the neurobiology of nicotine addiction: bridging the ...

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THE NEUROBIOLOGY OF NICOTINE ADDICTION: BRIDGING THE GAP FROM MOLECULES TO BEHAVIOUR Steven R. Laviolette and Derek van der Kooy Nicotine, the primary psychoactive component of tobacco smoke, produces diverse neurophysiological, motivational and behavioural effects through several brain regions and neurochemical pathways. Recent research in the fields of behavioural pharmacology, genetics and electrophysiology is providing an increasingly integrated picture of how the brain processes the motivational effects of nicotine. The emerging characterization of separate dopamine- and GABA (γ-aminobutyric acid)-dependent neural systems within the ventral tegmental area (VTA), which can mediate the acute aversive and rewarding psychological effects of nicotine, is providing new insights into how functional interactions between these systems might determine vulnerability to nicotine use.

Neurobiology Research Group, Department of Anatomy and Cell Biology, University of Toronto, Toronto, Canada, M5S 1A8, USA. email: [email protected] doi:10.1038/nrn1298

The addictive nature of nicotine remains a global health epidemic. Over three million smoking-related deaths are reported annually, worldwide. In the Western world, illness related to smoking is believed to be the cause of 20% of all deaths, making nicotine addiction the single largest cause of preventable mortality1,2. Despite these grim statistics, tobacco use is increasing in many developing countries3, with smoking-related mortalities predicted to exceed 10 million per year over the coming 30–40 years1. Although nicotine is generally not classified among ‘harder’ addictive drugs, such as cocaine or heroin, with continued use tobacco often becomes as difficult to abandon. As anybody who has ever struggled with repeated attempts at smoking cessation can attest to, nicotine is exceptionally intractable to quitting interventions. Since the identification of nicotine as the primary psychoactive component of tobacco smoke, a great amount of research has been undertaken to unravel the neuropharmacological, anatomical and behavioural underpinnings of its psychoactive effects. Various neural pathways and transmitter systems have emerged as compelling candidates for the processing of the psychoactive and addictive properties of nicotine. Here, we will examine research that implicates specific

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neurotransmitter systems, the potential roles of specific neuronal nicotinic acetylcholine receptor (nAChR) subtypes and specific neuroanatomical regions that have been implicated in mediating the addictive properties of nicotine. In particular, we will review the considerable body of evidence that implicates dopamine (DA) and non-DA neuronal substrates in the ventral tegmental area (VTA) as crucial for the rewarding and aversive motivational properties of nicotine. Whereas previous research has implicated DA-mediated neurotransmission as a direct mediator of a nicotine reward signal4–7, more recent evidence points to a more complex role for DA systems in the motivational effects of nicotine, including the aversive effects of nicotine and drug-induced plastic changes at the synapse8–10. We propose an integrated model that might account for the vulnerability to the rewarding and addictive properties of nicotine through acute actions on nonDA reward pathways. With continued nicotine exposure, plastic molecular alterations in central DA systems might underlie the continued propensity to consume nicotine by inducing craving, the aversive effects of withdrawal, and aberrant incentive-salience attribution to environmental stimuli that are associated with nicotine. VOLUME 5 | JANUARY 2004 | 5 5

©2004 Nature Publishing Group

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Figure 1 | The structure of neuronal nicotinic acetylcholine receptors (nAChRs). a | Although the precise molecular structure of nAChRs is not known, they are believed to be pentameric ion channels. Each nAChR is composed of five subunits arranged in either homomeric or heteromeric complexes of α- or β-subunit arrangements (left). Different subunit combinations confer unique functional properties to the ubiquitously distributed nAChRs throughout the brain. The schematic on the right shows the transmembrane topology of a single nAChR subunit. The transmembrane domains are labelled M1–M4. The larger amino-terminal domain contains the acetylcholine-binding site, whereas the M2 domain determines the ionic selectivity of the receptor and faces the inside of the channel pore. b | nAChRs are located at the soma, on presynaptic terminals and on postsynaptic boutons. This widespread localization confers the receptor with a wide range of functions, influencing neuronal signalling at the pre- and postsynaptic levels.

Nicotine signalling: pharmacology and anatomy

Nicotine acts on endogenous nAChRs that are found ubiquitously throughout the central (CNS) and peripheral nervous systems in almost all vertebrate and invertebrate species. The nAChRs are pentameric receptor complexes that serve as ligand-gated ion channels (FIG. 1). So far, 12 different neuronal nAChR subunits have been identified: α2–α10 and β2–β4 (REFS 11–15). The nAChR receptors form different combinations of α- and β-subunits. However, the α7–α9 subunits can also form homomeric nAChRs16,17. Functionally, the nAChR receptor complex can exist in three conformational states, which are dynamically regulated by exposure to the agonist: closed, open and desensitized11. When agonists bind to the nAChR, the receptor complex undergoes a conformational change in its structure, which allows the channel gate to open, permitting the passage of cations (such as Na+, K+ and also Ca2+, which might account for 1–10% of the nAChRmediated current18) through the channel pore. Ligand binding can produce a diverse range of neurophysiological effects. For example, nAChRs made of different subunit combinations can be located either on the soma and/or neuronal processes, enabling nAChR to act at the cell body and at the presynaptic and postsynaptic regions (FIG. 1). In v itro studies have examined the

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signalling properties of nicotine in various CNS regions. In particular, studies on the actions of nicotine on DA pathways, specifically within the VTA, have provided insights into how nicotine might modify signalling through DA and non-DA VTA systems.

The VTA and its input and output pathways. The mammalian VTA is a midbrain region that has been implicated in the rewarding motivational effects of a wide variety of addictive drugs, including cocaine19, alcohol20, opiates21,22 and nicotine8,23,24. Much evidence implicates the VTA and its associated efferent and afferent projections as an integrative centre for the psychoactive effects of nicotine. Within the VTA, DA neurons (designated as the A10 DA group), and their associated ascending projections to the nucleus accumbens and prefrontal cortex (PFC), comprise the well-characterized mesolimbic and mesocortical pathways. In addition, a population of VTA GABA neurons provide inhibitory input to the A10 DA neurons25,and there is anatomical evidence for descending projections to the brainstem mesopontine region, including the tegmental pedunculopontine nucleus (TPP)26,27 — a brain region that is important in DAindependent reward signalling. Both of these neuronal populations — the DA and GABA neurons — are involved in signalling reward19,21,28,29. The VTA also receives excitatory glutamatergic and cholinergic projections from both the TPP and the adjacent laterodorsal tegmental nucleus (LDT)25,30, as well as inhibitory GABA inputs from the TPP31. In FIG. 2, the ascending anatomical DA projections from the VTA to the nucleus accumbens and prefrontal cortex, as well as the VTA’s GABA connections with the TPP are shown. Recent electrophysiological work on brain slices has provided insights into the cellular mechanisms by which nicotine interacts with both of these neuronal populations in the VTA, and has implicated the VTA as a crucial site for central nicotine signalling through several pre- and postsynaptic substrates. Neurophysiolog y of nicotine signalling in the VTA. Neurons within the VTA have a wide variety of nAChRs17, and nicotine can activate both the DA and GABA neurons of the VTA32,33. The nAChR receptor profiles that are associated with these DA and GABA neurons differ considerably, and these differences might have important functional consequences for nicotine signalling in the mesolimbic system. For example, DA neurons of the VTA express the α2–α7 and β2–β4 subunits34,35, which can give rise to at least three pharmacologically distinct nAChR subtypes, of which one is probably a homomeric α7 receptor. Although less than half of the VTA neurons express nAChRs that contain α7 (REF. 17), this subunit is preferentially localized within the midbrain in the VTA, relative to the adjacent substantia nigra17. By contrast, less than 25% of the GABA neurons express the α3, α5, α6 and β4 subunits35, indicating that most nAChRs of these VTA neurons contain the α4 and β2 subunits. The administration of nicotine within concentration ranges that are readily self-administered in rodents and humans has been shown to increase DA release in the nucleus accumbens24,36. Furthermore, within the www.nature.com/reviews/neuro


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Figure 2 | The ventral tegmental area (VTA), and its efferent and afferent systems. a | Human (left) and rat (right) brains, showing the mesolimbic and mesocortical dopamine (DA) pathways, which originate in the VTA and send ascending projections to the nucleus accumbens (NAc) and prefrontal cortex (PFC), respectively. These pathways are strongly activated by nicotine and are implicated in its rewarding and aversive psychological properties. The VTA also sends a descending projection to the tegmental pedunculopontine nucleus (TPP), a brain region that is involved in non-DA-mediated reward signalling. The rewarding effects of nicotine are blocked by lesions69 or GABA (γ-aminobutyric acid)-mediated inhibition78 of this nucleus. Ascending cholinergic and glutamatergic projections from the TPP also influence VTA neuronal activity and can regulate the activity of DA neurons in the VTA46,47. b | Schematic showing the DA and GABA neuronal populations within the VTA. GABA neurons send descending projections to the TPP and provide inhibitory input to DA neurons. Both neuronal populations are activated by nicotine32,33,40. In addition, both neuronal populations receive excitatory glutamatergic inputs, which can regulate the relative activity of DA and GABA activity in the VTA.

physiological range of plasma nicotine concentrations that are obtained by smokers (~0.5 µM)37, nicotine potently activates DA neurons of the VTA37, an activation that is followed by desensitization of nAChRs after continued exposure to nicotine37. This observation indicates that, whereas the acute excitatory action of nicotine on DA neurons might signal its reinforcing, rewarding effect, the long-lasting desensitization of VTA nAChRs might represent a cellular basis of nicotine tolerance. Such a characterization seems reasonable, given the anecdotal reports that smokers tend to enjoy most the first cigarette of the day (at a time when nAChRs would not be in a state of prolonged desensitization). However, activation of DA neurons is not a scalar index of reward, nor is the corresponding increase in DA release in the target regions of the mesolimbic system a simple reinforcement signal (see later in text)38,39. As we will discuss, mesolimbic DA signalling also mediates aversive motivational events33 and is involved in associative learning processes38,39. Because of the important functional relationship between the DA and GABA neurons in the VTA25 (FIG. 2), it is imperative to examine the effects of nicotine on both of these neuronal populations. Recent studies on VTA slices have investigated the actions of nicotine on the NATURE REVIEWS | NEUROSCIENCE

activity of both DA and GABA neurons, leading to interesting findings about how nicotine affects the functional relationship between these two neuronal groups. Whereas the early, acute effects of nicotine in the VTA predominantly affect GABA neurons, the nAChRs that are associated with these cells desensitize rapidly9,33, leading to a long-lasting excitation of the DA neurons through removal of the inhibitory influence of GABA. In addition, the desensitization of inhibitory inputs to the DA system correlates with enhanced glutamatergic input to the DA neurons through the actions of nicotine on presynaptic nAChRs that are located on VTA glutamatergic terminals, which show a lesser degree of desensitization after nicotine exposure33. Extracellular recordings of DA neurons of the VTA in vivo after intravenous nicotine administration lead to a similar conclusion; nicotine can modify the activity of DA neurons through its actions on inhibitory GABA neurons40. Functionally, nicotine activation of GABA neurons would be expected to initially increase inhibitory input to the DA neurons (FIG. 2). However, with continued exposure to nicotine and the subsequent desensitization of the nAChRs of the GABA neurons, nicotine would presumably bypass these inhibitory cells and act directly on the DA neurons. These findings32,33,40 indicate that there might be a net shift in the activity level of DA neurons relative to the GABA cells in the VTA (FIG. 2) after prolonged in vitro or in vivo exposure to nicotine at concentrations that are comparable to those observed in the plasma of smokers. This shift would favour increased activity of the mesolimbic DA pathway. Although future studies are required to clarify these issues, the differences in desensitization kinetics between the distinct nAChR subunits and their divergent expression patterns on DA and GABA neurons in the VTA might account for the functional differences between these cells in response to nicotine exposure. In addition to the actions of nicotine on DA and GABA neurons, considerable evidence indicates that the actions of nicotine within the VTA might be mediated by glutamatergic transmission. Anatomically, the VTA receives substantial glutamatergic inputs from cortical and subcortical structures25. These excitatory inputs synapse on DA and GABA neurons25,30 (FIG. 2), and can therefore modulate the activity of both cell types.Various studies have indicated that α7-containing nAChRs might have a specific role in mediating the presynaptic actions of nicotine in the CNS, and, in particular, might regulate the release of glutamate41,42. Systemic nicotine has been shown to elevate glutamate levels in the VTA, and this effect is blocked by the relatively selective α7-subunit antagonist methyllycaconitine (MLA)43. In addition, lesions of the prefrontal cortex — a region that provides glutamatergic inputs to the VTA — reduce binding of the nAChR antagonist α-bungarotoxin in the VTA. This observation provides further evidence for the presynaptic localization of α7-containing nAChRs in the VTA, presumably in glutamatergic terminals43. Blockade of NMDA (N-methyl-D-aspartate) receptors and α7-containing nAChRs in the VTA diminishes the increase in mesolimbic DA release that is induced by VOLUME 5 | JANUARY 2004 | 5 7


REVIEWS nicotine44. This apparently unique role for the α7 subunit in the VTA might have important implications for the psychoactive effects of nicotine. Indeed, as we will discuss, various studies have indicated that functional interactions between DA, GABA and glutamate within the VTA are vital for the mediation of the motivational properties of nicotine.

MICRODIALYSIS

A technique that allows the sampling of neurochemicals in the brain of live animals. It commonly uses a small U-shaped cannula that serves a dual function: it allows the injection of molecules of interest to test their effect, and it provides a pathway for the flow and subsequent collection of perfusate from a small brain area. ANTISENSE KNOCKDOWN

Oligonucleotides with a sequence that is complementary to the mRNA of a given molecule can be used to block its translation. The subsequent temporary elimination of the protein of interest often provides useful information on its biological function. MEDIAL FOREBRAIN BUNDLE

Complex fibre tract that runs through the diencephalon. It contains descending fibres from telencephalic structures such as the basal olfactory regions, the periamygdaloid region and the septal nuclei, and ascending fibres from the aminergic brainstem nuclei. Intracranial stimulation along this tract can simulate motivational states and reinforce behaviour.

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Cholinergic modulation of DA function in the VTA through brainstem projections. The functional connections between the brainstem nuclei TPP and LDT with the VTA have been studied extensively (FIG. 2). Ascending inputs from the TPP and LDT to the VTA comprise cholinergic and glutamatergic fibres that synapse on DA and GABA neuronal populations of the VTA30. The cholinergic neurons of the TPP and LDT have been termed the Ch5 and Ch6 cell groups, respectively45, and these inputs can modulate the activity of the mesolimbic DA system. For example, electrical stimulation of the TPP elicits striatal DA efflux as measured by MICRODIALYSIS46, whereas LDT stimulation elicits a similar DA efflux in the nucleus accumbens through the activation of cholinergic and glutamatergic receptors in the VTA47. The behavioural effects of these ascending cholinergic inputs to the VTA seem to depend more importantly on signalling through muscarinic acetylcholine receptors than through nAChRs. For example, ANTISENSE KNOCKDOWN of muscarinic M5 receptors in the VTA of rats reduces the rewarding efficacy of stimulating the MEDIAL FOREBRAIN 48 BUNDLE . Similarly, pharmacological blockade of muscarinic receptors in the VTA is more effective than blockade of nAChRs at attenuating the rewarding effects of this stimulation49. These in vivo findings point to the behavioural complexity of cholinergic signalling in the VTA. Indeed, the functional balance between DA and GABA VTA neuronal substrates might have important implications for the central processing of the motivational properties of nicotine. The dual motivational effects of nicotine

We tend to think about drugs of abuse in terms of their ability to produce feelings of pleasure. Indeed, nicotine is known to induce feelings of pleasure and reward in humans and other species. But like many other addictive drugs, nicotine also has potent, aversive, unpleasant effects50–52. Nicotine can produce powerful anxiogenic effects systemically and centrally53,54 through activation of nAChR that contain α4, α7 or β2 subunits55. Many people experience noxious effects such as nausea, coughs and dizziness on their initial experience with tobacco56,57. Interestingly, tolerance to the aversive effects of nicotine develops with repeated exposure52,58. Although the precise neurobiological mechanism that underlies this tolerance to nicotine aversion is unknown, its existence indicates that chronic nicotine exposure might induce a functional alteration in neural systems that mediate the aversive and/or rewarding effects of nicotine. It has been suggested that relative sensitivity to the rewarding or aversive properties of nicotine might serve as a predictor of who might become addicted to tobacco59,60. So, an important goal in the study of nicotine


addiction is the delineation of the neuronal mechanisms that might be involved in transmitting the rewarding and aversive motivational effects of nicotine. Understanding how these neural systems interact might yield important clues as to how the brain initially responds to acute nicotine exposure (as a rewarding or an aversive stimulus), and how the continued exposure to the drug might eventually lead to dependence (compulsive nicotine craving and withdrawal symptoms).

Defining the role of DA: reward or aversion? For many years, a common theme in the literature on behavioural neuropharmacology has been the hypothesis that DA and its associated neural pathways serve as specific transducers of central reward signals. In its most simplistic form, this view of DA implies that any drug or stimulus that can produce reward as measured by behavioural reinforcement tests, such as conditioned place preference (CPP) or intravenous self-administration of the drug (BOX 1), does so by increasing levels of DA through activation of the mesolimbic pathway61. In this sense, DA was considered a direct, scalar index of reward. However, recent research has called into question this conceptualization of DA in motivational signalling, particularly in light of the fact that DA signalling also correlates with aversive, noxious stimuli38,39, and might serve to signal conditioned stimuli that predict reward (or errors in reward prediction), rather than the rewarding events per se 62. A role for DA-mediated transmission in such cognitive processes might transcend drug-naive versus drug-dependent states and might be relevant for motivationally important learning processes, independent of their rewarding or aversive emotional valence. However, a large body of research has supported the idea that the rewarding effects of several psychoactive drugs, including nicotine, are dependent on mesolimbic DA-mediated transmission. Indeed, DA neural systems have arguably received the greatest amount of experimental attention as potential mediators of the rewarding effects of nicotine. Using an intravenous procedure for the selfadministration of nicotine, several studies have reported that blocking DA-mediated transmission, pharmacologically or by lesions of the mesolimbic DA pathway, is sufficient to reduce or completely block the reinforcing effects of nicotine. For example, Corrigall et al.4 showed that pretreatment with specific antagonists of D1 or D2 DA receptor subtypes strongly attenuated nicotine selfadministration. Lesions of the mesolimbic DA system caused by 6-hydroxydopamine, a molecule that selectively destroys DA neurons, also attenuate nicotine self-administration5. Similarly, microinfusions of the nAChR antagonist dihydro-β-erythroidine directly into the VTA attenuate nicotine self-administration, but not the reinforcing effects of food or cocaine, implicating that the mesolimbic DA projection from the VTA is a crucial mediator of the reinforcing effects of nicotine6. Work using gene knockout technology has further implicated mesolimbic DA-mediated transmission in nicotine reward. Elimination of specific nAChR subunits has shown that the nAChR subunits that are www.nature.com/reviews/neuro


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Box 1 | Tasks used to study the motivational properties of nicotine a

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A behavioural test that is commonly used to study the motivational properties of nicotine and other drugs is Drug c Vehicle conditioned place preference (CPP; a). In this procedure, animals receive a specific drug and are placed in a unique environment that has a specific odour, colour and/or texture. On the next day, the animal receives the vehicle instead of the drug and is placed in another conditioning environment. After several such cycles, animals are given the opportunity to spend time in either the environment previously paired with the drug or with the vehicle. A key advantage of this task is that the experimenter controls Choice the precise amount and time course of exposure to the ? drug in question. More importantly, CPP allows the testing of both the rewarding properties of a drug (the animal can show a preference for the previously drugpaired environment) and its aversive properties (the animal can actively avoid an environment previously paired with the drug). An important drawback of CPP is that drugs are passively administered by the experimenter, instead of being self-administered, as is the case for real-life drug-taking behaviour. However, the associative learning that takes place during the CPP task might resemble the strong associations that smokers develop between the environmental cues that are associated with smoking and the reinforcing effects of nicotine111. Another commonly used model is the intravenous self-administration of drugs (b). Animals can be trained to reliably press a lever to receive a discrete infusion of drug. Lesions to specific brain regions or pre-treatments with specific pharmacological agents, such as dopamine (DA) receptor antagonists, can be performed to examine their effects on self-administration. This model has been successfully used to measure nicotine reinforcement in rodents and primates50. One of the primary advantages of the self-administration model is its resemblance to real-life drug-taking behaviour in humans: just like human smokers, animals trained in this task will consistently and compulsively selfadminister nicotine50. The lever presses are termed operant responses. Most studies on nicotine reinforcement rely on a ‘fixed-ratio’ schedule of operant responding in which the animal must make a fixed number of bar presses to receive a single infusion of nicotine. A third task that is used in studies on the motivational effects of nicotine is conditioned taste aversion (CTA; c). CTA is believed to tap directly into the aversive properties of a drug by taking advantage of the fact that animals seem intrinsically able to associate specific tastes with aversive states. By pairing a specific drug with a particular taste, animals might learn to avoid such a taste, as the unpleasant effects of the drug become associated with it. By contrast, a taste that is paired with the injection of vehicle is not avoided when the animal is given a choice between the two tastes. Many studies have used nicotine as a drug stimulus in this task and have reported that, like many other addictive drugs, it produces potent aversive effects8,10,50. Such reports, in combination with studies using the self-administration or CPP procedures, have conclusively shown the dual nature of the motivational effects of nicotine in animals, consistent with the reported aversive and rewarding psychological effects of nicotine in humans.


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Figure 3 | Different roles for dopamine (DA) signalling in the acute versus chronic phases of nicotine exposure. a | Nicotine self-administration is significantly attenuated in mice lacking the nicotinic acetylcholine receptor (nAChR) subunit β2 (nAChRβ2–/–), relative to wild-type (WT) animals. b | This attenuation is specific to nicotine, as the reinforcing effects of cocaine are unaffected in these mutant animals. Elimination of the nAChR β2 subunit also attenuates nicotine-induced DA release63, indicating that DA signalling might be essential for nicotine reinforcement. Reproduced, with permission, from Nature REF. 63  (1998) Macmillan Magazines Ltd. c | By contrast, the acute effects of nicotine produce biphasic motivational effects within the VTA. Whereas a low nicotine concentration produces aversion (as measured in the conditioned place preference task), high concentrations produce potent rewarding effects. d | Blockade of DA signalling with a systemic neuroleptic drug (α-flupenthixol) does not block the rewarding effects of high nicotine concentrations, potentiates the rewarding effects of middle-range nicotine doses, and switches the motivational effects of a low concentration from aversive to rewarding. Reproduced, with permission, from REF. 8  (2003) Macmillan Magazines Ltd. Asterisks in a and b indicate P< 0.05.

required for nicotine-induced DA release are also necessary for nicotine self-administration7,63. For example, mice that lack the β2 nAChR subunit showed decreased DA release in response to nicotine exposure, and this effect correlated with a strong attenuation of nicotine self-administration63, indicating that the ability of this subunit to produce nicotine reward might be tightly linked to its functional regulation of DA release in the mesolimbic pathway (FIG. 3a,b). Interestingly, these mutant mice also showed a strong reduction in nicotineinduced conditioned taste aversion (CTA)10 (BOX 1), indicating that a specific nAChR subunit that is linked to mesolimbic DA-mediated transmission is also involved in the aversive effects of nicotine. But studies using CPP, a test that is sensitive to both the aversive and rewarding properties of drugs, have disclosed a far more complex role for DA in the motivational properties of nicotine.

A common neural substrate for nicotine reward and aversion. Many early studies on the motivational effects of nicotine using the CPP test reported that, rather then producing a rewarding effect, nicotine produced aversive effects, as manifest by the development of conditioned aversions to environments paired with

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nicotine51, or no apparent motivational effects at all64. But a limitation of these studies is that they relied exclusively on systemic nicotine administration, which also targeted peripheral nAChRs. The activation of these receptors could potentially produce toxic and noxious effects, leading to the expression of such a conditioned place aversion. Intravenous nicotine self-administration also activates peripheral nAChRs, and high concentrations of intravenously administered nicotine do produce aversive effects50. An early study reported that infusions of the nicotinic agonist cytisine directly in the VTA to exclude peripheral effects could produce reward as measured in the CPP test65. A more recent study reported that both rewarding and aversive effects could be measured using the same test after microinfusions of nicotine itself into the VTA8. This study reported a dose-dependent, biphasic curve for the motivational effects of nicotine in the CNS; whereas a lower nicotine concentration in the VTA produced an aversive effect, higher concentrations produced potent rewarding effects (FIG. 3c). So, within a single brain region, nicotine can have rewarding or aversive effects as a function of nicotine concentration. Surprisingly, when the rewarding effects of higher nicotine concentrations were challenged by blocking DA-mediated transmission, either systemically or directly in the nucleus accumbens, there was no attenuation of the rewarding effects of nicotine. However, under these conditions, levels of nicotine in the VTA that previously produced no motivational effects now had rewarding effects, whereas the effects of lower nicotine concentrations switched from aversive to rewarding (FIG. 3d). In addition, when the aversive effects of nicotine were examined in the CTA test, DA receptor blockade prevented the aversive nicotine signal8. So, by contrast to previous studies of intravenous nicotine self-administration, these studies have indicated that the rewarding and aversive effects of nicotine are mediated within the VTA by separate and dissociable systems.Whereas the acute rewarding properties of nicotine were independent of DA signalling, the aversive effects of nicotine were crucially dependent on DA-mediated transmission8. Although these results from the CPP model point to an opposing functional role for DA signalling in the motivational effects of nicotine within the VTA, there are crucial differences between the CPP and the selfadministration studies. The most important of these differences is that in studies of self-administration4–6,63 the animals receive chronic exposure to nicotine, often over several weeks. By contrast, in most CPP studies, animals are naive to nicotine at the beginning of the experiment, and the drug is passively administered by the experimenter in controlled doses (BOX 1). So, whereas DA receptor blockade seems to attenuate nicotine reinforcement in animals that are chronically exposed to nicotine, the acute rewarding effects of nicotine can be mediated through a non-DA system in the VTA. In addition, when the acute aversive effects of nicotine are blocked by interfering with mesolimbic DA receptor signalling, the rewarding effects of nicotine are potentiated, presumably by the removal of an aversive signal8. As we will discuss, the motivational

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REVIEWS state — drug-naive, drug-dependent or withdrawn — might be vital to determine the role of DA in nicotine addiction. Beyond DA: new players in nicotine addiction

As DA does not exclusively transmit a nicotine reward signal, what other substrates are involved in the addictive properties of nicotine? As previously noted, nicotine produces the physiological activation of both DA and GABA neurons in the VTA32,33,40. As GABA neurons serve as a substrate for non-DA-mediated reward transmission28,29 and are acutely activated by nicotine32,33,40, this neuronal population is a good candidate for the mediation of nicotine reward signalling in the VTA. A recent report has found that GABA receptors in the VTA might be important mediators of the reinforcing properties of nicotine. Corrigall et al.23 reported that direct microinfusions of GABAA or GABAB receptor agonists into the VTA caused a significant reduction in nicotine self-administration, indicating that GABA receptors in the VTA can also mediate nicotine reinforcement. Whereas GABAA receptors in the VTA are predominantly localized to GABA neurons25,28, GABAB receptors are primarily localized on DA neurons and can strongly modulate the activity of the mesolimbic DA system25,66,67. Activation of GABAB receptors in the VTA can strongly decrease mesolimbic DA activity66,67 and attenuate nicotine-induced DA release in the mesolimbic pathway67,68. These data are consistent with a role for GABAB receptors in the control of VTA DA neurons. These results are also consistent with observations in animals that chronically self-administered nicotine, as blockade of DA signalling or direct inactivation of the mesolimbic DA system with a GABAB receptor agonist can block nicotine reward in this model.

The TPP. The TPP is crucially involved in the transmission of drug-related21,69,70 and natural reward information70,72. Notably, the TPP seems to be especially important in the early, acute phase of drug exposure. For example, lesions of the TPP block opiate reward, as measured in the CPP and self-administration tasks, only during the early stage of drug exposure21,71. Although the TPP has been implicated in the rewarding effects of opiates21,70, food70 and sex72, the rewarding properties of drugs such as cocaine73 do not seem to require this region. In the case of nicotine, several reports have implicated the TPP in the mediation of its rewarding effects. Anatomically, the TPP and VTA are connected by descending GABA inputs from the VTA, and by ascending cholinergic and glutamatergic inputs from the TPP (FIG. 2). Although these ascending cholinergic inputs from the TPP to the VTA DA neurons can influence the activity of the mesolimbic DA system46,47, it is unlikely that these inputs are directly involved in nicotine reward for several reasons. First, the cholinergic inputs to the VTA are topographically organized such that most of the cholinergic brainstem projections to the VTA arise from the adjacent LDT, rather than from the TPP, which projects more heavily to the adjacent substantia nigra74. Second, acutely administered nicotine predominantly activates noncholinergic TPP cells, including GABA and glutamate NATURE REVIEWS | NEUROSCIENCE

neurons75. In addition, intracranial self-stimulation activates GABA neurons of the TPP, rather than the cholinergic Ch6 cells76, further indicating that noncholinergic TPP mechanisms might be involved in reward signalling. Together, this evidence indicates that descending VTA inputs to the TPP might be the primary transducers of the nicotine reward signal. For example, bilateral excitotoxic lesions of the TPP block nicotine reward in the VTA and switch the motivational valence of nicotine from rewarding to aversive, as measured in the CPP model69. This result indicates that, whereas the TPP seems to selectively mediate a nicotine reward signal, the aversive effects of nicotine in the VTA (which are dependent on DA-mediated transmission in the acute state) remain intact after removal of the TPP pathway. An early report found that partial lesions of the dorsal TPP did not affect nicotine self-administration6, but a subsequent report from the same group claimed that more extensive and localized TPP lesions strongly attenuated nicotine self-administration77. Within the TPP, GABA receptors seem to be crucial for transmission of a nicotine reward signal. Corrigall et al.78 reported that GABAA and GABAB receptor agonists that were infused into the TPP attenuated nicotine self-administration on a fixedratio schedule of operant responding (see BOX 1). In addition, infusions of nicotine into the TPP induce CPP, further implicating the TPP as a non-DA system that is important for the motivational effects of nicotine79. So, increasing evidence indicates that non-DA neural substrates, including a GABA-mediated system within the TPP, might be important for the reinforcing and addictive properties of nicotine. Future studies are required to map out the functional pathways from the VTA to these non-DA neuronal reward substrates.

Emerging roles for the α7 subunit and glutamate. As described previously, α7-containing nAChRs might be preferentially involved in the regulation of the presynaptic effects of nicotine on glutamate release41–43. Such a role for the α7 subunit has been shown in various brain regions, including the VTA41–43. Until recently, no direct behavioural evidence had linked this particular subunit to the motivational effects of nicotine. However, several reports now indicate that α7 might be preferentially involved in the acute rewarding effects of nicotine. Panagis et al.80 reported that direct microinfusions of MLA in the VTA attenuated nicotine-induced potentiation of reward in an intracranial self-stimulation experiment. However, Grottick et al.81 found that blockade of α7-containing receptors had no effect on intravenous nicotine self-administration in animals chronically treated with nicotine, nor on the hyperlocomotor effects of chronic nicotine exposure, indicating that α7 might not be involved in the motivational signalling of nicotine after chronic exposure. Similarly, a recent study using MLA over a wide concentration range found that this antagonist blocked the acute effects of nicotine administered directly into the VTA, and switched its motivational valence from rewarding to aversive82. By contrast, β2containing nAChRs are implicated in both the rewarding VOLUME 5 | JANUARY 2004 | 6 1


REVIEWS and aversive effects of nicotine6,8,10,63, and seem to block its reinforcing effects in both the acute and chronic stages of exposure6,8,63. A caveat of these studies is that MLA at low concentrations interacts with nAChRs that do not contain the α7 subunit35. Several studies have found that blockade of glutamatergic transmission with specific NMDA receptor antagonists that are administered systemically or directly in the VTA can block the rewarding82–84 and aversive82 effects of nicotine. Interestingly, blockade of NMDA receptors is effective in reducing the reinforcing effects of nicotine in both the acute and chronic phases of nicotine exposure82–84. An integrated model of nicotine addiction

INCENTIVE SALIENCE

A psychological process whereby the perception of stimuli is transformed by increasing their salience, making them more attractive or wanted.

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The complexity of the motivational and physiological effects of nicotine makes it a formidable task to develop a unifying model of nicotine addiction. Owing to the ubiquity of central nAChR distribution, nicotine exerts multiple effects in many brain regions beyond the VTA. But as the studies that we reviewed in this article attest to, the VTA and its associated input and output pathways seem to be integral to the transmission of the motivational properties of nicotine. By comparing the results of molecular, electrophysiological and behavioural investigations on the central actions of nicotine, an integrated picture of the nicotine addiction process is beginning to emerge. A consistent theme is the dichotomy in the roles of DA in the motivational properties as a function of the stage of nicotine dependence. Indeed, whereas the rewarding effects of nicotine within the VTA can be mediated through DAindependent mechanisms in the early, acute phases of nicotine exposure8,69,75, blockade of DA-mediated transmission seems to attenuate its reinforcing properties once chronic exposure and dependence have taken place4–6,63. What mechanism could account for this apparent shift in the role of DA-mediated transmission as a function of nicotine exposure? Do the effects of DA receptor blockade in nicotine-dependent animals interfere with a rewarding effect of nicotine or with some other psychological process? FIGURE 4 presents a simplified model that summarizes some of the known functional differences between the role of DA and non-DA systems of the VTA in relation to the motivational effects of nicotine in the acute versus the chronic (addicted) state. We suggest that the shift from the acute effects of nicotine to the development of a dependence state involves a switch in the functional role of DA signalling in the VTA. In the acute state, activation of DA neurons by nicotine induces aversive effects, whereas GABA neurons and the associated descending inputs to the TPP mediate its rewarding effects8,69,75. Nicotine transiently activates GABA neurons, the nAChRs of which rapidly desensitize33. Simultaneously, nicotine potentiates glutamatergic transmission through nAChRs that show slower desensitization, leading to a functional shift in the actions of nicotine from the GABA to the DA neurons33. The balance between these separate systems might determine the initial vulnerability to the addictive properties of


nicotine by determining the relative sensitivity to the rewarding or aversive psychological effects of nicotine. But how do alterations in DA signalling during prolonged exposure ultimately lead to a role for DA in the motivational effects of nicotine in the drug-dependent state? Here, we consider two alternative possibilities. First, considerable evidence indicates that, rather than transmitting an acute reward signal during the early exposure to drugs, DA is specifically involved in the late phases of the drug-addiction process, and mediates drug craving or wanting. This view, proposed by Berridge and Robinson85,86, states that, after repeated drug exposure, sensitization of the DA systems, which signal the INCENTIVE SALIENCE of the drug (that is, how much the drug is craved), leads to a pathological amplification of this salience, leading to compulsive drug seeking and use. Indeed, repeated nicotine exposure induces sensitization of DA pathways87,88 and increases DA receptor expression in the projection areas of the VTA DA system89. In addition, specific blockade of D3 DA receptors has recently been shown to prevent nicotine-induced relapse to nicotine-seeking behaviours, indicating that alterations of this particular DA receptor subtype might be specifically involved in nicotine craving and relapse90. However, there is little evidence to suggest that the sensitized DA-mediated psychomotor responses to repeated nicotine represent an enhancement in the rewarding properties of nicotine per se. In addition, prolonged nicotine exposure might have profound effects on nAChR expression. Indeed, many studies have reported that chronic nicotine exposure increases the number of nAChR receptor subtypes in various brain regions91. Although it is not known if such nAChR receptor upregulation is related to an increased DA responsiveness after chronic nicotine exposure, upregulation of α4, β2 and α7 nAChR subunits92 within the VTA93 or other DA systems could conceivably contribute to such a heightened response. A related possibility is that plastic processes that increase DA responsivity to nicotine within the VTA might represent an aberrant form of drug-induced associative learning. Interestingly, exposing humans to imagery of stimuli that are associated with smoking (and so with the incentive properties of nicotine) activates neural regions that are linked to drug-induced DA sensitization processes, including the prefrontal cortex, amygdala and orbitofrontal cortex94. Although direct functional comparisons between in v ivo behavioural studies and in vitro neuronal recording studies are tenuous, it is possible that plastic alterations at DA synapses in the VTA lead to a prolonged activation of DA pathways. This activation might result in persistent nicotine craving owing to the pathological amplification of an incentivelearned association between nicotine and environmental stimuli that are associated with nicotine exposure (such as the sight of a cigarette or the smell of tobacco smoke). Blockade of this DA-mediated incentive learning signal in animals that are chronically exposed to nicotine would be expected to reduce nicotine self-administration, as indeed shown by most self-administration studies4–7,63. So, once dependence to nicotine has developed after



REVIEWS a VTA

Glutamate inputs

GABA

Acute nicotine aversion signal

Nucleus accumbens

b

Dopamine

GABA

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Aversive nicotine craving/withdrawal Sensitized incentive salience

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–

VTA

Glutamate inputs

Acute nicotine reward signal

– Dopamine

Desensitization of acute nicotine reward signal

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NMDA receptors α4β2-containing ACh receptors α7-containing ACh receptors

Figure 4 | An integrated model for nicotine reward signalling in the ventral tegmental area (VTA). a | In the acute stage, the initial activation by nicotine of GABA (γ-aminobutyric acid) neurons in the VTA32,33,40 produces rewarding effects through a GABA-dependent system that projects to the tegmental pedunculopontine nucleus (TPP)69,76,77. These effects might involve the activation of presynaptic nicotinic acetylcholine receptors (nAChRs) that contain the α7 subunit, as blockade of this subunit interferes with the acute rewarding effects of nicotine80,82, but leaves the aversive signal intact82. However, nicotine might also exert its motivational effects through direct actions on nAChRs containing the β2 subunit and located on GABA or dopamine (DA) neurons, as pharmacological blockade or genetic deletion of this subunit blocks both the aversive and rewarding effects of nicotine8,63,82. In this model, nAChRs are distributed on both VTA neuronal populations, and nicotineinduced activation of these receptors can therefore regulate the motivational effects of nicotine through either non-DA or DA systems. b | With repeated nicotine exposure, however, the GABA system that signals reward becomes desensitized, leading to a net shift in the action of nicotine to the DA neurons33,40. This shift is mediated at least partly by increased glutamatergic input to the DA system33. The shift in the functional balance between GABA and DA neuronal populations in the VTA might lead to a dysregulated DA signal in the VTA, which in turn leads to the aversive psychological effects of nicotine craving and withdrawal, and/or to the potentiation of the incentive salience of nicotine and its compulsive use. NMDA, N-methyl-D-aspartate.

NEUROLEPTIC

This term was originally coined to refer to the effects of early antipsychotic agents on cognition and behaviour.

prolonged nicotine exposure, the learned associations between its incentive motivational properties and the environmental stimuli that become associated with these effects might become dependent on DA signalling. However, this idea does not explain why DA-mediated transmission seems to carry a specific aversive signal in the acute phase of nicotine exposure8,10, and a reinforcing signal after chronic nicotine exposure. A second explanation for the role of DA-mediated signalling on the motivational effects of nicotine argues for a consistent role for DA in the aversive aspects of nicotine in both the acute and chronic phases of nicotine exposure. The underlying idea is that a dysregulation of DA-mediated signalling during nicotine dependence and withdrawal after chronic exposure might be responsible for the aversive effects of nicotine withdrawal. Whereas activation of the nAChRs of DA neurons can signal an aversive effect in the early phase of nicotine exposure8,10, the eventual desensitization of these nAChRs, which takes place after the desensitization of the nAChRs in VTA GABA neurons33, might account for the tolerance to the aversive properties of nicotine over time. Note, however, that it is not known whether the nAChR desensitization that is observed in v itro is analogous to the


behavioural tolerance to nicotine that has been reported in vivo. Indeed, whereas nAChR desensitization can take place in the order of seconds to minutes after nicotine exposure in vitro, behavioural nAChR tolerance in vivo develops over days or weeks. Chronic nicotine exposure and withdrawal can induce profound alterations in the mesolimbic DA system in rodents87–89,95 and humans96. It is possible that chronic nicotine causes a long-term reduction of the baseline level of DA-mediated signalling as a compensatory response after heightened DA-mediated transmission during continued nicotine exposure. Indeed, medications that increase DA concentrations have proven efficacious in preventing nicotine relapse and craving in smokers97. However, DA receptor antagonists and agonists have been reported to reduce nicotine intake in healthy smokers98 and in chronic smokers with schizophrenia99,100. In addition, DA receptor agonists and antagonists increase subjective measures of nicotine craving in chronic smokers101. If a lowered level of DA tone were responsible for the aversive nature of nicotine withdrawal, it seems unlikely that blocking or activating DA receptors would increase nicotine intake and subjective measures of craving. If a lowered baseline level of DA-mediated signalling were responsible for the aversive effects of nicotine withdrawal, it might be predicted that blocking DA-mediated transmission would worsen withdrawal and craving, in which case animals that self-administer nicotine might be expected to increase, rather than decrease, their intake. In addition, several studies have found that chronic nicotine does not lead to a decreased DA tone in response to subsequent nicotine exposure102,103, but rather to an enhanced DA release in response to nicotine after chronic nicotine exposure104. NEUROLEPTICS have also been reported to increase smoking rates in people with schizophrenia; this effect might represent a potentiation of the rewarding properties of nicotine, or a compensatory response owing to a decrease in such rewarding effects105. The exceedingly high rates of nicotine addiction that are observed in people with schizophrenia106 might indicate that abnormalities in DA-mediated signalling, which are considered to be a cardinal underlying pathology of schizophrenia, might increase the motivational salience and rewarding effects of nicotine8,106. These findings raise the possibility that by pharmacologically regulating DA-mediated transmission during nicotine withdrawal, the unpleasant psychological effects of withdrawal and craving after chronic nicotine exposure might be alleviated. By extension, the ability of DA receptor blockade to reduce nicotine self-administration in animals chronically exposed to nicotine might be due to a blockade of the aversive effects of nicotine withdrawal. In other words, by blocking the aversive effects of nicotine withdrawal and craving, self-administration of nicotine would be reduced. Interestingly, chronic opiate exposure and withdrawal also alter DA-mediated signalling107,108, and DA-mediated transmission is involved in the acute aversive effects of opiate exposure and withdrawal109,110, pointing to possible similarities in the functional role of DA in both nicotine and opiate addiction. Although future studies are required to VOLUME 5 | JANUARY 2004 | 6 3


REVIEWS examine more closely the functional role of DA-mediated signalling in the early phases of nicotine exposure versus the chronic state, the available studies in humans and animals consistently indicate that chronic nicotine exposure might lead to alterations in DA-mediated signalling, which might in turn lead to nicotine craving and compulsive drug-seeking behaviours. Conclusions and future directions

Despite the widespread effects of nicotine in the CNS, converging evidence at the behavioural, molecular, genetic and physiological levels points to the VTA and its associated DA and non-DA systems as crucial mediators of the motivational effects of nicotine. However, many important questions remain to be answered. What functional interactions take place between the systems that subserve the aversive and rewarding effects of nicotine during the development of nicotine dependence? Does this functional interaction determine the vulnerability

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to the addictive potential of nicotine? Studies on the neurophysiological effects of nicotine within the VTA provide new insights into how nicotine induces synaptic plasticity within the mesolimbic DA system. These studies might disclose a mechanism to account for the shift from non-DA-mediated reward signalling in the acute state to the involvement of DA-mediated transmission in the development of compulsive nicotine use. However, it will be important to examine how these in v it ro effects of nicotine might relate to what happens in vivo during the development of addiction. Finally, research that allows the dissection of the contribution of specific subunits of the nAChR to the motivational effects of nicotine and the development of addiction might ultimately lead to a clear understanding of which nAChR subunits are involved in the vulnerability to the rewarding properties of nicotine, and how alterations in specific nAChRs might lead to chronic nicotine use and dependence.

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Acknowledgements The authors thank CIHR for their support.

Competing interests statement The authors declare that they have no competing financial interests.

Online links DATABASES The following terms in this article are linked online to: LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/ nAChR FURTHER INFORMATION Encyclopedia of Life Sciences: http://www.els.net/ addiction | dopamine | nicotinic acetylcholine receptors Access to this interactive links box is free online.

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