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Am J Hum Genet 74:11–19 Nicotinic Receptors in Brain Diseases Jerry A. Stitzel Abstract The existence of neuronal nicotinic acetylcholine receptor (nAChRs) expression in the brain was discovered 30 years ago. Although the relevance of neuronal nAChRs at the time of their discovery was debated, it is now clear that nAChRs are expressed throughout the brain where they mainly serve a modulatory role. Neuronal nAChRs increasingly have become of interest due to the many obser- vations that various nAChR subtypes exhibit abnormal expression or function in a wide assortment of neurological diseases. In this review, the putative role of nAChRs in brain disease is discussed in several broad categories: (1) diseases associated with a loss of nAChRs, (2) diseases associated with innate differences in the expression of nAChRs, (3) diseases associated with genetic variability in genes that code for nAChR subunit proteins, and (4) diseases in which nAChRs are implicated based on the observation that nicotine has a therapeutic effect. Keywords Receptors, nicotinic · Parkinson’s disease · Alzheimer’s dis- ease · Schizophrenia · Autism · Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) · CHRNA5 · CHRNA3 · Nicotine dependence · Tourette’s syndrome · Down syndrome Contents 1 Introduction 758 1.1 Brief History 758 1.2 Activation, Desensitization, and Upregulation 759 2 Diseases Associated with Loss of Brain Nicotinic Receptors 760 2.1 Parkinson’s Disease 760 2.2 Alzheimer’s Disease 761 3 Diseases Associated with Innate Differences in the Expression of nAChRs 763 J.A. Stitzel (B) Department of Integrative Physiology, Institute for Behavioral Genetics, University of Colorado, Boulder, CO 80309, USA e-mail: stitzel@colorado.edu 757 J.P. Blass (ed.), Neurochemical Mechanisms in Disease, Advances in Neurobiology 1, DOI 10.1007/978-1-4419-7104-3_22, C Springer Science+Business Media, LLC 2011 758 J.A. Stitzel 3.1 Schizophrenia 763 3.2 Autism 765 4 Genetic Variants of nAChR Subunit Genes and Brain Disease 766 4.1 Autosomal Dominant Nocturnal Frontal Lobe Epilepsy (ADNFLE) 766 4.2 Other Genetic Variants in nAChR Subunit Genes and Their Relation to Diseases of the Brain 767 5 Diseases Where nAChRs Are Implicated by Therapeutic Effects of Nicotine 768 5.1 Tourette Syndrome 769 5.2 Down Syndrome 769 6 Conclusions 770 References 771 1 Introduction 1.1 Brief History Neuronal nicotinic acetylcholine receptors (nAChRs) are members of the cys- teine loop superfamily of ligand gated ion channels that includes ionotropic 5-HT, GABA, and glycine receptors. As their name implies, nAChRs are receptors for the endogenous neurotransmitter acetylcholine in the nicotinic branch of the choliner- gic system. The existence of nAChR in the brain was first demonstrated by ligand binding studies in the 1980s. Using radio-ligand binding techniques, several groups established that there were at least two distinct nAChR populations in the rodent brain: one that binds the ligand [125I]-α-bungarotoxin with high affinity (Marks and Collins, 1982; Morley et al., 1979; Oswald and Freeman, 1981) and one that binds the ligands [3H]-L-nicotine or [3H] acetylcholine with high affinity (Abood et al., 1980; Marks and Collins, 1982; Romano and Goldstein, 1980; Schwartz et al., 1982; Sershen et al., 1981). The two binding sites also were found to be expressed in overlapping yet distinct patterns in the brain (Clarke et al., 1985; Marks et al., 1986; Marks and Collins, 1982). At the time of their identification, the functional relevance of these binding sites in the brain was not clear (Abood et al., 1980, 1981; Sershen et al., 1981). However, from the mid-1980s through the early 1990s cDNAs for mul- tiple nAChR subunits were cloned from rat and chicken brain (Boyd, 1997). These studies not only led to the identification of 11 different genes (12 in chickens) that code for neuronal nAChR subunits but also demonstrated that various subunit com- binations could form functional nAChRs that could be activated by acetylcholine and nicotine. The subunit genes identified were named α2–α10 (α8 only found in chickens) and β2–β4 based on the presence (α subunit) or absence (β subunit) of vicinal cysteines in the N-terminal extracellular domain and the order in which they were cloned. Neuronal nAChRs, like nAChRs at the neuromuscular junction, also were found to be composed of five subunits that form a pentameric ring around a central cation pore. These early studies also demonstrated that some nAChRs are heteromeric, requiring both an α subunit (α2–α4, α6) and a β subunit (β2orβ4) in order to form a functional receptor in vitro. The most abundant heteromeric nAChR Nicotinic Receptors in Brain Diseases 759 in brain i s comprised of the subunits α4 and β2 (Flores et al., 1992; Whiting et al., 1991). The α4β2 ∗ (the asterisk indicates that other subunits such as α5 can contribute to α4β2 nAChRs) receptor exhibits high affinity for nicotinic agonists and has been demonstrated to be the [3H]-L nicotine binding site described in the early ligand- binding studies (Flores et al., 1992; Marubio et al., 1999; Picciotto et al., 1995; Whiting et al., 1991). Other nAChR α subunits were identified that could form func- tional pentameric receptors in vitro without a β subunit. The most prevalent of these so-called homomeric nAChRs in the brain is composed of α7 subunits. Homomeric α7 nAChRs exhibit low affinity for nicotinic agonists and immunological (Chen and Patrick, 1997) and genetic studies (Orr-Urtreger et al., 1997) demonstrated that α7 nAChRs are the previously described [125I]-α-bungarotoxin binding sites in brain. Although α4β2 ∗ nAChRs are the most abundant nAChR expressed in the brain, several other heteromeric nAChR subtypes exist in the brain. For example, within dopamine terminals there are at least five different heteromeric nAChRs composed of anywhere between two and four different subunits (Champtiaux et al., 2002; Cui et al., 2003; Klink et al., 2001; Marubio et al., 2003; Salminen et al., 2004). The nAChRs on dopamine terminals in the striatum include α4β2, α4β2α5, α6β2, α6β2β3, and α4α6β2β3. Data also indicate that the nAChRs in GABAergic ter- minals are α4β2 and α4β2α5 (Lu et al., 1998; McClure-Begley et al., 2009;Zhu and Chiappinelli, 1999), whereas nAChRs that modulate acetylcholine release in the interpeduncular nucleus are α3β4 and α3β3β4 heteromers (Grady et al., 2001, 2009). A combination of immunoprecipitation experiments and in situ hybridization studies also suggest the existence of additional heteromeric nAChR subtypes (Gotti et al., 2006b), including an α7β2 ∗ subtype (Liu et al., 2009) although the functional relevance of these potential nAChR subtypes remains to be determined. 1.2 Activation, Desensitization, and Upregulation Activation of nAChRs by agonists leads to the opening of a central channel that is permeable to cations including calcium (Mulle et al., 1992). Permeability to cal- cium is receptor subtype-dependent with α7 nAChRs exhibiting the greatest calcium permeability (Fucile et al., 2003; Fucile, 2004; Ragozzino et al., 1998). Although acute exposure to a nicotinic agonist activates nAChRs, continuous exposure to activating and even subactivating concentrations of agonist leads to receptor desen- sitization, a state in which the receptors become refractory to activation to agonists (Giniatullin et al., 2005; Quick and Lester, 2002). This property of nAChRs also is subtype-dependent with α7 nAChRs exhibiting the fastest rate of desensitization (Couturier et al., 1990; Seguela et al., 1993). However, due to the low sensitivity of α7 nAChRs to activation by nicotinic agonists, α7 nAChRs appear to remain active at nicotine concentrations in the range found in smokers (Mansvelder et al., 2002; Wooltorton et al., 2003). In contrast, α4β2 ∗ nAChRs appear to be desensi- tized at the same concentrations of nicotine (Mansvelder et al., 2002; Mansvelder and McGehee, 2000). The ability of nicotine to produce long-term desensitization 760 J.A. Stitzel of at least some nAChR subtypes at physiologically relevant concentrations have led some to refer to nicotine as a time-averaged antagonist (Hulihan-Giblin et al., 1990). Another property of at least some nAChR subtypes is upregulation of receptor numbers in response to long-term nicotine exposure. This phenomenon first was discovered in rodents by Marks et al. (1983) and Schwartz and Kellar (1983). Both of these groups demonstrated that chronic treatment of mice and rats led to upreg- ulation of what we now know are α4β2 ∗ nAChRs. High doses of nicotine also led to modest increases in α7 nAChRs (Marks et al., 1983). Upregulation of α4β2 ∗ nAChRs also is seen in brain tissue from smokers (Benwell et al., 1988; Breese et al., 1997; Perry et al., 1999). This upregulation of nAChRs has been termed paradoxical because the expected effect of chronic agonist exposure is receptor downregulation (Wonnacott, 1990). However, it has been postulated that the upregulation is due to the time-averaged antagonist property of nicotine. Support for this possibility comes from a study by Marks et al. (2004) that demonstrated that, despite the increase in numbers of α4β2 ∗ nAChRs following chronic nicotine treatment, the level of function of α4β2 ∗ nAChRs remained unchanged relative to controls. These findings suggest that upregulation serves as a homeostatic mechanism to maintain normal levels of receptor function in the presence of a “time-averaged” antagonist. Due to the widespread expression of nAChRs throughout the brain and their involvement in modulating the release of many neurotransmitters, it is not surprising that aberrant expression or function of nAChRs might contribute to a wide range of diseases. In the following sections, diseases of the brain in which nAChRs have been implicated are discussed in four broad categories, diseases associated with the loss of nicotinic receptors, diseases associated with innate differences in the expression of nicotinic receptors, diseases related to genetic variants in the genes that code for the nicotinic receptor subunits, and diseases in which nAChRs have been implicated due to a therapeutic effect of nicotine. 2 Diseases Associated with Loss of Brain Nicotinic Receptors The diseases associated with loss of nAChRs are typically neurodegenerative dis- eases. The two most studied diseases that are associated with the loss of nicotinic receptors in the brain are Parkinson’s and Alzheimer’s. 2.1 Parkinson’s Disease In Parkinson’s disease (PD), loss of nAChRs occurs in the nigrostriatal pathway (Aubert et al., 1992; Pimlott et al., 2004; Quik et al., 2004) as well as in the basal forebrain and cortex (Aubert et al., 1992; Lange et al., 1993; Perry et al., 1995; Rinne et al., 1991). Early studies indicated that high-affinity nAChRs are preferentially lost in PD. A combination of studies in both rodent and nonhuman primate models of PD suggests that the α6α4β2β3 nAChR is the most labile high affinity nAChR in response to nigrostriatal damage (Kulak et al., 2002; Quik et al., 2005). The loss of Nicotinic Receptors in Brain Diseases 761 this nAChR subtype also closely coincides with the loss of the dopamine transporter (Bordia et al., 2007; Gotti et al., 2006a; Quik et al., 2004). α4β2 ∗ nAChRs also appear to be lost in animal models of PD but only when lesions are severe (Bordia et al., 2007; Kulak et al., 2002; Quik et al., 2003). Similarly, α6β2 ∗ appear to be lost to a greater extent than α4β2 ∗ nAChRs in several brain areas of PD patients (Bohr et al., 2005; Bordia et al., 2007; Quik et al., 2004). In contrast, there appears to be no loss of α7 nAChRs in striatal tissue in both animal models and humans (Guan et al., 2002; Quik et al., 2005; Zoli et al., 2002). However, there may be a loss of α7 nAChRs in cortical regions of Parkinson’s disease patients (Banerjee et al., 2000; Burghaus et al., 2003) although this finding is not universal (Guan et al., 2002). It remains to be determined whether the loss of nAChRs in PD contributes to the development of the disease or simply is a marker of the disease and α6β2 ∗ nAChRs are simply present on the neurons most sensitive to damage. Data from knock-out mice demonstrate that the lack of any of the striatal expressed nAChR subunits does not lead to striatal neurodegeneration. Thus, the simple loss of these nAChRs alone is not sufficient to elicit a neurodegenerative state. Nonetheless, a role of nAChRs in PD is supported by epidemiological evidence that clearly demonstrates that there is an inverse relationship between smoking and the development of PD. Although the mechanism through which smoking delays the onset of PD remains to be elucidated, it is generally thought that nicotine acts as a neuroprotective agent via interaction with nAChRs. The ability of nicotine to be neuroprotective in general and in animal models of PD more specifically has been demonstrated in several in vitro and in vivo studies (Quik et al., 2008). Whether nicotine acts as a neuroprotective agent through activating or desensitizing nicotinic receptors is not clear. However, recent studies with mice possessing a hyperactive form of the α4 s ubunit suggest that heightened activity rather than loss of activity is neurodegenerative in the striatum (Labarca et al., 2001; Schwarz et al., 2006). Based on this and the fact that nAChR knock-out mice show no striatal neurodegeneration suggests that the neuroprotective proper- ties of nicotine may be through desensitization/inactivation of nAChRs rather than through activation. 2.2 Alzheimer’s Disease 2.2.1 Altered Expression of nAChRs Loss of nAChRs also is associated with Alzheimer’s disease (AD). The most pro- foundly affected nAChR subtype in AD is the α4β2 ∗ subtype. Results from both receptor-ligand binding assays (Nordberg et al., 1988; Nordberg and Winblad, 1986; Perry et al., 2000; Whitehouse et al., 1986, 1988) and immunological experiments (Burghaus et al., 2000; Gotti et al., 2006a; Guan et al., 2000; Martin-Ruiz et al., 1999; Wevers et al., 1999) indicate that α4β2 ∗ nAChRs are reduced by as much as 50% in cortical and hippocampal regions of postmortem brain tissue of AD patients. α4β2 ∗ nAChRs begin to decline in the earliest stages of AD (Marutle et al., 1999) and several recent studies have shown a significant correlation between the degree of loss of this nAChR subtype and cognitive deficits in early AD patients (Kadir 762 J.A. Stitzel et al., 2006; Sabri et al., 2008). Other studies have reported a correlation between the level of expression of cortical α4β2 ∗ nAChRs and degree of cognitive deficits in AD patients (Nordberg et al., 1995; Perry et al., 2000). However, not all studies have observed a significant correlation between the expression levels of α4β2 ∗ nAChRs and cognitive deficits in early AD patients (Ellis et al., 2008, 2009). Nonetheless, a putative role of α4β2 ∗ nAChRs in AD-related neurodegeneration is supported by the observation that β2 nAChR-null mutant mice exhibit elevated age-related neu- rodegeneration in cortical brain areas and hippocampus and increased age-related cognitive deficits (Zoli et al., 1999). Some studies also have found alterations in the expression of other nAChR subunits, including α3 and α7, in postmortem brain tissue of AD patients (Guan et al., 2000; Mousavi et al., 2003; Wevers et al., 1999) and animal models (Bednar et al., 2002; Jones et al., 2006;Mousavietal.,2004). However, these findings gen- erally are not consistent. In the case of α7, some studies have found no change in expression of this subunit (Gotti et al., 2006a; Martin-Ruiz et al., 1999), oth- ers have found a decrease in expression of this subunit ( Burghaus et al., 2000; Engidawork et al., 2001; Guan et al., 2000; Wevers et al., 1999), and a few stud- ies have reported an increase in the expression of this nAChR subunit (Counts et al., 2007; Hellstrom-Lindahl et al., 1999; Teaktong et al., 2003). This apparent dispar- ity in the relationship between α7 expression and AD may be explained by a recent study by Jones et al. (2006) in which α7 expression was assessed in a transgenic mouse possessing a mutant form of the human amyloid precursor protein (APP) that results in familial AD. Results of this study demonstrated that α7 expression increases progressively to levels three- or fourfold higher than normal control brain by 9 months of age. However, by 12 months of age the transgenic mice expressed lowerlevelsofα7 than controls. Therefore, the relationship between α7 expression and AD may be age and/or disease state-dependent. It is of interest to note that despite the fact that several studies have demonstrated a reduction in α4β2 ∗ and potentially other nAChRs in AD patients, changes in RNA levels for these receptor subunits generally have not been observed in AD patients (Mousavi et al., 2003; Terzano et al., 1998). This observation suggests that the loss of nAChRs in AD is mediated posttranscriptionally. 2.2.2 Interaction of nAChRs with β Amyloid nAChRs also have been implicated in the etiology of AD via interactions with amy- loid β (Aβ), a 39–43 amino acid polypeptide that is thought to play a critical role in the pathogenesis of AD. Several studies have shown that Aβ 1-42 binds with high affinity to both α4β2 ∗ and α7 nAChRs. In addition, nicotinic receptors are impli- cated in neuroprotection from Aβ toxicity by the observations that nicotine reduces Aβ accumulation and neurotoxicity both in vitro (Kihara et al., 1998, 1999, 2001; Liu and Zhao, 2004; Zamani et al., 1997) and in animal models (Gahring et al., 2003; Hellstrom-Lindahl et al., 2004; Nordberg et al., 2002; Zhang et al., 2006). Moreover, the deposition of Aβ is significantly reduced in postmortem brain from AD patients who were smokers (Hellstrom-Lindahl et al., 2004). However, there are conflicting Nicotinic Receptors in Brain Diseases 763 data regarding whether the interaction of Aβ with various nAChR subtypes acti- vates (Chin et al., 2006; Dineley et al., 2001, 2002; Fu and Jhamandas, 2003)or inhibits (Grassi et al., 2003; Lamb et al., 2005; Liu et al., 2001, 2009; Magdesian et al., 2005; Soderman et al., 2008; Tozaki et al., 2002; Wu et al., 2004) the function of the receptors. In addition, a recent paper reported that oligomeric Aβ 1-42 at low concentrations (1 nM) selectively inhibits a novel and putatively α7β2nAChR(Liu et al., 2009). It also has been reported that there is a physical interaction between Aβ and α7 nAChRs (Wang et al., 2000a,b) and that the interaction between Aβ and the α7 nAChR facilitates the internalization of Aβ in neurons (Nagele et al., 2002). This reported internalization of Aβ by α7 nAChRs may explain the observation that Aβ and α7 nAChRs have been found to be colocalized in neurons of AD patients (Nagele et al., 2002; Wang et al., 2000a,b; Wevers et al., 1999). It has been postu- lated that the excessive intraneuronal accumulation of Aβ via internalization by the α7 nAChR leads to neuronal death (Nagele et al., 2002). However, this hypothesis is not consistent with the observation that α7 nAChRs are not preferentially lost in AD. 3 Diseases Associated with Innate Differences in the Expression of nAChRs Although there obviously is individual variability in the expression of brain nAChRs in the population, altered expression of some nAChRs relative to healthy controls is associated with several neuropsychiatric disorders. The best-studied example of low nAChR expression in brain and disease is schizophrenia. A second example discussed is autism. 3.1 Schizophrenia Schizophrenia is characterized by multiple symptoms including, but certainly not limited to, psychosis, apathy, and cognitive impairment (Austin, 2005; Mueser and McGurk, 2004). Another common feature of schizophrenia is poor sensory inhibi- tion including the inability to “filter” repetitive stimuli (Baker et al., 1987; Boutros et al., 1999; Braff et al., 2001; Clementz et al., 1998; Holzman, 2000; Kelley and Bakan, 1999; Lee and Williams, 2000). The inability to filter repetitive stim- uli is believed to lead to personality decompensation (Venables, 1964, 1992) and almost certainly contributes to the cognitive deficits associated with schizophrenia (Erwin et al., 1998; Simosky et al., 2002). The first evidence that nicotinic recep- tors may be involved in schizophrenia was the observation that either smoking or nicotine-normalized deficits in sensory inhibition, as measured by P50 auditory gat- ing, in schizophrenic patients (Adler et al., 1992). In addition, smoking, nicotine, or nicotinic agonists more recently have been shown to i mprove cognitive perfor- mance in schizophrenic patients (Freedman et al., 2008; Harris et al., 2004; Olincy et al., 2006; Sacco et al., 2005). These apparent “beneficial” effects of nicotinic agents in schizophrenics may explain the extremely high rate of smoking in this 764 J.A. Stitzel population. It is estimated that anywhere between 50 and 90% of schizophrenic patients smoke (Dalack et al., 1998; Hughes et al., 1986; Lohr and Flynn, 1992). In contrast, smoking rates i n individuals with other mental illnesses are around 25% and the prevalence of smoking in the general population is about 20% (Dalack et al., 1998; Williams and Ziedonis, 2004). Moreover, schizophrenic patients exhibit altered smoking behaviors that allow them to extract significantly more nicotine per cigarette than nonschizophrenic smokers (Tidey et al., 2005). The first direct evidence that alterations in nicotinic receptor expression might contribute to schizophrenia was from a study by Freedman and colleagues (1995) who demonstrated that schizophrenic patients had lower levels of α7nAChRsas measured by 125 I-αBTX binding and lower levels of α4β2 ∗ nAChRs as measured by [3H] cytisine binding in hippocampus relative to controls. The reduced binding was the result of both fewer labeled cells and diminished labeling per cell. In addi- tion to reduced expression in the hippocampus, α7 nAChRs also have been shown to be decreased in other brain areas of schizophrenic subjects including the reticu- lar thalamic nucleus (Court et al., 1999) and multiple cortical regions (Guan et al., 1999; Marutle et al., 1999, 2001). However, the data regarding the expression of high affinity (predominantly α4β2 ∗ nAChRs) is less clear. Results suggest that in schizophrenic patients, there is a reduction in high-affinity nAChRs in hippocampus (Breese et al., 2000; Freedman et al., 1995) and no change from controls in thalamus (Breese et al., 2000;Courtetal.,1999). However, there are conflicting results on the expression of high-affinity nAChRs in the striatum and cortex of schizophrenic sub- jects. Some studies indicate that there is an increase in high-affinity receptors in these brain regions of schizophrenic subjects (Court et al., 2000; Martin-Ruiz et al., 2003; Marutle et al., 2001) whereas other reports show that high-affinity receptor binding is lower in these brain regions of schizophrenic patients (Breese et al., 2000; Durany et al., 2000). Despite the equivocal results for high-affinity receptor expression in schizophrenia, there is evidence that regulation of this nAChR popula- tion is abnormal. As mentioned previously, smoking generally leads to a significant upregulation of α4β2 ∗ nAChRs in brain. However, depending upon brain region, upregulation of high-affinity nAChRs is either absent or substantially reduced in schizophrenic brain relative to controls (Breese et al., 2000). Support for a role of both α4β2 ∗ and α7 nAChRs in schizophrenia also comes from pharmacological and animal model data. For example, the α4β2 ∗ selective agonists ABT-418 (Stevens and Wear, 1997) and A-85380 (Wildeboer and Stevens, 2008) and the α7 selective agonist DMXB-A (O’Neill et al., 2003; Simosky et al., 2001; Stevens et al., 1998) improve innate and drug-induced deficits in auditory gating in rodents. DMXB-A also has been shown to improve sensory gating and cognitive function and to reduce negative symptoms in two recent clinical trials (Freedman et al., 2008; Olincy et al., 2006). Additional support for nAChRs in schizophrenia largely is based on mouse genetic models. Mice heterozygous for a null mutation in Chrna7, the gene that codes for the α7 subunit, exhibit reduced expression of the α7 subunit, poor auditory gating, and other functional deficits in the hippocampus similar to those observed in schizophrenic patients (Adams et al., 2008). In addition, a naturally occurring . PARK8 locus in autosomal dominant parkinsonism: confirmation of linkage and further delineation of the disease- containing interval. Am J Hum Genet 74:11–19 Nicotinic Receptors in Brain Diseases Jerry. acetylcholine in the nicotinic branch of the choliner- gic system. The existence of nAChR in the brain was first demonstrated by ligand binding studies in the 1980s. Using radio-ligand binding techniques,. [125I]-α-bungarotoxin binding sites in brain. Although α4β2 ∗ nAChRs are the most abundant nAChR expressed in the brain, several other heteromeric nAChR subtypes exist in the brain. For example, within dopamine