Genetics of nicotinic acetylcholine receptors: relevance to nicotine addiction

Abstract

Human twin studies have suggested that there is a substantial genetic component underlying nicotine dependence, ongoing smoking and ability to quit. Similarly, animal studies have identified a number of genes and gene products that are critical for behaviors related to nicotine addiction. Classical genetic approaches, gene association studies and genetic engineering techniques have been used to identify the gene products involved in nicotine dependence. One class of genes involved in nicotine-related behavior is the family of nicotinic acetylcholine receptors (nAChRs). These receptors are the primary targets for nicotine in the brain. Genetic engineering studies in mice have identified a number of subunits that are critical for the ability of nicotine to activate the reward system in the brain, consisting of the dopaminergic cell bodies in the ventral tegmental area and their terminals in the nucleus accumbens and other portions of the mesolimbic system. In this review we will discuss the various lines of evidence suggesting that nAChRs may be involved in smoking behavior, and will review the human and animal studies that have been performed to date examining the genetic basis for nicotine dependence and smoking.

INTRODUCTION

Tobacco smoking is expected to be the single biggest cause of mortality worldwide by 2030, killing about 10 million people per year [1]. This projection may seem paradoxical given the increased public awareness about the danger of smoking; however, dependence on nicotine, the primary psychoactive component of tobacco, leads many people to continue to smoke despite their desire to quit. It is therefore critical to understand the genetic basis underlying the mechanisms involved in nicotine reinforcement and dependence, so that novel treatments can be developed to fight this epidemic.

The primary targets for nicotine are the nicotinic acetylcholine receptors (nAChRs), ligand-gated ion channels with a central cation pore. nAChRs are highly conserved across species [2], and are expressed in most tissues and organs, including the brain. nAChRs can be detected on presynaptic terminals, cell bodies and dendrites of many neuronal subtypes (see [3] for a review). The endogenous ligand for nAChRs is acetylcholine (ACh) [4], and activation of nAChRs can potentiate neurotransmitter release and neuronal excitability throughout the brain. As a result, nAChRs can modulate a large number of behaviors, ranging from basic physiological functions such as pain sensation, sleep pattern and feeding, to more complex processes involved in learning, affect and reward [4–10]. Moreover, nAChRs affect brain development, through their effects on synaptic transmission and plasticity [11], as well as aging, through their neuroprotective effects (for review, see [10]).

NICOTINIC RECEPTOR SUBTYPES: LOCALIZATION, COMPOSITION AND PHARMACOLOGY

Several homologous nAChR subtypes are expressed in distinct but overlapping patterns in the brain (reviewed in [12]). Each nAChR is composed of five subunits and can either be homomeric or heteromeric. While very close in structure, each receptor subtype is also unique based on its ligand selectivity, affinity, and pharmacokinetics. In mammals, contrary to what has been recently reported in insects whereby alternative splicing could potentially provide different subunits [13], each subunit is encoded by a single gene (α2-10 and β2-4 subunits encoded by CHRNA2-10 and CHRNB2-4) [12]. One major nAChR subtype is sensitive to α-bungarotoxin (αBTX; homomeric and heteromeric receptors containing α7-α10 subunits), whereas αBTX-insensitive receptors are heteromeric and contain α2-α6 and β2-β4 subunits [7]. In the brain the most abundant and widespread subtypes are α7 and α4/β2-containing (α4/β2*, where * indicates other, as yet unidentified subunits) nAChRs [14, 15]. Homomeric nAChRS are composed of α subunits with five ligand-binding sites (one at each subunit interface) while α/β-containing nAChRs have only two binding sites (at the α-β interface) [16]. Further, heteromeric nAChR composition obeys the “rule of three” defined in [12]: two α subunits defining the main binding sites; II: two complementary non-α subunits; III: a structural subunit not involved in acetylcholine binding.

The structure and localization of the different nAChR subtypes has been established using a number of complementary techniques, including in situ hybridization and PCR for specific subunit RNAs, immunoprecipitation for protein subunits, imaging by autoradiography, PET and SPECT, and functional electrophysiological or neurotransmitter release assays. A number of limitations are inherent to these different techniques (false positives, lack of quantification, limited specificity of antibodies and ligands, etc.) [17, 18] and therefore studies using genetically modified animals have been very important in characterizing and/or confirming the localization, composition, pharmacology and functionality of the various nAChR subtypes [19, 20]. Further, while a number of studies historically focused on the biochemical properties and molecular composition of nAChRs, the development of genetic animal models with knockout, knockin or selective expression of nAChR subunits has allowed for the investigation of the role of specific nAChRs in complex behaviors, including those related to addiction.

GENETIC EXPLORATION OF NICOTINIC RECEPTOR POLYMORPHISMS

Family and twin studies

Several twin studies as early as the 1950’s concluded that smoking behavior is determined by underlying genetic factors [21–23]. It seems clear, however, that environmental actors are more critical for smoking initiation whereas genetic factors contribute strongly smoking persistence and difficulty in quitting [24, 25].

The identification of the potential gene(s) and allele variant(s) responsible for smoking has been very challenging: first, smoking behavior is not a single trait and involves exposure to the drug, habit formation, cued reinforcement, nicotine metabolism and withdrawal. Second, nicotine addiction is a complex trait, meaning that many genes with relatively small effects are likely to modify smoking behavior. Third, unpredictable interactions between genetic and environmental factors add another level of complexity. However, nicotine dependence is a very reliable and robust disorder in humans, and is easy to quantify compared to other complex brain disorders, thus leading to precise phenotypical profile that cannot be obtained in other models. Furthermore, the development of powerful large-scale genetic techniques and the cross-communication between several fields including animal studies, biochemical investigations and molecular biology have provided several candidate genes for smoking. Nevertheless, the majority of the genes likely to be involved in smoking behaviors are still to be identified.

Candidate gene approaches and Single Nucleotides Polymorphism (SNP) analyses

A promising hypothesis-driven approach to identifying genes involved in nicotine addiction involves identifying polymorphisms in genes known to interact directly with nicotine. The enzymes that metabolize nicotine and clear it from the blood stream have been particularly interesting targets. For instance, two studies have shown that polymorphisms in the cytochrome CYP2A6 is associated with smoking [26, 27], although another study did not confirm this finding [28]. Polymorphisms in other cytochromes such as CYP2D6 did not reveal any association in several independent cohorts [29, 30].

Since nAChRs are the primary targets for nicotine in the brain, several studies have looked for associations between SNPs in genes encoding nAChR subunits and smoking behavior. Analyses of SNPs in CHRNA4 and CHRNB2 subunits detected a haplotypic association with an α4 polymorphism but not a β2 polymorphism, and this association was only found in women [31]. Another study showed no correlation between β2 subunit polymorphisms and smoking [32]. This does not necessarily suggest that the α4/β2 nAChRs are not important for smoking behavior (see also “animal models” section), since the currently identified SNPs do not appear to result in functional changes in these nAChR subunits.

Recent studies have suggested that the less abundant β4* nAChRs could play a significant role in cigarette smoking [33]. The analysis of several variants of CHRNB4 revealed that specific point mutations were responsible for differential sensitization of β4* nAChRs, possibly resulting in differential sensitivity to nicotine.

Linkage analyses

A number of whole-genome linkage analyses have been performed to try to identify novel genes associated with smoking and nicotine dependence. Numerous genomic loci have shown linkage with smoking-related behaviors. For instance, associations with smoking have been found for loci located on chromosomes 1, 2, 6, 9, 11, 14, 17, 18, 19, 21 [34–38]. In the case of the locus identified on chromosome 17, fine mapping coupled with targeted SNP analyses revealed that CHRNB1, the gene encoding the muscle nAChR β1 subunit, was associated with smoking [39]. This outcome appears paradoxical as CHRNB1 is not thought to be expressed in the brain; however there may be a contribution of peripheral nAChRs to smoking behavior that is not yet understood. It is also possible that the same chromosomal region contains other genes that may also account for smoking-related behaviors [40]. Despite these advances, many of these loci do not show consistent linkage across different independent studies and cohorts, cannot be replicated, and/or lack power to undertake satisfactory meta-analyses [41]. There is therefore still a lot to be done to understand the genetic susceptibility to smoking. In addition, ethical concerns have been raised regarding the implications of identifying genetic susceptibility to smoking, and this has become the object of debate [42, 43].

MODELING AND TESTING NICOTINE ADDICTION IN ANIMALS

The American Psychiatric Association defines dependence (“addiction”) as persistent drug use despite the motivation to quit, withdrawal symptoms when the drug is not readily available [44]. In addition, relapse and reinstatement of drug dependence even after a long period of abstinence generally accompany the aforementioned symptoms While several of these symptoms can be modeled in animals, this clinical profile cannot be completely recapitulated (for instance, it is difficult to model the motivation to quit). In addition, dose-reponse relationships can very greatly across species, and this is particularly true for nicotine [45]. Instead, animal studies have focused on reproducing specific endophenotypes, which can be reliably measured in animals.

Like many other drugs of abuse, nicotine is believed to act in part through activation of the mesocorticolimbic system. Activation of nAChRs on dopaminergic neurons of the ventral tegmental area (VTA) increases their firing rate and stimulate dopamine (DA) release from their terminals in the nucleus accumbens [46–48]. Lesions of, or infusion of nicotinic antagonists into, the VTA can prevent the development of behaviors related to nicotine addiction [46, 49].

Nicotine self-administration, a paradigm in which an animal can work to receive infusions of nicotine, is one test with relatively high face-validity for human nicotine intake (see [50] for a detailed review); however, concerns about the reproducibility of nicotine self-administration and its ability to predict the efficacy of therapeutics for smoking cessation have been raised [51–54]

Another paradigm, conditioned place preference (CPP), assesses the ability of a drug to induce an association between its rewarding effects and environmental cues. Response to smoking-related cues is a critical component of nicotine addiction in humans, and similar effects have been observed in animals [55]. There is a very narrow dose range for nicotine CPP in rodents [56], perhaps because rodents are highly susceptible to stress-nicotine interactions (for example, mice must be pre-handled to show nicotine-induced CPP [57]). It is believed that this procedure would decrease stress levels in the experimental animals which might diminish stress-induced behavioral confounds.

Locomotor activation is frequently used as a measure of psychostimulant effects related to stimulation of the dopaminergic system. Similarly, locomotor sensitization is thought to depend on neuroadaptations that occur in the mesolimbic DA system following repeated drug exposure [58]. Many studies have demonstrated that nicotine increases extracellular DA levels in the nucleus accumbens leading, in turn, to increased locomotor activity [58–61]

Over time, smokers become less sensitive to the effects of nicotine, a phenomenon known as tolerance. The mechanisms underlying nicotine tolerance are not completely clear because chronic nicotine can induce desensitization of nAChRs, but also results in nAChR upregulation [62]. Upregulation is still observed in abstinent smokers at least 14 days after smoking cessation [63], and tolerance can also be observed without changes in nicotine binding [64]. In animals, this pharmacological response can be assessed using nicotine hypothermia and hyperalgesia. When injected with nicotine, body temperature decreases, due in part to vasoconstriction. Repeated nicotine injection leads to tolerance to the hypothermic effects of nicotine but sensitization to pain induced by footshock or tail pinch [65]. While there are likely to be differences human responses and animal models, partly due to difficulties in assessing this complex phenotype, upregulation of high-affinity nicotinic receptors is observed following chronic nicotine administration or tobacco use across species examined to date (see for instance, [66–69]).

Combining behavioral assays related to addiction with pharmacological and/or genetic manipulations of nAChRs can provide insight into the neurobiology of nicotine addiction and potential pharmacotherapies for smoking cessation.

GENETIC ANIMAL MODELS USED TO STUDY THE EFFECTS OF NICOTINE

Genetically-modified animals have been very valuable for identifying the physiological and behavioral roles of individual nAChR subtypes [5, 9]. This has been particularly important because most pharmacological agents that modulate nAChR function are not specific for individual subtypes. Invertebrates such as Drosophila. and C. elegans can be useful in studying the functional properties of nAChRs [13, 70, 71] because these organisms are easily genetically manipulated; however these invertebrate models do have limitations such as less complex brain structures and different nAChR subtypes and expression patterns as compared to mammalian species. Also, fundamental differences in nicotinic receptors (expression, pharmacology) have been observed across the animal kingdom, and this is particularly true between mammals and invertebrates [72]).

Genetically-manipulated mice are more difficult to generate and maintain than flies or worms, but the mouse is a mammalian organism with a fully sequenced genome, and exhibits behaviors that can be related to human addiction. A genetically-manipulated mouse line should show reproducibility of phenotypes across time and hopefully across laboratories [73]. Further, a genetic animal model should rely on the same basic underlying mechanisms involved in addiction-related phenotypes. It is worth noting, however, that animal phenotypes that resemble human traits (face validity) may not always reflect similar underlying mechanisms.

Inbred/selected strains

Inbred mouse strains, lines of mice that are genetically identical as a result of inbreeding, have been very useful in demonstrating the genetic basis for nicotine sensitivity, physiological response and behavior [74]. Comprehensive batteries of behavioral tests have been used to further define the effects of nicotine across a large number of inbred strains [75], and have shown that response to nicotine cannot simply be explained by nAChR levels in the brain [76]. Further investigations have shown that mice selected for ethanol sensitivity also show differential susceptibility to nicotine-induced locomotor depression [77]. Further, susceptibility for nicotine-induced seizures was inversely correlated to oral nicotine intake across several mouse strains, suggesting a possible protective mechanism against nicotine-induced toxicity [78].

Similar studies have been performed in inbred rat strains. The Roman High and Low Avoidance strains of rats [79] display differential responses to nicotine in various assays [80] including locomotor activation [81], although other responses to nicotine, such as avoidance behavior, were not different between the two lines [82]. Variability in function of the adrenergic system seems to underlie many of the differences between these two lines [83]. Similarly, the Flinder sensitive rat line [84] is hypersensitive to anticholinesterase agents and shows alterations in central nAChR number and function [85, 86].

As in human linkage studies, it has been difficult to identify specific genes responsible for differential responses to nicotine in these inbred lines. A point mutation in the α4 gene, found in DBA mice and not in C57BL/6 mice, has been linked to response to nicotine. This confirms that differences in α4* nAChR function across mouse strains cannot be attributed to variation in nAChR expression levels but rather, to the biophysical properties of polymorphic α4* nAChRs [87].

The improvement in molecular techniques has permitted extensive molecular dissection of nAChR functions (see fig. 1), including their roles in to behaviors related to addiction. The first genetic modifications of nAChRs in mice were developed by deleting (“knocking out”, abbreviated KO) DNA sequences encoding individual nAChR subunits (see for instance [88, 89]. While this approach defined links between specific nicotinic receptor subunits and behaviors related to nicotine addiction, constitutive knockout of a gene can result in molecular and physiological alterations, particularly during development [90]. In addition, nAChRs are expressed in many neuronal subtypes throughout the brain. Thus, KO mice lacking nAChR subunits in all cell types cannot be used to identify the anatomical locus of nAChR function. Thus, experiments using transgenic animals should always be interpreted with caution.

Figure 1. Genetically-manipulated mouse models and their relevance for nicotine addiction.

Timeline of the reported knockout, knockin of mutations, for various nAChR subunits. Major phenotypes related to nicotine addiction are shown. Please note that the date for generation of the knockout lines may not match the date that the individual phenotypes were reported (see text for detailed paradigms and references). Abbreviations: ↑, increased compared to wild-type controls; ↓, decreased compared to wild-type controls; =, similar to wild-type controls; αCTX MII, α-conotoxin MII; CPP, nicotine conditioned place preference; DA, dopamine; DAT, dopamine transporter; KO, knockout mouse line; KI, knockin mouse line; Loc, chronic nicotine induced locomotor activation; Nic, nicotine; PA, passive avoidance; Self-admin., nicotine self-administration.

Targeted point mutations (“knockin”) in nAChR subunits have been used to alter the sensitivity of both in α4 [91, 92] and α7 [93] subunits in vivo. Cell-specific and inducible transgenic expression systems can also provide information about the time in development or the neuronal subpopulations that are essential for particular behavioral effects of nicotine [94, 95]. The use of viral vectors to express nAChR subunits in specific cells/brain loci on the background of a constitutive knockout has been extremely powerful in identifying the anatomical basis for behavioral and neurochemical effects of nicotine [54, 96]. Similarly, antisense oligonucleotides have been used to decrease the expression of specific nAChRs subunits in adulthood [97], although this approach has only been used for the α6 subunit.

The β2subunit [88]

The β2-subunit, along with its partner α4, is the most widely-expressed nAChR subunit in the brain [98]. β2* nAChRs have a high affinity for nicotine [88] and are essential for the control of dopaminergic neuron firing as they allow these neurons to switch from a resting to an active state [99]. In knockout mice lacking the β2 subunit, binding to α4/β2 agonists and antagonists is abolished [88, 98], but the animals survive, are able to breed and appear grossly normal in most behavioral tasks [88]; however, most responses to nicotine appear to be absent in β2 subunit KO mice, including nicotine-induced DA release, nicotine-elicited increases in the firing rate of DA neurons and nicotine-elicited DA release in the striatum [100, 101]. The inability of nicotine to stimulate the DA system in β2 KO mice is consistent with the absence of nicotine self-administration in these animals [100]. Several aspects of nicotine response associated with DA system activation are also abolished in β2 KO mice, including locomotor activation [95, 102], nicotine discrimination [103], conditioned place preference [104] and increase in conditioned reinforcement following chronic nicotine exposure [105]. In addition, β2 KO animals have increased incentive for food reinforcement [105], and are not sensitive to the enhancing effects of nicotine or ethanol in contextual learning [106, 107] and startle reflex [108].

Interestingly, the threshold for cocaine conditioned place preference is increased in β2 KO mice [109] and the ability of repeated cocaine exposure to result in sensitization of DA release is decreased in β2 KO mice treated with an α7 antagonist [110]. While there was a blunting of cocaine-induced CPP, β2 KO mice readily self-administer morphine, but not nicotine, into the VTA [54]. In contrast to acute measurements of nicotine reward and reinforcement, somatic signs of nicotine withdrawal are still observed in β2 KO mice [111, 112].

Conditional rescue approaches have been important in defining the brain areas and times in development that β2* nAChRs are necessary for particular behaviors. For example, β2 KO mice show enhanced passive avoidance to a mild footshock [88] and tetracycline-inducible, localized transgenic rescue in corticothalamic glutamatergic neurons showed that this was due to a developmental role for β2* nAChRs [95]. Similarly, viral vector-mediated re-expression of the β2 subunit in the VTA rescued DA release in the accumbens, neuronal firing in the VTA and intra-VTA nicotine self-administration [54], indicating that nAChRs in the VTA are critical for nicotine reinforcement during adulthood.

The α4 subunit [113, 114]

The α4 subunit is the partner of the β2 subunit in the majority of heteromeric nAChRs in the brain. Animal studies have also shown that a functional polymorphism in the α4 subunit modulates nicotine intake in different mouse strains [115], possibly due to an alteration of the ratio between high and low affinity α4/β2* nAChRs [116, 117].

Like β2 KO mice, α4 null mutant mice do not show nicotine-elicited increase in striatal extracellular DA levels [118] and further abnormalities include failure to properly modulate dopaminergic transmission and impaired dopamine transporter levels [119]. These mice are also less sensitive to the locomotor depressant effects of nicotine while paradoxically, α4 KO mice had higher baseline locomotor activity compared to control animals. α6/β2* and α4β2* nAChRs appear to be the predominant nAChR subtypes that mediate endogenous cholinergic modulation of DA release [19]. Thus, increased function of α6/β2* nAChRs may be responsible for the baseline increase in locomotion in α4 KO mice.

Introduction of a point mutation resulting in a hypersensitive α4* nAChR receptor [91, 120] resulted in nicotine-dependent locomotor sensitization and conditioned place preference at much lower doses of nicotine than were effective in wild type mice [92]. This increase in sensitivity results in potential activation of these nAChRs by endogenous choline, and this may explain the loss of midbrain dopaminergic neurons in these α4 knockin mouse lines [91, 121]. α4 knockin mice also showed tolerance to a low dose of nicotine as measured by nicotine-induced hypothermia. These data strongly suggest that α4* nAChRs are both necessary and sufficient for behaviors related to nicotine addiction. Since the α4 subunit requires the β2 subunit for assembly, these studies using genetically modified mice suggest that α4/β2* nAChRs are critical for nicotine-related reward, motivation and tolerance.

The α7 subunit [89]

Like most of the other nAChR subunit KO mice, mice lacking the α7 subunit do not show gross anatomical or behavioral abnormalities [122]. The pharmacology and cellular responses of heteromeric nAChR subtypes are not altered in α7 KO mice [123]. There are, however, estrous cycle disturbances and lower rate of survival of pups born to α7 KO mothers [124]. Despite the broad expression of the α7 subunit in the brain, studies in α7 KO mice have not yet identified a role for the α7 subunit in nicotine reward and conditioning. Indeed, α7 KO animals show normal nicotine-conditioned place preference [104], lever-pressing behavior, nicotine-induced locomotor depression, and nicotine tolerance [125]. However, α7 KO animals do not show hyperalgesia following nicotine withdrawal [65]. It is clear that α7* nAChRs are important for long-term potentiation in the VTA [126], thus the absence of a phenotype related to nicotine reward in α7 KO mice may be due to compensation by other nAChR subunits. Consistent with this possibility, mice lacking both the α7 and the β2 subunits show greatly impaired passive avoidance learning [127].

Mice with a knockin of a point mutation resulting in hypersensitive α7 nAChRs are much more impaired than any of the knockout lines. Hypersensitive α7 knockin mice are barely viable since homozygotes die almost immediately after birth. Breeding of heterozygous animals showed that mice with hypersensitive α7 nAChR function showed nicotine-induced seizures at low doses (inversely correlated with oral nicotine intake in inbred strains of mice), whereas α7 null-mutants showed normal nicotine-induced seizure thresholds [128, 129].

The α3 subunit [130]

The α3 subunit is expressed at high levels throughout the autonomic ganglia [131], in the medial habenula and superior colliculus [98] but low levels of CHRNA3 mRNA is also found in DA neurons [132–134]. α3 KO mice die very early, possibly due to autonomic dysfunction and impaired growth [130]. In addition, α3 KO mice show megacystis in the colon, which is analogous to the hypoperistalsis that has been observed in a human subject with an α3 deletion mutation [135]. α3 KO mice are resistant to nicotine-induced seizure and hypolocomotion [136]. Taken together, these data suggest that the α3 subunit is critical for ganglionic function but may also contribute to central responses to nicotine, such as nicotine-induced locomotor depression.

The β4 subunit [137]

The β4 subunit co-localizes with the α3 subunit in both the brain and the autonomic ganglia, but the β2 subunit may partially substitute for the loss of β4 in the autonomic ganglia since β4 KO mice were relatively normal, whereas β4/β2 double KO mice had a roughly similar phenotype to α3 KO animals [137]. These results confirm that α3/β4* nAChRs are the major nAChR subtype in the autonomic nervous system. β4* nAChRs appear to be responsible for the development of both somatic signs of nicotine withdrawal (such as increased grooming, chewing, scratching, and tremors) [111] and hyperalgesia [112], although the withdrawal syndrome in human smokers is normally characterized by more emotional processes, such as anhedonia and affective dysfunction, rather than somatic responses [138, 139].

The α5 subunit [140, 141]

α5 null mutant mice do not show brain or anatomical abnormalities; moreover, these KO mice express normal levels of mRNAs encoding other nAChR subunits and have unchanged [¹²⁵I]-epibatidine and [¹²⁵Iα-bungarotoxin binding in the brain compared to wild-type animals. Despite having relatively unchanged nicotinic binding, α5 KO mice are less sensitive to nicotine-induced seizures and are also more resistant to the hypolocomotion induced by acute nicotine in specific mouse strains [140, 141].

The α6 subunit [133]

α6 knockout mice, like many other nAChR null mutant lines, did not exhibit any obvious neurological or behavioral deficits, even in areas with high levels of α6 subunit expression, such as the visual and dopaminergic pathways [133]. One possibility is that expression of α4 subunits at least partially compensates for the loss of the α6 subunit in the dopamine system [133]. Knockout mouse studies were critical in demonstrating the that the α6, rather than the α3, subunit partners with β2 in mediating α-conotoxin MII-sensitive, nicotine-induced DA release [133], suggesting that α6* nAChRs play a potential role in addiction-related behaviors.

GENETICALLY-MANIPULATED MOUSE MODELS: BEYOND ADDICTION

The development of genetically-modified animals with altered expression of nAChR subunit has identified many functions relevant to nicotine addiction; however, nicotinic receptors are involved in many functions in the nervous system, and also are important for basic physiological mechanisms.

It is clear that both anxiety and depression are related to smoking behavior in human subjects [9]. Consistent with a role for nAChRs in regulation of anxiety-like behavior, mice lacking either the α4 subunit or the α7 subunit show less anxiety-like behaviors [122, 142]. With respect to depression-like behavior, β2 KO mice do not respond to classical and atypical antidepressants, emphasizing the relationship between the cholinergic and the monoaminergic systems in mood disorders [143–145]. Interestingly, these mice are also less immobile in the forced swim and tail suspension tests at baseline, suggesting that β2* nAChRs may normally have a role in regulating mood.

CONCLUSIONS

Nicotine addiction is a continuing public health problem, and the need for therapeutics continues to drive research on nAChRs and their functions. To date, research into the role of human nAChR polymorphisms in smoking behavior has not resulted in significant associations, but this may be because functional alterations in nAChRs results in more profound dysfunction, such as nocturnal frontal lobe epilepsies [146, 147] for a review see [148]. Limited power analyses, population variations and variable phenotypic definitions of nicotine dependence may also contribute to the lack of progress in the human genetics of vulnerability to smoking. Knockin mice bearing human ADNFLE mutations will be extremely useful in determining the functional consequences of these alteration in nAChR subunits, and may reveal neurochemical and behavioral consequences of these mutations that have not yet been examined in human subjects.

Genetically-manipulated mice have been useful for identifying nAChR subtypes that are important for behaviors related to nicotine addiction. The combination of genetic and pharmacological techniques has highlighted specific targets for therapeutics to help people to quit smoking. In addition, the genetically-manipulated mouse models have contributed to a greater understanding of the role of nAChRs in behaviors such as cognition and affect that may contribute to ongoing smoking, as well as to fundamental contributions of nAChRs to neuronal excitability. These studies have provided new targets for the investigation of nicotine-related mechanisms linked to addiction