The Neurobiology of Nicotine Addiction: Bridging the Gap from Molecules to Behaviour

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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.

The addictive nature of nicotine remains a global health neurotransmitter systems, the potential roles of specific epidemic. Over three million smoking-related deaths neuronal nicotinic acetylcholine receptor (nAChR) are reported annually, worldwide. In the Western world, subtypes and specific neuroanatomical regions that illness related to smoking is believed to be the cause of have been implicated in mediating the addictive prop- 20% of all deaths, making nicotine addiction the single erties of nicotine. In particular, we will review the con- largest cause of preventable mortality1,2.Despite these siderable body of evidence that implicates dopamine grim statistics, tobacco use is increasing in many devel- (DA) and non-DA neuronal substrates in the ventral oping countries3,with smoking-related mortalities tegmental area (VTA) as crucial for the rewarding and predicted to exceed 10 million per year over the coming aversive motivational properties of nicotine. Whereas 30–40 years1.Although nicotine is generally not classi- previous research has implicated DA-mediated neuro- fied among ‘harder’ addictive drugs, such as cocaine transmission as a direct mediator of a nicotine reward or heroin, with continued use tobacco often becomes as signal4–7,more recent evidence points to a more com- difficult to abandon. As anybody who has ever struggled plex role for DA systems in the motivational effects of with repeated attempts at smoking cessation can attest nicotine, including the aversive effects of nicotine and to, nicotine is exceptionally intractable to quitting drug-induced plastic changes at the synapse8–10. interventions. We propose an integrated model that might account Since the identification of nicotine as the primary for the vulnerability to the rewarding and addictive psychoactive component of tobacco smoke, a great properties of nicotine through acute actions on non- Neurobiology Research Group, Department of amount of research has been undertaken to unravel the DA reward pathways. With continued nicotine expo- Anatomy and Cell Biology, neuropharmacological, anatomical and behavioural sure, plastic molecular alterations in central DA University of Toronto, underpinnings of its psychoactive effects. Various systems might underlie the continued propensity to Toronto, Canada, neural pathways and transmitter systems have emerged consume nicotine by inducing craving, the aversive M5S 1A8, USA. as compelling candidates for the processing of the effects of withdrawal, and aberrant incentive-salience email: [email protected] psychoactive and addictive properties of nicotine. attribution to environmental stimuli that are associated doi:10.1038/nrn1298 Here,we will examine research that implicates specific with nicotine.

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a signalling properties of nicotine in various CNS regions. Ligand binding site In particular, studies on the actions of nicotine on DA pathways, specifically within the VTA, have provided H2N NH2 H N HOOC COOH 2 insights into how nicotine might modify signalling through DA and non-DA VTA systems. COOH Extracellular The VTA and its input and output pathways. The mam- M1 M2 M3 M4 malian VTA is a midbrain region that has been implicated in the rewarding motivational effects of a wide variety of Cytoplasmic 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 inte- grative centre for the psychoactive effects of nicotine. Presynaptic Postsynaptic Within the VTA, DA neurons (designated as the A10 DA b nAChRs nAChRs 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 descend- Preterminal ing projections to the brainstem mesopontine region, nAChRs including the tegmental pedunculopontine nucleus (TPP)26,27 — a brain region that is important in DA- Figure 1 | The structure of neuronal nicotinic acetylcholine receptors (nAChRs). independent reward signalling. Both of these neuronal a | Although the precise molecular structure of nAChRs is not known, they are believed to be populations — the DA and GABA neurons — are pentameric ion channels. Each nAChR is composed of five subunits arranged in either homomeric involved in signalling reward19,21,28,29.The VTA also or heteromeric complexes of α- or β-subunit arrangements (left). Different subunit combinations receives excitatory glutamatergic and cholinergic projec- confer unique functional properties to the ubiquitously distributed nAChRs throughout the brain. tions from both the TPP and the adjacent laterodorsal The schematic on the right shows the transmembrane topology of a single nAChR subunit. The tegmental nucleus (LDT)25,30,as well as inhibitory GABA transmembrane domains are labelled M1–M4. The larger amino-terminal domain contains the 31 acetylcholine-binding site, whereas the M2 domain determines the ionic selectivity of the receptor inputs from the TPP .In FIG. 2,the ascending anatomical and faces the inside of the channel pore. b | nAChRs are located at the soma, on presynaptic DA projections from the VTA to the nucleus accumbens terminals and on postsynaptic boutons. This widespread localization confers the receptor with a and prefrontal cortex, as well as the VTA’s GABA connec- wide range of functions, influencing neuronal signalling at the pre- and postsynaptic levels. tions 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 Nicotine signalling: pharmacology and anatomy these neuronal populations in the VTA, and has impli- Nicotine acts on endogenous nAChRs that are found cated the VTA as a crucial site for central nicotine sig- ubiquitously throughout the central (CNS) and periph- nalling through several pre- and postsynaptic substrates. eral nervous systems in almost all vertebrate and inver- tebrate species. The nAChRs are pentameric receptor Neurophysiology of nicotine signalling in the VTA. complexes that serve as ligand-gated ion channels (FIG. 1). Neurons within the VTA have a wide variety of nAChRs17, So far, 12 different neuronal nAChR subunits have been and nicotine can activate both the DA and GABA neu- identified: α2–α10 and β2–β4 (REFS 11–15).The nAChR rons of the VTA32,33.The nAChR receptor profiles that receptors form different combinations of α- and are associated with these DA and GABA neurons differ β-subunits. However, the α7–α9 subunits can also form considerably, and these differences might have important homomeric nAChRs16,17. functional consequences for nicotine signalling in the Functionally, the nAChR receptor complex can exist mesolimbic system. For example, DA neurons of the in three conformational states, which are dynamically VTA express the α2–α7 and β2–β4 subunits34,35,which regulated by exposure to the agonist: closed, open and can give rise to at least three pharmacologically distinct desensitized11.When agonists bind to the nAChR, the nAChR subtypes, of which one is probably a homomeric receptor complex undergoes a conformational change α7 receptor. Although less than half of the VTA neurons in its structure, which allows the channel gate to open, express nAChRs that contain α7 (REF.17), this subunit is permitting the passage of cations (such as Na+,K+ and preferentially localized within the midbrain in the VTA, also Ca2+,which might account for 1–10% of the nAChR- relative to the adjacent substantia nigra17.By contrast, less mediated current18) through the channel pore. than 25% of the GABA neurons express the α3, α5, α6 Ligand binding can produce a diverse range of neuro- and β4 subunits35,indicating that most nAChRs of these physiological effects. For example, nAChRs made of VTA neurons contain the α4 and β2 subunits. different subunit combinations can be located either on The administration of nicotine within concentration the soma and/or neuronal processes, enabling nAChR to ranges that are readily self-administered in rodents and act at the cell body and at the presynaptic and postsynap- humans has been shown to increase DA release in tic regions (FIG. 1). In vitro studies have examined the the nucleus accumbens24,36.Furthermore, within the

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© 2004 Nature Publishing Group REVIEWS a activity of both DA and GABA neurons, leading to inter- PFC VTA esting findings about how nicotine affects the functional NAc TPP 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 b VTA Glutamate DA neurons of the VTA in vivo after intravenous nicotine inputs administration lead to a similar conclusion; nicotine can GABA modify the activity of DA neurons through its actions on inhibitory GABA neurons40. TPP Functionally, nicotine activation of GABA neurons would be expected to initially increase inhibitory input Nucleus Dopamine accumbens 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 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) presumably bypass these inhibitory cells and act directly 32,33,40 pathways, which originate in the VTA and send ascending projections to the nucleus accumbens on the DA neurons. These findings indicate (NAc) and prefrontal cortex (PFC), respectively. These pathways are strongly activated by nicotine that there might be a net shift in the activity level of DA and are implicated in its rewarding and aversive psychological properties. The VTA also sends a neurons relative to the GABA cells in the VTA (FIG. 2) descending projection to the tegmental pedunculopontine nucleus (TPP), a brain region that is after prolonged in vitro or in vivo exposure to nicotine at involved in non-DA-mediated reward signalling. The rewarding effects of nicotine are blocked by concentrations that are comparable to those observed in 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 the plasma of smokers. This shift would favour increased can regulate the activity of DA neurons in the VTA46,47. b | Schematic showing the DA and GABA activity of the mesolimbic DA pathway. Although future neuronal populations within the VTA. GABA neurons send descending projections to the TPP and studies are required to clarify these issues, the differences provide inhibitory input to DA neurons. Both neuronal populations are activated by nicotine32,33,40. in desensitization kinetics between the distinct nAChR In addition, both neuronal populations receive excitatory glutamatergic inputs, which can regulate subunits and their divergent expression patterns on DA the relative activity of DA and GABA activity in the VTA. and GABA neurons in the VTA might account for the functional differences between these cells in response to nicotine exposure. physiological range of plasma nicotine concentrations In addition to the actions of nicotine on DA and that are obtained by smokers (~0.5 µM)37, nicotine GABA neurons, considerable evidence indicates that the potently activates DA neurons of the VTA37,an activation actions of nicotine within the VTA might be mediated by that is followed by desensitization of nAChRs after con- glutamatergic transmission. Anatomically, the VTA tinued exposure to nicotine37.This observation indicates receives substantial glutamatergic inputs from cortical that, whereas the acute excitatory action of nicotine on and subcortical structures25.These excitatory inputs DA neurons might signal its reinforcing, rewarding effect, synapse on DA and GABA neurons25,30 (FIG. 2),and can the long-lasting desensitization of VTA nAChRs might therefore modulate the activity of both cell types.Various represent a cellular basis of nicotine tolerance. Such a studies have indicated that α7-containing nAChRs might characterization seems reasonable, given the anecdotal have a specific role in mediating the presynaptic actions reports that smokers tend to enjoy most the first cigarette of nicotine in the CNS, and, in particular, might regulate of the day (at a time when nAChRs would not be in a the release of glutamate41,42.Systemic nicotine has been state of prolonged desensitization). However, activation shown to elevate glutamate levels in the VTA, and this of DA neurons is not a scalar index of reward, nor is the effect is blocked by the relatively selective α7-subunit corresponding increase in DA release in the target regions antagonist methyllycaconitine (MLA)43.In addition, of the mesolimbic system a simple reinforcement signal lesions of the prefrontal cortex — a region that provides (see later in text)38,39.As we will discuss, mesolimbic DA glutamatergic inputs to the VTA — reduce binding of the signalling also mediates aversive motivational events33 nAChR antagonist α-bungarotoxin in the VTA. This and is involved in associative learning processes38,39. observation provides further evidence for the presynaptic Because of the important functional relationship localization of α7-containing nAChRs in the VTA, between the DA and GABA neurons in the VTA25 (FIG. 2), presumably in glutamatergic terminals43. it is imperative to examine the effects of nicotine on both Blockade of NMDA (N-methyl-D-aspartate) receptors of these neuronal populations. Recent studies on VTA and α7-containing nAChRs in the VTA diminishes the slices have investigated the actions of nicotine on the increase in mesolimbic DA release that is induced by

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nicotine44.This apparently unique role for the α7 subunit addiction is the delineation of the neuronal mechanisms in the VTA might have important implications for the that might be involved in transmitting the rewarding and psychoactive effects of nicotine. Indeed, as we will discuss, aversive motivational effects of nicotine. Understanding various studies have indicated that functional interactions how these neural systems interact might yield important between DA, GABA and glutamate within the VTA clues as to how the brain initially responds to acute nico- are vital for the mediation of the motivational properties tine exposure (as a rewarding or an aversive stimulus), of nicotine. and how the continued exposure to the drug might even- tually lead to dependence (compulsive nicotine craving Cholinergic modulation of DA function in the VTA and withdrawal symptoms). through brainstem projections. The functional connections between the brainstem nuclei TPP and LDT with Defining the role of DA: reward or aversion? For many the VTA have been studied extensively (FIG. 2).Ascending years, a common theme in the literature on behavioural inputs from the TPP and LDT to the VTA comprise neuropharmacology has been the hypothesis that cholinergic and glutamatergic fibres that synapse on DA DA and its associated neural pathways serve as specific and GABA neuronal populations of the VTA30.The transducers of central reward signals. In its most cholinergic neurons of the TPP and LDT have been simplistic form, this view of DA implies that any drug or termed the Ch5 and Ch6 cell groups, respectively45, and stimulus that can produce reward as measured by behav- these inputs can modulate the activity of the mesolimbic ioural reinforcement tests, such as conditioned place DA system. For example, electrical stimulation of the TPP preference (CPP) or intravenous self-administration elicits striatal DA efflux as measured by MICRODIALYSIS46, of the drug (BOX 1),does so by increasing levels whereas LDT stimulation elicits a similar DA efflux in the of DA through activation of the mesolimbic pathway61. nucleus accumbens through the activation of cholinergic In this sense, DA was considered a direct, scalar index and glutamatergic receptors in the VTA47. of reward. However, recent research has called into ques- The behavioural effects of these ascending cholinergic tion this conceptualization of DA in motivational sig- inputs to the VTA seem to depend more importantly on nalling, particularly in light of the fact that DA signalling signalling through muscarinic acetylcholine receptors also correlates with aversive, noxious stimuli38,39, and than through nAChRs. For example, ANTISENSE KNOCKDOWN might serve to signal conditioned stimuli that predict of muscarinic M5 receptors in the VTA of rats reduces reward (or errors in reward prediction), rather than the the rewarding efficacy of stimulating the MEDIAL FOREBRAIN rewarding events per se 62.A role for DA-mediated trans- BUNDLE48.Similarly, pharmacological blockade of mission in such cognitive processes might transcend MICRODIALYSIS muscarinic receptors in the VTA is more effective than drug-naive versus drug-dependent states and might be A technique that allows the blockade of nAChRs at attenuating the rewarding effects relevant for motivationally important learning sampling of neurochemicals in 49 the brain of live animals. of this stimulation .These in vivo findings point to processes, independent of their rewarding or aversive It commonly uses a small the behavioural complexity of cholinergic signalling emotional valence. However, a large body of research U-shaped cannula that serves a in the VTA. Indeed, the functional balance between has supported the idea that the rewarding effects of dual function: it allows the DA and GABA VTA neuronal substrates might have several psychoactive drugs, including nicotine, are injection of molecules of interest to test their effect, and it important implications for the central processing of the dependent on mesolimbic DA-mediated transmission. provides a pathway for the flow motivational properties of nicotine. Indeed, DA neural systems have arguably received the and subsequent collection of greatest amount of experimental attention as potential perfusate from a small brain The dual motivational effects of nicotine mediators of the rewarding effects of nicotine. area. We tend to think about drugs of abuse in terms of their Using an intravenous procedure for the self-

ANTISENSE KNOCKDOWN ability to produce feelings of pleasure. Indeed, nicotine is administration of nicotine, several studies have reported Oligonucleotides with a known to induce feelings of pleasure and reward in that blocking DA-mediated transmission, pharmacolog- sequence that is complementary humans and other species. But like many other addictive ically or by lesions of the mesolimbic DA pathway, is suf- to the mRNA of a given drugs, nicotine also has potent, aversive, unpleasant ficient to reduce or completely block the reinforcing molecule can be used to block its 50–52 4 translation. The subsequent effects .Nicotine can produce powerful anxiogenic effects of nicotine. For example, Corrigall et al. showed 53,54 temporary elimination of the effects systemically and centrally through activation of that pretreatment with specific antagonists of D1 or D2 protein of interest often provides nAChR that contain α4, α7 or β2 subunits55.Many peo- DA receptor subtypes strongly attenuated nicotine self- useful information on its ple experience noxious effects such as nausea, coughs and administration. Lesions of the mesolimbic DA system biological function. dizziness on their initial experience with tobacco56,57. caused by 6-hydroxydopamine, a molecule that selec-

MEDIAL FOREBRAIN BUNDLE Interestingly, tolerance to the aversive effects of nicotine tively destroys DA neurons, also attenuate nicotine Complex fibre tract that runs develops with repeated exposure52,58.Although the precise self-administration5.Similarly, microinfusions of the through the diencephalon. It neurobiological mechanism that underlies this tolerance nAChR antagonist dihydro-β-erythroidine directly into contains descending fibres from to nicotine aversion is unknown, its existence indicates the VTA attenuate nicotine self-administration, but not telencephalic structures such as the basal olfactory regions, the that chronic nicotine exposure might induce a functional the reinforcing effects of food or cocaine, implicating periamygdaloid region and the alteration in neural systems that mediate the aversive that the mesolimbic DA projection from the VTA is a septal nuclei, and ascending and/or rewarding effects of nicotine. crucial mediator of the reinforcing effects of nicotine6. fibres from the aminergic It has been suggested that relative sensitivity to the Work using gene knockout technology has further brainstem nuclei. Intracranial rewarding or aversive properties of nicotine might serve implicated mesolimbic DA-mediated transmission in stimulation along this tract can simulate motivational states and as a predictor of who might become addicted to nicotine reward. Elimination of specific nAChR sub- reinforce behaviour. tobacco59,60.So, an important goal in the study of nicotine units has shown that the nAChR subunits that are

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

a Drug Choice b

Vehicle ?

A behavioural test that is commonly used to study the motivational properties of nicotine and other drugs is c Drug 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 drug- paired 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 self- administer 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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51 64 a 50 b 100 nicotine ,or no apparent motivational effects at all .But WT nicotine nAChRβ2–/– 90 a limitation of these studies is that they relied exclusively 40 Cocaine WT saline 80 on systemic nicotine administration, which also targeted 30 * peripheral nAChRs. The activation of these receptors Cocaine 70 * could potentially produce toxic and noxious effects, lead- 20 60 per hour ing to the expression of such a conditioned place aversion. * 50 * Intravenous nicotine self-administration also activates 10 Discrimination index Nose-poke reponses * (% active responses) Naive40 Naive * peripheral nAChRs, and high concentrations of intra- * 0 30 venously administered nicotine do produce aversive Baseline 1 2 3 4 5 Baseline1 2 3 45 effects50. Self-administration, daily sessions Self-administration, daily sessions An early study reported that infusions of the nicotinic

cd300 300 agonist cytisine directly in the VTA to exclude peripheral effects could produce reward as measured in the CPP 200 200 test65.A more recent study reported that both rewarding

100 100 and aversive effects could be measured using the same 8 Preference (+) Preference (+) test after microinfusions of nicotine itself into the VTA . 0 0 Aversion (–) Aversion (–) This study reported a dose-dependent, biphasic curve for Difference score Difference Difference score Difference –100 –100 Neuroleptic the motivational effects of nicotine in the CNS; whereas Control a lower nicotine concentration in the VTA produced –200 –200 0 2 8 an aversive effect, higher concentrations produced 24 48 80 48 0.8 0.8 8.0 0.08 0.08 potent rewarding effects (FIG. 3c).So, within a single brain 0.008 0.008 0.0008 0.0008 Intra-VTA nicotine dose Intra-VTA nicotine dose region, nicotine can have rewarding or aversive effects as (nmol/hemisphere) (nmol/hemisphere) a function of nicotine concentration. Figure 3 | Different roles for dopamine (DA) signalling in the acute versus chronic phases of Surprisingly, when the rewarding effects of higher nicotine exposure. a | Nicotine self-administration is significantly attenuated in mice lacking the nicotine concentrations were challenged by blocking nicotinic acetylcholine receptor (nAChR) subunit β2 (nAChRβ2–/–), relative to wild-type (WT) animals. DA-mediated transmission, either systemically or directly b | This attenuation is specific to nicotine, as the reinforcing effects of cocaine are unaffected in these in the nucleus accumbens, there was no attenuation of the 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 rewarding effects of nicotine. However, under these permission, from Nature REF.63  (1998) Macmillan Magazines Ltd. c | By contrast, the acute effects conditions, levels of nicotine in the VTA that previously of nicotine produce biphasic motivational effects within the VTA. Whereas a low nicotine produced no motivational effects now had rewarding concentration produces aversion (as measured in the conditioned place preference task), high effects, whereas the effects of lower nicotine concentra- concentrations produce potent rewarding effects. d | Blockade of DA signalling with a systemic tions switched from aversive to rewarding (FIG. 3d).In α neuroleptic drug ( -flupenthixol) does not block the rewarding effects of high nicotine concentrations, addition, when the aversive effects of nicotine were exam- potentiates the rewarding effects of middle-range nicotine doses, and switches the motivational ined in the CTA test, DA receptor blockade prevented the effects of a low concentration from aversive to rewarding. Reproduced, with permission, from REF.8 8  (2003) Macmillan Magazines Ltd. Asterisks in a and b indicate P< 0.05. aversive nicotine signal .So, by contrast to previous studies of intravenous nicotine self-administration, these studies have indicated that the rewarding and aversive required for nicotine-induced DA release are also neces- effects of nicotine are mediated within the VTA by sepa- sary for nicotine self-administration7,63.For example, rate and dissociable systems. Whereas the acute rewarding mice that lack the β2 nAChR subunit showed decreased properties of nicotine were independent of DA signalling, DA release in response to nicotine exposure, and this the aversive effects of nicotine were crucially dependent effect correlated with a strong attenuation of nicotine on DA-mediated transmission8. self-administration63,indicating that the ability of this Although these results from the CPP model point subunit to produce nicotine reward might be tightly to an opposing functional role for DA signalling in the linked to its functional regulation of DA release in the motivational effects of nicotine within the VTA, there mesolimbic pathway (FIG. 3a,b).Interestingly, these are crucial differences between the CPP and the self- mutant mice also showed a strong reduction in nicotine- administration studies. The most important of these induced conditioned taste aversion (CTA)10 (BOX 1), indi- differences is that in studies of self-administration4–6,63 the cating that a specific nAChR subunit that is linked to animals receive chronic exposure to nicotine, often over mesolimbic DA-mediated transmission is also involved several weeks. By contrast, in most CPP studies, animals in the aversive effects of nicotine. But studies using CPP,a are naive to nicotine at the beginning of the experiment, test that is sensitive to both the aversive and rewarding and the drug is passively administered by the experi- properties of drugs, have disclosed a far more complex menter in controlled doses (BOX 1).So, whereas DA recep- role for DA in the motivational properties of nicotine. tor blockade seems to attenuate nicotine reinforcement in animals that are chronically exposed to nicotine, the acute A common neural substrate for nicotine reward and rewarding effects of nicotine can be mediated through a aversion. Many early studies on the motivational effects non-DA system in the VTA. In addition, when the acute of nicotine using the CPP test reported that, rather aversive effects of nicotine are blocked by interfering with then producing a rewarding effect, nicotine produced mesolimbic DA receptor signalling, the rewarding effects aversive effects, as manifest by the development of of nicotine are potentiated, presumably by the removal of conditioned aversions to environments paired with an aversive signal8.As we will discuss, the motivational

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state — drug-naive, drug-dependent or withdrawn — neurons75.In addition, intracranial self-stimulation might be vital to determine the role of DA in nicotine activates GABA neurons of the TPP, rather than the addiction. cholinergic Ch6 cells76,further indicating that non- cholinergic TPP mechanisms might be involved in Beyond DA: new players in nicotine addiction reward signalling. As DA does not exclusively transmit a nicotine reward Together, this evidence indicates that descending VTA signal, what other substrates are involved in the addictive inputs to the TPP might be the primary transducers of properties of nicotine? As previously noted, nicotine pro- the nicotine reward signal. For example, bilateral excito- duces the physiological activation of both DA and GABA toxic lesions of the TPP block nicotine reward in the neurons in the VTA32,33,40.As GABA neurons serve as a VTA and switch the motivational valence of nicotine substrate for non-DA-mediated reward transmission28,29 from rewarding to aversive, as measured in the CPP and are acutely activated by nicotine32,33,40, this neuronal model69.This result indicates that, whereas the TPP population is a good candidate for the mediation of nico- seems to selectively mediate a nicotine reward signal, the tine reward signalling in the VTA.A recent report has aversive effects of nicotine in the VTA (which are depen- found that GABA receptors in the VTA might be impor- dent on DA-mediated transmission in the acute state) tant mediators of the reinforcing properties of nicotine. remain intact after removal of the TPP pathway. An early Corrigall et al.23 reported that direct microinfusions of report found that partial lesions of the dorsal TPP did 6 GABAA or GABAB receptor agonists into the VTA caused not affect nicotine self-administration ,but a subsequent a significant reduction in nicotine self-administration, report from the same group claimed that more extensive indicating that GABA receptors in the VTA can also and localized TPP lesions strongly attenuated nicotine 77 mediate nicotine reinforcement. Whereas GABAA recep- self-administration .Within the TPP,GABA receptors tors in the VTA are predominantly localized to GABA seem to be crucial for transmission of a nicotine 25,28 78 neurons , GABAB receptors are primarily localized on reward signal. Corrigall et al. reported that GABAA and DA neurons and can strongly modulate the activity of the GABAB receptor agonists that were infused into the 25,66,67 mesolimbic DA system .Activation of GABAB recep- TPP attenuated nicotine self-administration on a fixed- tors in the VTA can strongly decrease mesolimbic DA ratio schedule of operant responding (see BOX 1). In activity66,67 and attenuate nicotine-induced DA release in addition, infusions of nicotine into the TPP induce CPP, the mesolimbic pathway67,68.These data are consistent further implicating the TPP as a non-DA system that is 79 with a role for GABAB receptors in the control of VTA important for the motivational effects of nicotine . DA neurons. These results are also consistent with obser- So, increasing evidence indicates that non-DA neural vations in animals that chronically self-administered substrates, including a GABA-mediated system within nicotine, as blockade of DA signalling or direct inactiva- the TPP,might be important for the reinforcing and

tion of the mesolimbic DA system with a GABAB receptor addictive properties of nicotine. Future studies are agonist can block nicotine reward in this model. required to map out the functional pathways from the VTA to these non-DA neuronal reward substrates. The TPP. The TPP is crucially involved in the transmission of drug-related21,69,70 and natural reward Emerging roles for the α7 subunit and glutamate. As information70,72.Notably, the TPP seems to be especially described previously, α7-containing nAChRs might be important in the early, acute phase of drug exposure. preferentially involved in the regulation of the presynap- For example, lesions of the TPP block opiate reward, as tic effects of nicotine on glutamate release41–43.Such a role measured in the CPP and self-administration tasks, only for the α7 subunit has been shown in various brain during the early stage of drug exposure21,71.Although the regions, including the VTA41–43.Until recently, no direct TPP has been implicated in the rewarding effects of behavioural evidence had linked this particular subunit opiates21,70,food70 and sex72, the rewarding properties to the motivational effects of nicotine. However, several of drugs such as cocaine73 do not seem to require this reports now indicate that α7 might be preferentially region. In the case of nicotine, several reports have impli- involved in the acute rewarding effects of nicotine. cated the TPP in the mediation of its rewarding effects. Panagis et al.80 reported that direct microinfusions of Anatomically, the TPP and VTA are connected by MLA in the VTA attenuated nicotine-induced potentia- descending GABA inputs from the VTA, and by ascend- tion of reward in an intracranial self-stimulation experi- ing cholinergic and glutamatergic inputs from the TPP ment. However, Grottick et al.81 found that blockade of (FIG. 2).Although these ascending cholinergic inputs from α7-containing receptors had no effect on intravenous the TPP to the VTA DA neurons can influence the activity nicotine self-administration in animals chronically of the mesolimbic DA system46,47, it is unlikely that these treated with nicotine, nor on the hyperlocomotor effects inputs are directly involved in nicotine reward for several of chronic nicotine exposure, indicating that α7 might reasons. First, the cholinergic inputs to the VTA are topo- not be involved in the motivational signalling of nicotine graphically organized such that most of the cholinergic after chronic exposure. Similarly, a recent study using brainstem projections to the VTA arise from the adjacent MLA over a wide concentration range found that this LDT, rather than from the TPP, which projects more antagonist blocked the acute effects of nicotine adminis- heavily to the adjacent substantia nigra74.Second, acutely tered directly into the VTA, and switched its motivational administered nicotine predominantly activates non- valence from rewarding to aversive82.By contrast, β2- cholinergic TPP cells, including GABA and glutamate containing nAChRs are implicated in both the rewarding

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and aversive effects of nicotine6,8,10,63,and seem to block its nicotine by determining the relative sensitivity to the reinforcing effects in both the acute and chronic stages of rewarding or aversive psychological effects of nicotine. exposure6,8,63.A caveat of these studies is that MLA at But how do alterations in DA signalling during pro- low concentrations interacts with nAChRs that do not longed exposure ultimately lead to a role for DA in the contain the α7 subunit35. motivational effects of nicotine in the drug-dependent Several studies have found that blockade of glutama- state? Here, we consider two alternative possibilities. tergic transmission with specific NMDA receptor antago- First, considerable evidence indicates that, rather nists that are administered systemically or directly in the than transmitting an acute reward signal during the VTA can block the rewarding82–84 and aversive82 effects of early exposure to drugs, DA is specifically involved in nicotine. Interestingly, blockade of NMDA receptors the late phases of the drug-addiction process, and medi- is effective in reducing the reinforcing effects of nico- ates drug craving or wanting. This view, proposed by tine in both the acute and chronic phases of nicotine Berridge and Robinson85,86, states that, after repeated exposure82–84. drug exposure, sensitization of the DA systems, which signal the INCENTIVE SALIENCE of the drug (that is, how An integrated model of nicotine addiction much the drug is craved), leads to a pathological ampli- The complexity of the motivational and physiological fication of this salience, leading to compulsive drug effects of nicotine makes it a formidable task to develop seeking and use. Indeed, repeated nicotine exposure a unifying model of nicotine addiction. Owing to the induces sensitization of DA pathways87,88 and increases ubiquity of central nAChR distribution, nicotine exerts DA receptor expression in the projection areas of the multiple effects in many brain regions beyond the VTA. VTA DA system89.In addition, specific blockade of But as the studies that we reviewed in this article attest D3 DA receptors has recently been shown to prevent to,the VTA and its associated input and output path- nicotine-induced relapse to nicotine-seeking behav- ways seem to be integral to the transmission of the iours, indicating that alterations of this particular motivational properties of nicotine. DA receptor subtype might be specifically involved in By comparing the results of molecular, electrophysi- nicotine craving and relapse90.However, there is little ological and behavioural investigations on the central evidence to suggest that the sensitized DA-mediated actions of nicotine, an integrated picture of the nicotine psychomotor responses to repeated nicotine represent addiction process is beginning to emerge. A consistent an enhancement in the rewarding properties of nicotine theme is the dichotomy in the roles of DA in the moti- per se.In addition, prolonged nicotine exposure might vational properties as a function of the stage of nicotine have profound effects on nAChR expression. Indeed, dependence. Indeed, whereas the rewarding effects of many studies have reported that chronic nicotine expo- nicotine within the VTA can be mediated through DA- sure increases the number of nAChR receptor subtypes independent mechanisms in the early, acute phases of in various brain regions91.Although it is not known if nicotine exposure8,69,75,blockade ofDA-mediated trans- such nAChR receptor upregulation is related to an mission seems to attenuate its reinforcing properties increased DA responsiveness after chronic nicotine once chronic exposure and dependence have taken exposure, upregulation of α4, β2 and α7 nAChR place4–6,63.What mechanism could account for this subunits92 within the VTA93 or other DA systems could apparent shift in the role of DA-mediated transmission conceivably contribute to such a heightened response. as a function of nicotine exposure? Do the effects of DA A related possibility is that plastic processes that receptor blockade in nicotine-dependent animals inter- increase DA responsivity to nicotine within the VTA fere with a rewarding effect of nicotine or with some might represent an aberrant form of drug-induced asso- other psychological process? ciative learning. Interestingly, exposing humans to FIGURE 4 presents a simplified model that summa- imagery of stimuli that are associated with smoking (and rizes some of the known functional differences between so with the incentive properties of nicotine) activates the role of DA and non-DA systems of the VTA in rela- neural regions that are linked to drug-induced DA tion to the motivational effects of nicotine in the acute sensitization processes, including the prefrontal cortex, versus the chronic (addicted) state. We suggest that the amygdala and orbitofrontal cortex94.Although direct shift from the acute effects of nicotine to the develop- functional comparisons between in vivo behavioural ment of a dependence state involves a switch in the studies and in vitro neuronal recording studies are tenu- functional role of DA signalling in the VTA. In the acute ous, it is possible that plastic alterations at DA synapses in state, activation of DA neurons by nicotine induces the VTA lead to a prolonged activation of DA pathways. aversive effects, whereas GABA neurons and the associ- This activation might result in persistent nicotine craving ated descending inputs to the TPP mediate its rewarding owing to the pathological amplification of an incentive- effects8,69,75.Nicotine transiently activates GABA neu- learned association between nicotine and environmental rons, the nAChRs of which rapidly desensitize33. stimuli that are associated with nicotine exposure (such Simultaneously, nicotine potentiates glutamatergic as the sight of a cigarette or the smell of tobacco smoke). INCENTIVE SALIENCE transmission through nAChRs that show slower desen- Blockade of this DA-mediated incentive learning signal A psychological process whereby sitization, leading to a functional shift in the actions of in animals that are chronically exposed to nicotine would the perception of stimuli is nicotine from the GABA to the DA neurons33.The bal- be expected to reduce nicotine self-administration, as transformed by increasing their 4–7,63 salience, making them more ance between these separate systems might determine indeed shown by most self-administration studies . attractive or wanted. the initial vulnerability to the addictive properties of So, once dependence to nicotine has developed after

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© 2004 Nature Publishing Group REVIEWS a behavioural tolerance to nicotine that has been reported Glutamate VTA in vivo.Indeed, whereas nAChR desensitization can take inputs Acute nicotine place in the order of seconds to minutes after nicotine reward signal GABA TPP exposure in vitro,behavioural nAChR tolerance in vivo develops over days or weeks. Chronic nicotine exposure and withdrawal can induce – Acute nicotine profound alterations in the mesolimbic DA system in aversion signal Nucleus 87–89,95 96 Dopamine rodents and humans .It is possible that chronic accumbens nicotine causes a long-term reduction of the baseline level of DA-mediated signalling as a compensatory response after heightened DA-mediated transmission b Glutamate VTA Desensitization of during continued nicotine exposure. Indeed, medications inputs acute nicotine reward signal that increase DA concentrations have proven efficacious GABA TPP in preventing nicotine relapse and craving in smokers97. However, DA receptor antagonists and agonists have

– been reported to reduce nicotine intake in healthy smok- Aversive nicotine NMDA receptors ers98 and in chronic smokers with schizophrenia99,100.In Nucleus craving/withdrawal α4β2-containing Dopamine addition, DA receptor agonists and antagonists increase accumbens ACh receptors Sensitized α7-containing subjective measures of nicotine craving in chronic smok- incentive salience ACh receptors ers101.Ifa lowered level of DA tone were responsible for Figure 4 | An integrated model for nicotine reward signalling in the ventral tegmental area the aversive nature of nicotine withdrawal, it seems (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 unlikely that blocking or activating DA receptors would tegmental pedunculopontine nucleus (TPP)69,76,77. These effects might involve the activation of increase nicotine intake and subjective measures of crav- presynaptic nicotinic acetylcholine receptors (nAChRs) that contain the α7 subunit, as blockade of ing. If a lowered baseline level of DA-mediated signalling this subunit interferes with the acute rewarding effects of nicotine80,82, but leaves the aversive signal were responsible for the aversive effects of nicotine with- intact82. However, nicotine might also exert its motivational effects through direct actions on nAChRs drawal, it might be predicted that blocking DA-mediated containing the β2 subunit and located on GABA or dopamine (DA) neurons, as pharmacological transmission would worsen withdrawal and craving, in blockade or genetic deletion of this subunit blocks both the aversive and rewarding effects of which case animals that self-administer nicotine might be nicotine8,63,82. In this model, nAChRs are distributed on both VTA neuronal populations, and nicotine- induced activation of these receptors can therefore regulate the motivational effects of nicotine expected to increase, rather than decrease, their intake. through either non-DA or DA systems. b | With repeated nicotine exposure, however, the GABA In addition, several studies have found that chronic system that signals reward becomes desensitized, leading to a net shift in the action of nicotine to nicotine does not lead to a decreased DA tone in response the DA neurons33,40. This shift is mediated at least partly by increased glutamatergic input to the DA to subsequent nicotine exposure102,103,but rather to an 33 system . The shift in the functional balance between GABA and DA neuronal populations in the VTA enhanced DA release in response to nicotine after chronic might lead to a dysregulated DA signal in the VTA, which in turn leads to the aversive psychological nicotine exposure104. NEUROLEPTICS have also been reported effects of nicotine craving and withdrawal, and/or to the potentiation of the incentive salience of to increase smoking rates in people with schizophrenia; nicotine and its compulsive use. NMDA, N-methyl-D-aspartate. this effect might represent a potentiation of the rewarding properties of nicotine, or a compensatory response 105 prolonged nicotine exposure, the learned associations owing to a decrease in such rewarding effects . The between its incentive motivational properties and the exceedingly high rates of nicotine addiction that are environmental stimuli that become associated with these observed in people with schizophrenia106 might indicate effects might become dependent on DA signalling. that abnormalities in DA-mediated signalling, which are However, this idea does not explain why DA-mediated considered to be a cardinal underlying pathology of transmission seems to carry a specific aversive signal in schizophrenia, might increase the motivational salience the acute phase of nicotine exposure8,10,and a reinforcing and rewarding effects of nicotine8,106. signal after chronic nicotine exposure. These findings raise the possibility that by pharmaco- A second explanation for the role of DA-mediated logically regulating DA-mediated transmission during signalling on the motivational effects of nicotine argues nicotine withdrawal, the unpleasant psychological effects for a consistent role for DA in the aversive aspects of of withdrawal and craving after chronic nicotine expo- nicotine in both the acute and chronic phases of nicotine sure might be alleviated. By extension, the ability of DA exposure. The underlying idea is that a dysregulation of receptor blockade to reduce nicotine self-administration DA-mediated signalling during nicotine dependence and in animals chronically exposed to nicotine might be due withdrawal after chronic exposure might be responsible to a blockade of the aversive effects of nicotine with- for the aversive effects of nicotine withdrawal. Whereas drawal. In other words, by blocking the aversive effects of activation of the nAChRs of DA neurons can signal an nicotine withdrawal and craving, self-administration aversive effect in the early phase of nicotine exposure8,10, of nicotine would be reduced. Interestingly, chronic the eventual desensitization of these nAChRs, which opiate exposure and withdrawal also alter DA-mediated takes place after the desensitization of the nAChRs in signalling107,108, and DA-mediated transmission is NEUROLEPTIC VTA GABA neurons33,might account for the tolerance involved in the acute aversive effects of opiate exposure This term was originally coined to the aversive properties of nicotine over time. Note, and withdrawal109,110,pointing to possible similarities in to refer to the effects of early antipsychotic agents on however, that it is not known whether the nAChR desen- the functional role of DA in both nicotine and opiate cognition and behaviour. sitization that is observed in vitro is analogous to the addiction. Although future studies are required to

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examine more closely the functional role of DA-mediated to the addictive potential of nicotine? Studies on the signalling in the early phases of nicotine exposure versus neurophysiological effects of nicotine within the VTA the chronic state, the available studies in humans and provide new insights into how nicotine induces synaptic animals consistently indicate that chronic nicotine plasticity within the mesolimbic DA system. These exposure might lead to alterations in DA-mediated sig- studies might disclose a mechanism to account for the nalling, which might in turn lead to nicotine craving shift from non-DA-mediated reward signalling in and compulsive drug-seeking behaviours. the acute state to the involvement of DA-mediated transmission in the development of compulsive nicotine Conclusions and future directions use. However, it will be important to examine how Despite the widespread effects of nicotine in the CNS, these in vitro effects of nicotine might relate to what converging evidence at the behavioural, molecular, happens in vivo during the development of addiction. genetic and physiological levels points to the VTA and its Finally, research that allows the dissection of the associated DA and non-DA systems as crucial mediators contribution of specific subunits of the nAChR to the of the motivational effects of nicotine. However, many motivational effects of nicotine and the development of important questions remain to be answered. What func- addiction might ultimately lead to a clear understanding tional interactions take place between the systems that of which nAChR subunits are involved in the vulnera- subserve the aversive and rewarding effects of nicotine bility to the rewarding properties of nicotine, and how during the development of nicotine dependence? Does alterations in specific nAChRs might lead to chronic this functional interaction determine the vulnerability nicotine use and dependence.

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