent footshock as a stressor is also an effective method of reinstating nicotine-seeking behavior – even after periods ranging from 5 days to 2 weeks (e.g., Buczek et al. 1999). This supports the clinical observation that stress can be a major precipitant of relapse; continued work in this area is crucial as this is a major therapeutic intervention point for smoking-cessation medications.
Animal models of the “psychological” experience of smoking A number of recent studies have attempted to model the wide variety of experiences and use patterns that smokers report. Areas of investigation have included psychological mood states, such as depression and anxiety, and the cooccurring use of other substances, such as caffeine and alcohol. The relationship between nicotine and depression has been one of growing interest. Studies have shown that cigarette smoking is quite common among depressed individuals and that individuals with a long history of depression are less likely to succeed at smoking cessation (Tizabi et al. 1999). In fact, buproprion, an antidepressant, is prescribed to patients seeking to discontinue smoking (NIDA 1998). Semba et al. (1998) used a “learned helplessness” model that involved shock and an escape test in male rats and found that chronic administration of nicotine may have an apparent antidepressant effect, and that mecamylamine, a nicotinic antagonist, reversed this effect of nicotine. Similarly, Tizabi et al. (1999) examined the apparent antidepressant effects of nicotine in the “forced swim” model of depression with two related rat lines. The Flinders Sensitive Line (FSL) has been specifically bred to be a “depression-prone” rat, and the Flinders Resistant Line (FRL) has been bred to be a “depressionresistant” rat. After both acute and chronic (14-day) administration of nicotine, immobility was decreased in the “depressed” FSL rat but not in the FRL rats. Djuricˇ et al. (1999), also using the forced swim-test with female FSL and FRL rats (found after 14 days of oral nicotine ingestion), an antidepressant effect of nicotine that did not interact with genetic background. Another area of investigation is the relationship between smoking and anxiety, focusing on the anxiolytic/
anxiogenic effects of nicotine and nicotine withdrawal. Ouagazzal et al. (1999) found that nicotine had no effect at the lower doses, but was anxiogenic in the elevated plus-maze test at higher doses. Irvine et al. (2001a) in a study with male Wistar rats examined the effects of nicotine in the social interaction test after 4 weeks of nicotine SA and then, again, 24 to 72 h after its cessation. Rats that self-administered nicotine showed less interaction with other rats in the social interaction test than the control group (an apparent nicotine-induced, anxiety-like effect). These group differences disappeared during the nicotine cessation period. Irvine et al. (2001a) concluded that “nicotine self administration is not maintained because of its anxiolytic effect, but despite or because of, its anxiogenic effect.” They also pointed out that the findings of the animal studies were in conflict with common assumptions about the behavior of human smokers. The cessation of nicotine administration also appears to induce anxiety in animals, and this has been proposed as paralleling the withdrawal symptoms that serve as negative reinforcers working to maintain the use of tobacco. Classical rodent models of “anxiety,” such as the black/white box test (Costall et al. 1989), the elevated plus-maze test (Irvine et al. 2001b), and the social interaction test (Irvine et al. 1999), have been used to evaluate the nicotine withdrawal state. An anxiogenic response first appeared within 8–96 h following the cessation of chronic experimenter-delivered nicotine in these settings. However, anxiogenic effects were not detected 1 or 3 days following the end of a 4-week period of intravenous nicotine (0.03 mg/kg per injection) SA (Irvine et al. 2001a; see above). To summarize, the overall relationship between nicotine and anxiety remains complex and unclear. Tobacco use and cigarette smoking often occur concurrently with the use of other substances. Recent studies have attempted to examine two common combinations – nicotine and caffeine and nicotine and alcohol. Shoaib et al. (1999) reported that caffeine markedly increased the reinforcing properties of nicotine using an SA model, and its consequent removal diminished the reinforcing properties of nicotine. This finding supports a role for caffeine as a possible contributor to nicotine SA and relapse (Shoaib et al. 1999). Other studies examined the interaction between nicotine and alcohol. Here the emphasis has been on the effect of nicotine on alcohol consumption. Nadel and Samson (1999), using male Long-Evans rats, found that nicotine had only a slight effect on increasing alcohol SA. Lê et al. (2000), using male Wistar rats, found a complex relationship between exposure to nicotine and alcohol consumption. Nicotine only impacted alcohol SA at higher doses (0.8 mg/kg), and the effect was biphasic, as it actually suppressed alcohol consumption at first and then increased it. The authors suggested that this biphasic effect of nicotine on alcohol consumption might explain conflicting reports about the relationship between these substances in the literature.
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Gender and genetics as mediating variables in the effects of nicotine In a comprehensive review, Perkins et al. (1999) discussed the emerging evidence that men and women differ in their responses to smoking in general. The human data point to a pattern in which the relative importance of “nonnicotine” factors that are involved in smoking behavior is greater for women than for men. This may include such factors as pleasure from the smoke itself, social reinforcements for smoking, comfort from having something to manipulate in social situations, and gratification from oral stimulation. Faraday et al. (1999) have also pointed out that women are more inclined to see smoking as a useful mechanism for coping with social situations or unpleasant affective states. Perkins et al. (1999) went on to point out that these differences have had troubling consequences for women smokers who are seeking to quit; mainly that women have been less successful than men in studies using nicotine-replacement interventions. Interestingly, women respond better than men in studies in which such nonnicotine medications as clonidine or buproprion are used (Perkins et al. 1999). The animal literature has supported this pattern to some degree as well. Perkins et al. (1999) note that nicotine appears to be equally reinforcing for males and females, but that aspects of the experimental environment that may become associated with nicotine may contribute a more significant role in the maintenance of nicotine SA for females than for males. Faraday et al. (1999) examined the interaction of gender and housing (single versus group) on nicotine-induced locomotion in Long-Evans rats in a Plexiglass arena. The emerging patterns showed that there was an interaction between gender and housing, and nicotine affected the arousal and exploration behavior of males, while it appeared to have had an “anxiolytic-like” effect on the female rats. Donny et al. (2000), examined sex differences in SA using Sprague-Dawley rats. In this study they used both fixed-ratio schedules, which they postulated reflect the hedonic aspect of nicotine, and progressive-ratio schedules, which they suggest better reflect “drug craving” or incentive motivation (Markou et al. 1993). Their finding was that while that there was no significant sex difference in the fixed-ratio schedule, females had higher breaking points in the progressive-ratio schedule. The females, in both contingency paradigms, also demonstrated a shorter latency to first infusion, and this may be interpreted as another index reflecting nicotine incentive motivation. Lastly, in an attempt to look at the interplay between genetics and the SA of drugs of abuse, Todte et al. (2001) examined the propensity of Brown-Norway rats (BNR) and Wistar-Kyoto rats (WKR) to orally self-administer ethanol, nicotine, cocaine, and morphine. The WKR strain is known for its higher emotionality and its lower plasma stress catecholamine responses (Todte et al. 2001). They also examined age as an important mediating
Fig. 2 Anatomical sites of action for nicotine in the rat brain. This sagittal section illustrates the distribution of high-affinity binding sites for [3H]nicotine. SC Superior colliculus, AV anteroventral nucleus, VL ventrolateral nucleus, Rt reticular nucleus, Cpu dorsal striatum, Acb nucleus accumbens, SNC/VTA substantia nigra compacta/ventral tegmental area. (Slightly modified from Clarke et al. 1985, copyright by the Society for Neuroscience)
variable. Both strain and age differences were found, with the WKR consuming more ethanol and nicotine than the BNR, and the older BNR consumed less nicotine and ethanol than the younger (Todte et al. 2001). These and other (Robinson et al. 1996; Stolerman et al. 1999) recent studies therefore support future investigation of the influence of genetic factors in nicotine SA.
Primary molecular site of action: the neuronal nicotinic acetylcholine receptors Nicotine specifically binds to the nicotinic receptors (nAChRs), which mediate initially its effects, and are widely distributed in the brain of many species including humans (for a review see Paterson and Nordberg 2000). A precise map of the distribution of nAChR (Fig. 2) was first established using quantitative autoradiography with [3H]nicotine (Clarke et al. 1984, 1985), and later with other approaches such as immunohistochemistry (Deutch et al. 1987; Swanson et al. 1987) and in situ hybridization (Dineley-Miller and Patrick 1992; Flores et al. 1992; Wada et al. 1989). Due to the wide distribution of nAChRs nicotine addiction or chronic administration may alter the function of most, if not all, of the brain regions and various neurotransmitter systems. Although nAChRs are located in many peripheral tissues, the rewarding properties of nicotine are mediated by the interaction of this alkaloid with nAChRs in the CNS. In animals, systemic administration of a nicotinic antagonist which crosses the blood-brain barrier (mecamylamine) attenuates the intravenous SA of nicotine (Corrigall and Coen 1989; Goldberg et al. 1981; Risner and Goldberg 1983). Similarly, mecamylamine pretreatment blocks the effects of nicotine in a conditioned place preference assay (Fudala et al. 1985) and inhibits the stimulant effect of nicotine on locomotor activity, in chronically treated rats (Benwell et al. 1995). During
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precipitated nicotine withdrawal, recent data demonstrate that central nAChRs mediate elevated brain stimulation reward thresholds, whereas both central and peripheral nAChRs appear to mediate the somatic withdrawal syndrome (Watkins et al. 2000a, 2000b). Neuronal nAChRs are pentameric and allosteric membrane protein complexes. They are members of the superfamily of ligand-gated ion channels permeable to cations (Na+/K+, Ca2+; see Changeux et al. 1984), and this ionic flux leads to a depolarization and an excitatory response of neurons. Each neuronal nAChR subunit is encoded by a separate gene. To date, of the 12 distinct genes cloned, the expression of nine has been found in the brain of rat or primate. These subunits have been named α2, α3, α4, α5, α6, α7, β2, β3, and β4. Therefore, multiple combinations of some but not all of these five subunits can form functional heteromeric (α with β) or homomeric (α) nAChRs with distinct pharmacophysiological features (Lukas et al. 1999; Zoli et al. 1998). In addition to this molecular complexity, the regional distribution of the different subunits differs among brain areas and among neuronal populations. In brain, the most widely and concurrently expressed subunits are α4 and β2. In contrast, the patterns of α7, α3, and α5 subunit expression are more limited in both their density and localization. The α2, α6, β3, and β4 subunits are limited to only a few brain regions. Although the molecular diversity of the initial site of action for nicotine is well explored, it is still unknown whether one (or several) particular subunit combination of nAChR preferentially mediates the reinforcing effects of nicotine, or whether a particular genotype(s) is involved in tobacco consumption behavior. Studies with subtype-selective nAChR antagonists, focusing on their ability to modulate nicotine’s reinforcing (and other) effects would therefore be of value. For example, a recent study has shown that the α4/β2 nAChR selective antagonist erysodine blocked the discriminative and reinforcing effects of nicotine in rats (Mansbach et al. 2000). Studies using transgenic mice that lack specific subunits of the nAChR could also help determine which subunits play a fundamental role in nicotine addiction (for reviews see Cordero-Erausquin et al. 2000; Marubio and Changeux 2000; Picciotto et al. 2000). Several (but not all possible) mice lacking one or two subunits have been generated. While some of these appear to have multiorgan dysfunctions and impaired survival, others reach adulthood (Bansal et al. 2000; Caldarone et al. 2000; Franceschini et al. 2000; Marubio et al. 1999; Paylor et al. 1998; Ross et al. 2000; Rossi et al. 2001; Vetter et al. 1999; Xu et al. 1999a, 1999b). Their behavior has not been fully explored in the context of nicotine reinforcement. While α7 subunit-deficient mice have no detectable abnormalities in the density of [3H]nicotine sites (Orr-Urtreger et al. 1997), α4 and β2-subunit deficient mice have lost almost all their sites in all brain regions (Marubio et al. 1999; Ross et al. 2000). Interestingly, β2 subunit-deficient mice which have been trained to selfadminister cocaine cease their nose-poking behavior
when nicotine is substituted for cocaine (Epping-Jordan et al. 1999; Picciotto et al. 1998). Similar studies with α4- or α7-deficient and viable mice have not been described to date. Nicotine-induced place preference experiments using these three types of genetically altered mice have also not been documented, although a recent report suggests that the β2 subunit contributes to the motivational effect of cocaine (Zachariou et al. 2001). Taken together, it is therefore possible that the reinforcing and motivational properties of nicotine are mediated by a neuronal nAChR made of at least one β2 subunit. In contrast to many other receptors that are downregulated following a chronic exposure to their agonist, nAChR are unexpectedly up-regulated after chronic nicotine administration (Marks et al. 1983; Schwartz and Kellar 1983; for a review see Wonnacott 1990). Instead of a decrease in the number of nicotine binding sites, an increase following chronic nicotine administration has been observed in postmortem brain studies of rodents (Marks et al. 1992; Pauly et al. 1991, 1996; Zhang et al. 1994) and humans (Benwell et al. 1988; Breese et al. 1997; Court et al. 1998) as well as in the living brain of baboons (Kassiou et al. 2001). Chronic exposure of rats to mainstream cigarette smoke for more than 3 months resulted in an increase in nicotine binding sites in the cerebral and cerebellar cortices and more modestly in the striatum but not in the thalamus or hippocampus where nAChR are also densely expressed (Yates et al. 1995). With few exceptions studies using multiple injections of various doses of nicotine have shown increases in nicotine binding sites in the cortex, the striatum, and the hippocampus (Collins et al. 1988; Coutcher et al. 1992; el-Bizri and Clarke 1994; Flores et al. 1997; Ksir et al. 1987; Lapchak et al. 1989; Nordberg et al. 1989; Romanelli et al. 1988; Rowell and Li 1997; Ulrich et al. 1997; Wall et al. 2000; Zhang et al. 1994). The up-regulation of nicotine binding sites, which was also found in the brains of rats that self-administered nicotine (Donny et al. 2000), occurs largely in specific cerebral regions. This may reflect a differential responsiveness of nAChR to chronic exposure to nicotine, possibly related to a differential subunit composition. Immunoprecipitation protocols or binding studies using specific subtype ligands suggest that the subunit composition of the up-regulated nAChRs is made up of α4 and β2 subunits in the cortex and the hippocampus of rats chronically treated with nicotine and not of α3 or β4 subunits (Flores et al. 1992, 1997). Chronic nicotine exposure in mice also led to an increase in α-bungarotoxin sites (thought to represent nAChR that contain the α7 subunit; Marks et al. 1983; Pauly et al. 1991), although this has not been found in the postmortem brains of human smokers (Court et al. 1998). It has been proposed that simultaneously with an increased number of nicotine sites a functional desensitization of the nAChR occurs during chronic exposure to nicotine. Indeed, the nAChR is an allosteric protein complex with at least three conformational states. The interaction of nicotine or acetylcholine (low affinity) with the
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nAChR induces a quick transition from the resting state (channel closed) to the active state (channel opened with high affinity). If nicotine exposure is prolonged, the receptor slowly enters into a desensitized state (channel closed) that is unresponsive to the agonist. The increased number of nicotine sites could be a neuronal response to the accumulation of receptors desensitized after long-term nicotine exposure. Desensitization of nAChR populations may be relevant in human smokers, since prolonged exposure to low nicotine concentrations (such as may be observed in “trough” periods or at nighttime) may result in receptor desensitization (see “Chronopharmacokinetic characteristics of nicotine” above; see also Marks et al. 1996; Rosecrans and Karan 1993) In mice regardless of the nicotine administration protocol used changes in nAChR numbers are not attributable to transient or sustained changes in nAChR subunit mRNA levels (Pauly et al. 1996). The nature of the mechanisms involved in the up-regulation of nicotine receptors is therefore likely posttranscriptional, although increased synthesis may not be totally excluded (Ryan and Loiacono 2001; Yu et al. 1996). Since the reported up-regulation of nAChR is also accompanied by enhanced behavioral responses such as locomotor activity or cognitive performance, this molecular event might be related to the behavioral sensitization observed during chronic nicotine exposure (Abdulla et al. 1996; Ksir et al. 1985, 1987; Nisell et al. 1996). Of greater salience to the field of nicotine addiction is the question of whether changes in the density of nicotinic sites are a crucial molecular event in the early abstinence phase. It could be hypothesized that as soon as the concentration of nicotine is no longer sufficient to desensitize nAChR, the desensitized nAChR return to their resting state. Consequently, when nicotine exposure is terminated, the total number of functional nAChR potentially sensitive to nicotine (or acetylcholine) should be greatly increased at the cell surface, thus rapidly altering the physiology of neuronal circuits. However, in adult rodents the return of the up-regulated receptors to basal values takes at least 1 week after the cessation of nicotine administration (although not in all brain regions; Collins et al. 1990; Koylu et al. 1997; Pietila et al. 1998). In addition, a more robust and longer lasting receptor up-regulation was observed in rats exposed to nicotine during the adolescent period (Trauth et al. 1999). Overall, it is possible that the persistent up-regulation of nicotine binding sites during cessation may play a role in the intensity of the early withdrawal symptoms and therefore the likelihood of relapse. Knowledge of the neurobiological and behavioral consequences of the increase in nicotine binding sites in various brain regions is therefore important to extend our understanding of altered function in active smokers as well as in others exposed to nicotine (such as nonsmokers, newborns exposed during pregnancy, and ex-smokers therapeutically exposed to nicotine through nasal spray, transdermal patches, or gum).
Downstream sites of action: the mesolimbic dopamine neurons Although the function of numerous neurotransmitter systems expressing nAChR could be altered by nicotine, the initial reinforcing properties of this compound are thought to be mediated primarily through the mesolimbic and mesocortical dopamine (DA) systems. These neuronal pathways consist primarily of the DA neurons from the ventral tegmental area (VTA) that project to various cerebral regions, notably the nucleus accumbens and various limbic cortices. These brain areas are involved in reward functions and mediate the actions of natural reinforcers as well as many drugs of abuse. To our knowledge, SA of nicotine directly into those DA terminal areas has not been described to date. However, direct administration of nicotine in the mesolimbic and mesocortical regions substitutes for the discriminative stimulus of nicotine established through subcutaneous injection (Miyata et al. 1999). The importance of those regions, especially the nucleus accumbens, was also supported by the finding that bilateral lesions of DA neurons led to a reduction in intravenous nicotine SA (Corrigall et al. 1992; Singer et al. 1982). A bilateral intra-accumbens microinjection of 6-hydroxydopamine into the accumbens also abolished the locomotor stimulant effect of subcutaneous nicotine in rats (Clarke et al. 1988). Similarly, the blockade of DA transmission by selective DA antagonists in nonlesioned rats also reduced nicotine SA (Corrigall and Coen 1991b) and completely blocked nicotine-conditioned place preference (Acquas et al. 1989) and nicotine-induced ICSS (Huston-Lyons et al. 1993). These observations, in sum, suggest a major role for the DA neurons, especially from the mesolimbic system, in mediating the reinforcing effects of nicotine. Nicotinic AChRs are localized on the cell bodies of DA neurons in the ventral mesencephalon as well as on DA terminals in the nigrostriatal and mesolimbic pathways (Clarke and Pert 1985; Clarke et al. 1984; London et al. 1985). Most of those DA neurons express abundantly the α6 and β3 nAChR subunit mRNA (Le Novere et al. 1996) and also at moderate levels α3, α4, α5, and β2 subunit mRNA (Dineley-Miller and Patrick 1992; Duvoisin et al. 1989; Sorenson et al. 1998; Wada et al. 1989). Although there is no unique expression pattern of these various nAChR subunits within DA neurons from the VTA, the activation of nAChR from this area alone could explain some of the behavioral effects seen after repeated systemic administration of nicotine. Indeed, infusion of a nicotinic antagonist into the VTA prior to intravenous nicotine SA sessions significantly decreases the number of nicotine infusions obtained (Corrigall et al. 1994). Moreover, repeated applications of a nicotinic agonist (cytisine) directly into the VTA induced a place preference for the cytisine paired compartment (Museo and Wise 1994), and repeated applications of nicotine showed a progressive increase in the locomotor response in rats (Panagis et al. 1996; Reavill and Stolerman 1990).
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These data support the hypothesis that the α3, α4, α5, α6, β2, and β3 subunit containing nAChR, which are localized on the mesolimbic system, contribute to both the reinforcing properties and the psychostimulant effects of nicotine. The interaction of nicotine with nAChR localized on the VTA-accumbens pathway causes profound modifications in the functioning of DA neurons. Acute as well as chronic intermittent administration of nicotine leads to an increase in the activity of midbrain DA neurons by accelerating the firing of neurons and by switching their activity from regular to burst firing patterns (Grenhoff et al. 1986; Lichtensteiger et al. 1982; Mereu et al. 1987; Nisell et al. 1996). Moreover, cessation of continuous nicotine administration by subcutaneous osmotic minipump (6 mg/kg for 12 days) also alters firing rates of midbrain DA neurons (Rasmussen and Czachura 1995). These changes in electrophysiological activity, especially burst firing, could be associated with greater neurotransmitter release in the terminal fields of DA neurons, initiating neurochemical changes within those neurons or within neurons from projection areas. An acute systemic injection of nicotine, such as with other drugs of abuse, increases the extracellular concentrations of DA in the terminal fields of the mesolimbic system (Imperato et al. 1986; Maisonneuve and Glick 1999; Mifsud et al. 1989; Pontieri et al. 1996; for a review see Di Chiara 2000), the nigrostriatal pathway (Di Chiara and Imperato 1988; Toth et al. 1992), and the mesocortical pathway (Bassareo et al. 1996; Summers and Giacobini 1995; Toth et al. 1992). Repeated nicotine injections in rats (0.4 mg/kg nicotine tartrate subcutaneously for 7 days) increased the dopamine release caused by local nicotine administration in the striatum, as measured with in vivo microdialysis (Marshall et al. 1997). Interestingly, this effect was not observed if the same daily nicotine dose was administered as a continuous infusion (Marshall et al. 1997). In other experiments with chronically treated rats, a systemic injection of nicotine increased DA release in both the prefrontal cortex (Nisell et al. 1996) and the nucleus accumbens (Benwell et al. 1994; Nisell et al. 1997; Reid et al. 1996). The nicotineinduced DA release is of the same amplitude or higher in chronically (as compared to acutely) injected rats. Rats treated daily with nicotine may have both higher basal extracellular DA concentrations in the nucleus accumbens, as well as an increased DA release in response to a further nicotine challenge (Benwell and Balfour 1992; Benwell et al. 1994, 1995; Damsma et al. 1989). Local intrategmental mecamylamine injection reduced DA overflow in the ipsilateral nucleus accumbens of rats treated chronically with nicotine (by osmotic minipump) for 14 days (Hildebrand et al. 1999). A similar effect was observed after the termination of nicotine administration (Fung et al. 1996). These observations suggest that dynamic DA transmission is preserved (and may be enhanced) in the mesolimbic and corticolimbic systems during intermittent nicotine administration, and may be decreased during withdrawal.
Nicotinic receptors localized on DA cell bodies or terminals could initiate nicotine-induced DA release. In fact, in vivo microdialysis studies have shown that the local infusion of nicotine directly into the VTA is sufficient to increase the DA extracellular concentration in the nucleus accumbens (Marshall et al. 1997; Mifsud et al. 1989; Nisell et al. 1994). However, although persistent effects are observed when nicotine is administered locally to the DA cell bodies, local administration to the terminal fields of DA neurons can also stimulate (albeit transiently) DA release (Mifsud et al. 1989; Nisell et al. 1994). VTA or somatodendritic nAChRs have been implicated in the effects of nicotine in SA, conditioned place preference assays, or on spontaneous activity. Thus nAChRs of the VTA have a crucial role in both nicotineinduced DA release and in the reinforcing properties of nicotine. A possibly contrasting line of evidence involves the finding that schizophrenic patients receiving haloperidol (a dopaminergic D2-like antagonist) smoke at high rates (e.g., McEvoy 1995). It is possible that this behavior continues because high levels of tobacco use may allow the individual to compensate for decreases in dopaminebased reinforcement caused by haloperidol (see also Rose and Corrigal 1997) and because the individual may be receiving continued reinforcement from nonnicotine stimuli involved in smoking. Several investigators have also suggested that the maintenance of drug SA (and particularly nicotine or tobacco) may also depend critically on cues associated with smoking, which may acquire “conditioned reinforcer” status by their pairing with self-administered nicotine and its secondary dopamine release (Balfour et al. 2000; Di Chiara 2000; Donny et al. 1999; see also above). Of particular relevance to this hypothesis is the temporally contingent relationship between smoking behavior (e.g., puff inhalation) and its associated sensory stimuli or “cues”, with a rapid rise in intra-arterial nicotine levels (see “Chronopharmacokinetic characteristics of nicotine”, above). It has also been suggested that the maintenance of nicotine or tobacco SA is especially dependent on possible secondary reinforcers, due to the presumed desensitization of nAChRs after prolonged exposure (Balfour et al. 2000).
The endogenous opioid system and its connection to the withdrawal process Preclinical studies on nicotine reinforcement exploring the involvement of the endogenous opioid system – µ-, δ-, or κ-opioid receptors or their endogenous agonist peptides, the enkephalins, dynorphins, and β-endorphin – appear to be rare. In addition, in most animal studies no conclusive involvement has been found (for a review see Pomerleau 1998). For example, a preinjection with κ/µ-opioid receptor antagonist naltrexone prior to SA sessions did not affect the reinforcing properties of nicotine (Corrigall and Coen 1991b), and an injection of naloxone
a b c
Dhatt et al. (1995) Hollt and Horn (1992) Houdi et al. (1998)
Early withdrawal (24 h) Mu receptor Chronic nicotine effect (≤2 h)
Dynorphin system Chronic nicotine effect (≤2 h)
Late withdrawal (7 d–14 d)
Early withdrawal (24 h–≤3 d)
Enkephalin system Chronic nicotine effect (≤2 h)
Late withdrawal (7 d–21 d)
Early withdrawal (4 h–≤3 d)
POMC system Chronic nicotine effect (
