Neuronal nicotinic receptors in the human brain

Progress in Neurobiology 61 (2000) 75±111

www.elsevier.com/locate/pneurobio

Neuronal nicotinic receptors in the human brain David Paterson, Agneta Nordberg* Department of Clinical Neuroscience, Occupational Therapy and Elderly Care Research, Division of Molecular Neuropharmacology, Karolinska Institute, Huddinge University Hospital, S-14186 Huddinge, Sweden Received 20 July 1999

Abstract Neuronal nicotinic acetylcholine receptors (nAChRs) are a family of ligand gated ion channels which are widely distributed in the human brain. Multiple subtypes of these receptors exist, each with individual pharmacological and functional pro®les. They mediate the e€ects of nicotine, a widely used drug of abuse, are involved in a number of physiological and behavioural processes and are additionally implicated in a number of pathological conditions such as Alzheimer's disease, Parkinson's disease and schizophrenia. The nAChRs have a pentameric structure composed of ®ve membrane spanning subunits, of which nine di€erent types have thus far been identi®ed and cloned. The multiple subunits identi®ed provide the basis for the heterogeneity of structure and function observed in the nAChR subtypes and are responsible for the individual characteristics of each. A substantial amount of information on human nAChR structure and function has come from studies on neuroblastoma cell lines which naturally express nAChRs and from recombinant nAChRs expressed in Xenopus oocytes. In vitro brain nAChR distribution can be mapped with a number of appropriate agonist and antagonist radioligands and subunit distribution may be mapped by in situ hybridization using subunit speci®c mRNA probes. Receptor distribution in the living human brain can be studied with noninvasive imaging techniques such as PET and SPECT, with a signi®cant reduction in nAChRs in the brains of Alzheimer's patients having been identi®ed with ‰11 CŠ nicotine in PET studies. Despite the signi®cant body of knowledge now accumulated about nAChRs, much remains to be elucidated. This review will attempt to describe the current knowledge on the nAChR subtypes in the human brain, their functional roles and neuropathological involvement. # 2000 Elsevier Science Ltd. All rights reserved.

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Nicotinic receptor structure . . . . . . . . 1.2. Ligand binding sites . . . . . . . . . . . . . 1.2.1. ACh binding site . . . . . . . . . . 1.2.2. Allosteric binding sites. . . . . . 1.3. Transition states of nicotinic receptors 1.4. Nicotinic receptor upregulation . . . . .

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Abbreviations: aBTX, Alpha bungarotoxin; Ach, Acetylcholine; nAChRs, nicotinic acetylcholine receptors; AChRs, nicotinic receptor sites that bind nicotinic agonists with high anity; ACTH, Adrenocorticotropic hormone; AD, Alzheimer's disease; ADNFLE, Autosomal dominant frontal lobe epilepsy; APP, Amyloid precursor protein; bA4, b amyloid; BTXRs, nicotinic receptor sites that bind bungarotoxin with high anity; CGRP, calcitonin gene related peptide; ChAT, Choline acetyltransferase; CNS, Central nervous system; DA, Dopamine; DHbE, Dihydrob-erythroidine; DMEA, Dimethylethanolamine; DMPP, 1,1-dimethyl-4-phenylpiperazanium; EEG, Electro encephalograph; GABA, gamma amino butyric acid; 5-HT, 5 hydroxy tryptamine (serotonin); kBTX, kappa bungarotoxin; Kd, Dissociation constant; MLA, Methylcaconitine; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropine; MRI, Magnetic resonance imaging; NA, Noradrenaline; NBM, Nucleus basalis of Meynert; NMDA, N-methyl-D-aspartame; PCP, phencyclidine; PD, Parkinson's disease; PET, Positron emission tomography; PKA, Protein kinase A; PKC, Protein kinase C; SPECT, Single photon emission computed tomography; TS, Gilles de Tourette syndrome. * Corresponding author. Tel: +46-8-58585467; fax: +46-8-6899210. E-mail address: [email protected] (A. Nordberg). 0301-0082/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 1 - 0 0 8 2 ( 9 9 ) 0 0 0 4 5 - 3

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D. Paterson, A. Nordberg / Progress in Neurobiology 61 (2000) 75±111

2.

Neuronal nicotinic receptor subtypes in the human brain . . . . . . . . . . . . . . . . . 2.1. Ligand binding studies on human brain tissue . . . . . . . . . . . . . . . . . . . . 2.2. Binding studies in human neuroblastoma cell lines . . . . . . . . . . . . . . . . . 2.3. Nicotinic receptor subunit expression in oocytes and transfected cell lines 2.3.1. Heteromeric nicotinic receptors . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2. Homomeric nicotinic receptors . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Electrophysiological studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Evidence for four functional subtypes of nicotinic receptor . . . . . . . . . . .

3.

Distribution of nicotinic receptors in the human brain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87 3.1. Ligand binding studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88 3.2. Subunit mRNA distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90

4.

Imaging of nicotinic receptors with PET and SPECT . 4.1. PET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1.1. [18 FŠ NFEP or ‰18 FŠ FPH . . . . . . . . . . 4.1.2. [18 FŠ A-85380 and ‰11 CŠ A-8548. . . . . . 4.1.3. [11 CŠ MPA . . . . . . . . . . . . . . . . . . . . 4.1.4. [76 BrŠ BAP . . . . . . . . . . . . . . . . . . . . 4.2. SPECT . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

5.

Nicotinic receptor function in the CNS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 5.1. Functional and behavioural e€ects of nicotine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96 5.2. Role of nicotinic receptors in cognitive and memory functions . . . . . . . . . . . . . . . . . . 96

6.

Pathology of neuronal nicotinic receptors . 6.1. Epilepsy . . . . . . . . . . . . . . . . . . . . 6.2. Alzheimer's disease . . . . . . . . . . . . 6.2.1. Alzheimer's disease therapy 6.3. Parkinson's disease . . . . . . . . . . . . 6.4. Schizophrenia . . . . . . . . . . . . . . . . 6.5. Tourette's syndrome . . . . . . . . . . . 6.6. Anxiety and depression . . . . . . . . .

7.

Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

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91 92 93 94 95 95 95

97 97 98 99 100 101 101 102

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

1. Introduction Neuronal nicotinic acetylcholine receptors (nAChRs) are transmitter gated ion channels which belong to a gene super family of homologous receptors including GABA, glycine and 5-hydroxy tryptamine (5-HT) (Karlin and Akabas, 1995). A number of di€erent subtypes of nAChR exist each with individual pharmacological and physiological pro®les and distinct anatomical distribution in the brain. By analogy with their muscle counterparts neuronal nAChRs are believed to have a pentameric structure consisting of ®ve membrane spanning regions around a central ionchannel. This structure is composed of a number of subunits of which there are multiple subtypes, the

genes for all or most of which have been cloned and expressed. Although much is now known about the structure and functional properties of neuronal nAChRs (mainly from expression studies), relatively little is understood about their physiological role in man. Evidence suggests that nAChRs do not appear to function in the classical postsynaptic, directly excitatory manner of their muscle counterparts. Their location in the brain is not limited to postsynaptic but also to pre-, peri- and extrasynaptic sites where they may modulate neuronal function by a variety of actions (LindstroÈm, 1997). To this end, neuronal nAChRs are involved in a number of functional processes including cognition, learning and memory, arousal, cerebral blood ¯ow and metabolism, and a

D. Paterson, A. Nordberg / Progress in Neurobiology 61 (2000) 75±111

growing list of pathological conditions (Levin and Simon, 1998). For example, mutations in one of the nAChR subunit genes is responsible for a speci®c form of epilepsy, autosomal dominant frontal lobe epilepsy (ADNFLE), and it has be suggested that an nAChR subunit gene mutation may be among the factors predisposing to schizophrenia (Chini et al., 1994; Steinlein et al., 1995; Freedman et al., 1997; Levin and Simon, 1998). In Alzheimer's disease (AD), there is a signi®cant loss of high anity nAChR sites (Whitehouse and Au, 1986; Nordberg and Winblad, 1986; Nagata et al., 1996; Whitehouse et al., 1988a) which is believed to re¯ect the pathophysiological changes underlying the cognitive decline and dementia observed in this condition. High anity nAChR sites are also reduced in the brains of Parkinson's disease (PD) patients (Whitehouse et al., 1983, 1988a), and thus, nAChRs may also be involved in the dementia which is associated with this condition. Neuronal nAChRs are also potential therapeutic targets in a number of CNS disorders with nicotine observed to be bene®cial in AD, PD and Tourette's syndrome (Jones et al., 1992; Vidal, 1996; Newhouse et al., 1997). Thus, there is great interest in the development of selective nAChR agonists as therapies (Sershen et al., 1987; Madhok et al., 1995; Dursun and Reveley, 1997; Newhouse et al., 1997; Maggio et al., 1998; Sabbagh et al., 1998). Nicotine and nAChRs may also apparently be involved in the pathophysiology of both anxiety and depression. Nicotine has anxiolytic properties in animal models and retrospective and prospective clinical studies have demonstrated a relationship between smoking and major depression; persons with major depression are more likely to smoke and more likely to develop severe depressive episodes upon cessation of smoking (Covey et al., 1997, 1998). The most obvious role of neuronal nAChRs is mediation of tolerance and addiction to nicotine in chronic tobacco users and the symptoms of withdrawal experienced upon cessation of use (Benowitz, 1996). Neuronal nAChRs are therefore involved in a complex range of functions in which their exact role and full potential as therapeutic targets has yet to be elucidated. This review will attempt to describe the subtypes of neuronal nAChR present in human brain, their individual functional roles and involvement in behaviour and pathological conditions. 1.1. Nicotinic receptor structure Biochemical investigations with Torpedo receptor (Galzi and Changeux, 1995) and with neuronal receptors (Anand et al., 1991) have established that both peripheral (e.g. muscle type) and neuronal nAChRs are comprised of hetero-oligomers consisting of ®ve membrane spanning subunits which form a barrel like

77

structure in the membrane around a central ion channel (Cartaud et al., 1973). Molecular cloning studies in chick, rat and human have identi®ed multiple genes that encode various subtypes of subunit that allow assembly of a wide variety of receptor oligomers with di€erent distribution and distinct pharmacological pro®les (Sargent, 1993). Peripheral nAChRs, such as those found at the neuromuscular junction, are made up of a1, b1, g and d or e subunits (in adult and fetal forms of the receptor, respectively) in the ®xed stoichiometry of 2.1.1.1.1 (i.e. 2a1, 1b1, 1d or 1e). Neuronal nAChRs di€er from those in the periphery as they have no g, d, or e subunits in their make up and consist of various complements of a2±a9 and b2±b4 subunits. Presently, six a (a2±a7) and three b (b2±b4) subunits have been identi®ed and cloned from human brain (Sargent, 1993; Galzi and Changeux, 1995; McGehee and Role, 1995; Elliott et al., 1996; Gotti et al., 1997). In contrast to muscle type receptors, neuronal nAChR subunits assemble according to a general 2a3b stoichiometry, with the possibility of more than one a subunit subtype within a pentamer (Conroy et al., 1992). However, a7, a8 and a9 subunits are known to form functional homo-oligomers consisting of a single a subunit subtype (Couturier et al., 1990). For more information on nomenclature of nAChRs and their subunits, the reader is referred to the recent IUPHAR Subcommittee report (Lukas et al., 1999). Analysis of the amino acid sequences of nAChRs reveals signi®cant homology between the neuronal nAChR subtypes and peripheral nAChRs. In general, the nicotinic receptor sequence consists of: (1) a large hydrophillic amino terminal domain, (2) a compact hydrophobic domain split into three segments of 19± 27 amino acids termed M1±M3, (3) a small highly variable hydrophillic domain and (4) a hydrophobic C terminal domain of approximately 20 amino acids termed M4. Fig. 1. It is thought that the large hydrophillic domain containing the amino terminal contains phosphorylation sites and is exposed to the synaptic cleft where it plays a role in ligand binding. The small hydrophobic domain exposed to the cytoplasm contains glycosylation sites, and the four hydrophobic domains (M1±M4) comprise the transmembrane segments of the receptor, some of which line the ion channel (Galzi and Changeux, 1995). 1.2. Ligand binding sites A diverse range of compounds are known to be pharmacologically active at nAChRs, several of which are listed in Table 1 (for a detailed exploration of the properties of these compounds at nAChRs, the reader is referred to the recent review of Gotti et al., 1997). Drugs acting at nAChRs can be divided into three main classes: (1) agonists, (2) antagonists and (3) allo-

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tor-ion channel complex, the nature of which will now be discussed.

Fig. 1. Nicotinic receptor structure. (A) Pentameric structure, subunit stoichiometry and number of binding sites of muscle type, neuronal heteromeric a4b2 and a3b2b4a5 types and homomeric a7, a8 and a9 nicotinic receptors. (B) Nicotinic receptor sequence showing the hydrophilic extracellular domain containing the ACh binding site, four transmembrane segments M1±M4, the intracellular hydrophobic domain and the small C terminal domain. Highlighted in the box is transmembrane segment M2 which is thought to form the lining of the ion channel.

steric ligands Ð both activating and inhibitory. These compounds produce their e€ects by action at one of a number of ligand binding sites that exist on the recep-

1.2.1. ACh binding site From studies on Torpedo electric organ, it was elucidated that the nAChR present in this tissue and that at the neuromuscular junction carried two ACh binding sites (Reynolds and Karlin, 1978; LindstroÈm et al., 1979). Both sites interact in a positively co-operative manner and thus, both sites must be occupied by ACh or a nicotinic agonist to induce channel activation. A number of experiments have been performed with Torpedo, muscle and neuronal nAChRs to determine the location of the ACh binding sites in the receptor structure (see Galzi and Changeux, 1995). The amino acids which contribute to the ACh binding sites in muscle and Torpedo were located at the interfaces between a and d subunits, and involve cysteine residues 192 and 193 (Kao and Karlin, 1986). Similarly, in hetero-oligomeric neuronal nicotinic receptors, two ACh binding sites are thought to exist at the interface between a and b subunits (Alkondon and Albuquerque, 1993). However, in the homo-oligomeric a7, a8 and a9 receptors, ®ve identical ACh binding sites are formed due to the identical nature of the a subunits making up the receptor protein (Wang et al., 1996). Fig. 2 shows the location of the classical ACh and a number of allosteric binding sites on the nAChR complex. 1.2.2. Allosteric binding sites The function of neuronal nAChRs is subject to modulation by a variety of compounds including physostigmine, steroids, ethanol and Ca2+ ion channel blockers that do not bind to the classical ACh sites, but to a number of structurally distinct allosteric sites which are in turn insensitive to ACh. These binding

Table 1 Activators and inhibitors of nicotinic receptors Agonists

Antagonists

Allosteric activators

Allosteric inhibitors

Channel blockers

ACh Nicotine Epibatidine ABT-418 Cytisine GTS-21 (+)-Anatoxin Anabaseine RJR-2403 SIB-1765F

DHbE MLA d-Tubocurarine aBTX nBTX Strychnine a-Conotoxin IMI a-Conotoxin MII

Physostigmine Galanthamine Tacrine Benzoquinonium Codeine 5-HTa

Ethanolb Phencyclidineb MK801b Chlorpromazineb Progesteronec Corticosteronec Dexamethasonec Nimodipined Nifedipined

Mecamylamine Chlorisondamine Hexamethonium

a

Also inhibits in the mM range. Binds to the negative allosteric site. c Binds to the steroid site. d Binds to the dihydropyridine site. b

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79

binding of ACh at the classical site. In M10 cells tacrine produces a concentration dependent increase of a4b2 nAChR sites, an e€ect that was blocked by the nAChR antagonist mecamylamine, without increasing either a4 or b2 mRNA levels (Svensson and Nordberg, 1996). When tacrine (10ÿ7 M) treatment was combined with nicotine (10ÿ6 M) the e€ect was additive suggesting that the upregulation of nAChRs induced by tacrine occurs through activation of the receptor complex via a site distinct to that of the classical ACh binding site.

Fig. 2. Schematic cross section of a nicotinic receptor showing the ion channel, the ACh binding site and multiple allosteric sites distributed throughout the extracellular part of the protein (modi®ed from Lena and Changeux, 1993). Allosteric sites shown include the noncompetitive allosteric activator site (NCA); non-competitive negative allosteric sites (NCB); binding sites for Ca2+ and steroids and phosphorylation sites (P).

sites and the compounds which activate them will now be brie¯y discussed. 1.2.1.1. Non competitive allosteric activator site. As with the classical ACh binding site, this positively acting allosteric site is located on the a subunit of the receptor protein. Compounds that bind to this site are termed channel activators as they enhance channel opening and ion conductance (Pereira et al., 1993) and include the cholinesterase inhibitors physostigmine, tacrine and galanthamine and the muscle relaxant benzoquinonium (Svensson and Nordberg, 1996). There is evidence to suggest that 5-HT also binds to this site increasing ion conductance by increasing the frequency of channel opening (Schrattenholz et al., 1996). In cultured M10 and PC12 cells, these compounds can activate single channel activity, an action which is una€ected by the application of competitive nicotinic antagonists, thus con®rming their action at a site distinct from that of ACh on the receptor-ion channel (Pereira et al., 1994; Storch et al., 1995). However, channel activation was only observed on a small scale, suggesting that the primary function of this class of receptor is to enhance nAChR activity induced by the

1.2.1.2. Non competitive negative allosteric site. In contrast to the non-competitive allosteric activator site, ligand binding to this receptor site inhibits ion channel function. A diverse range of compounds including chlorpromazine, phencyclidine, MK801, local anaesthetics, ethanol and barbiturates can activate this receptor type to produce a negative e€ect on nAChR ion channel function without directly a€ecting ACh binding (Lena and Changeux, 1993). These non-competitive blockers act on two distinct sites that di€er from those of competitive blockers. The ®rst high anity site, which binds ligands in the nanomolar range, is thought to be located within the ion channel and is composed of amino acids of the M2 segment in each of the ®ve subunits making up the receptor protein. Binding of ligands to this site is facilitated by agonist activation of the receptor and produces a rapid reversible channel blockade with ion conductance blocked by simple steric hindrance (Valenzuela et al., 1994). The second site binds ligands with low anity (>100 mM) and is postulated to be located at the interface between the receptor protein and the lipid membrane. Multiple sites exist for each receptor (10±20) with binding of ligands accelerating desensitization of the receptor-ion channel. In cultured PC12 cells with single channel recording ethanol has been observed to reduce the mean open time of channels and accelerate the decay phase of ACh induced currents (Nagata et al., 1996). Additionally, in oocytes expressing a7 receptors, ethanol inhibited ACh induced currents without a€ecting the anity of ACh for the receptor. The e€ect of ethanol treatment on nAChRs expressed in M10 and SH-SY-5Y cells has also been examined (Gorbounova et al., 1998). Ethanol produces a dose related decrease in nAChR number as measured with ‰3 HŠ nicotine in M10 cells and ‰3 HŠ epibatidine in SH-SY-5Y cells. Chronic ethanol (100 mM) treatment of M10 cells also partly attenuated nAChR upregulation produced by treatment of the cells with nicotine. In these same cells, ethanol also signi®cantly decreased a3 and increased a4 and a7 mRNA levels while having no e€ect on b2 mRNA levels.

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1.2.1.3. Steroid binding site. Steroids have the ability to desensitize nicotinic receptors by action at a site located in the extracellular hydrophillic domain that is distinct from the ACh site (Bertrand et al., 1991; Inoue and Kuriyama, 1991). Progesterone, corticosterone, and dexamethasone are potent inhibitors of a3 subtype containing ganglionic receptors expressed in SH-SY5Y cells while having no a€ect on ACh binding (Ke and Lukas, 1996). Corticosterone is known to desensitize nAChRs and to produce tolerance to the e€ects of nicotine, while chronic administration in mice reduces the number of brain ‰125 IŠ a-bungarotoxin binding sites (Grun et al., 1992; Pauly and Collins, 1993; Robinson et al., 1996; Stitzel et al., 1996; Caggiula et al., 1998). Furthermore, high steroid concentrations (mM range) have been observed to displace ‰125 IŠ a-bungarotoxin binding from rat brain membranes and reduce the anity of nicotine for this site (Lena and Changeux, 1993). 1.2.1.4. Dihydropyridine site. L-type Ca2+ channel antagonists such as nimodipine and nifedipine are capable of blocking agonist induced activation of nicotinic receptors (Lopez et al., 1993) and are able to inhibit noradrenaline release from chroman cells in a reversible non-voltage dependent manner (Gandia et al., 1996). The mechanism of action of these compounds is unknown but the binding site is proposed to exist within the ion channel. Interestingly, Ca2+ ions themselves modulate nicotinic receptor-ion channel function. Multiple Ca2+ binding sites exist on both muscle type and neuronal nicotinic receptors (Fairclough et al., 1993), which when activated produce a voltage sensitive decrease in conductance. A further category of Ca2+ binding site, found only on neuronal nicotinic receptors, potentiates agonist activation of ion currents in a voltage insensitive manner (Mulle et al., 1992). 1.2.1.5. Additional allosteric modulation of nicotinic receptors. Phosphorylation by protein kinase A, protein kinase C or by tyrosine kinase of de®ned residues within the cytoplasmic loop results in desensitization of the receptor-ion channel (Huganir and Greengard, 1990). A number of pharmacologically active substances indirectly enhance nAChR desensitization via phosphorylation. This generally occurs through induced changes in intracellular Ca2+ concentration and activation of Ca2+ sensitive protein kinases such as those above. For example, the neuropeptide CGRP (calcitonin gene related peptide) and substance P both enhance nicotinic receptor desensitization through activation of phosphorylating enzymes (Miles et al., 1989; Simmons et al., 1990).

1.3. Transition states of nicotinic receptors Nicotinic receptors can exist in at least one of four interconvertible functionally distinct conformational states at any one time. These states can be interpreted in terms of the ``conformational scheme'' of Katz and Thesle€ (1957) and consist of: (1) a resting state R, (2) an activated state A, where the channel opens on a microsecond to millisecond timescale when activated but which has a low anity for ACh (10±1 mM), and (3) and (4), one of two desensitized closed channel states I and D that are refractory to activation on a millisecond±minute timescale, but exhibit high anity for ACh (10 nM±1 mM) and nicotinic ligands (Galzi and Changeux, 1995). Binding of ligands to the nAChR structure either at the ACh site or any of the allosteric sites can modify the equilibrium between the di€erent conformational states of the receptor at any one time. Additionally, ligands binding to the nAChR can be considered to di€erentially stabilize the conformational state to which they preferentially bind (Lena and Changeux, 1993). 1.4. Nicotinic receptor upregulation Nicotinic receptors go against convention in that prolonged exposure to agonists results in an increase in receptor number, a contradiction of the generally accepted paradigm that over exposure to agonists produces receptor down regulation and overexposure to antagonists, receptor upregulation. Long term exposure to nicotine results in increased number of nAChRs in the brain of several species including humans. Postmortem binding studies have revealed increased ‰3 HŠ nicotine and ‰3 HŠ ACh binding sites in the brains of smokers compared to non-smokers with a dose dependent correlation observed between increased binding sites and the number of cigarettes smoked (Benwell et al., 1988; Breese et al., 1997; NybaÈck et al., 1989). Furthermore, the number of binding sites observed in the brains of ex-smokers was lower than that of non-smokers. It is proposed that the desensitization and upregulation of nAChRs following chronic nicotine exposure is the basis of tolerance to nicotine displayed by smokers as well as being in¯uential in producing withdrawal symptoms on cessation of smoking (Benwell et al., 1988; Balfour and Fagerstrom, 1996; Dani and Heinemann, 1996). In rats, subchronic treatment (0.45 mg/kg twice daily) with nicotine results in an increase in the number of high anity nAChR sites in the cortex, while the proportion of low anity sites is reduced. In addition, there was a signi®cant reduction in agonist anity of both types of nAChR (Romanelli et al., 1988). The rationale behind nAChR upregulation is thought to lie in their rapid desensitization and consequent inacti-

D. Paterson, A. Nordberg / Progress in Neurobiology 61 (2000) 75±111

vation following chronic agonist exposure, putatively resulting in a de®cit in cholinergic function, which is then counteracted by an increase in receptor number (Schwartz and Kellar, 1985). Upregulation, desensitization and the eventual inactivation of nAChRs appear to be dependent upon the length of agonist exposure and upon the nature of the agonist itself (Rowell and Duggan, 1998; Reitstetter et al., 1999). The rate of recovery from desensitization of muscle type nAChRs expressed in TE67/RD cells was recently observed to be signi®cantly faster following a short exposure to nicotine or ACh, with functional recovery from nicotine observed to be consistently more rapid than recovery from ACh (Reitstetter et al., 1999). These observations indicate that more than one state of receptor desensitization exists, and that, agonists vary in their ability to induce these di€erent states. Individual nAChR subtypes also vary in their sensitivity to desensitization and inactivation following agonist exposure. It appears that a4b2 and a7 nAChRs are more sensitive to upregulation and desensitization than other subtypes. Chronic exposure of oocyte expressed human a4b2, a7 and a3 (formed from combinations of a3, b2, b4 and a5 subunits) receptors to submicromolar concentrations of nicotine results in irreversible inactivation of the majority of a4b2 and a7 receptors but substantially fewer a3 subtype (Olale et al., 1997). Similar results were observed by Hsu et al. (1996) with oocyte expressed a4b2 and a3b2 nAChRs. Functional responses in a4b2 nAChRs could be completely abolished following a 48 h incubation with nicotine, while responses in a3b2 nAChRs could only be reduced by 50±60%. Additionally, the half-time for recovery of a3b2 nAChRs was faster than that of a4b2, at 7.5 and 21 h, respectively. These observations suggest that the behavioural e€ects of nicotine (e.g. tolerance, withdrawal) are predominantly mediated through a4b2 and a7 nAChRs. Furthermore, both of these receptor types when expressed in HEK 293 cells, M10 cells and oocytes are upregulated, following chronic nicotine exposure (Gopalakrishnan et al., 1996; Eilers et al., 1997; Molinari et al., 1998). Chronic exposure of human a7 nAChRs expressed in HEK 293 cells to 100 mM nicotine produced a 2.5 fold increase in ‰125 IŠ alphabungarotoxin (aBTX) sites, an e€ect that was observed to be concentration dependent (Molinari et al., 1998). The agonists epibatidine, anabaseine and 1,1-dimethyl4-phenylpiperazinium (DMPP) also increased ‰125 IŠ aBTX sites. A similar range of agonists increased the number of ‰3 HŠ epibatidine binding sites in M10 cells expressing a4b2 receptors, while the antagonists dihydro-b-erythroidine (DHbE), methylcaconitine (MLA) and d-tubocuraine had no e€ect (Eilers et al., 1997). Additionally, evidence suggests that the various nAChR subtypes display di€erent properties in their response to chronic agonist stimulation, with the a-

81

nity of each subtype for a particular agonist in¯uencing the magnitude of receptor upregulation observed (Warpman et al., 1998). Nicotine induced upregulation of nAChR sites in M10 cells expressing a4b2 nAChRs di€ers from that in SH-SY5Y neuroblastoma cells expressing a3, a5, b2 and b4 subunits (a3b2, a3b4, a3b4a5, a3b2a5 and a3b2b4a5 possible receptor combinations) with 100 times greater concentration of nicotine required to induce a similar magnitude of nAChR upregulation in SH-SY5Y as in M10 cells (Warpman et al., 1998). In this study, the anity of nicotine for nAChRs in SH-SY5Y (a3 nAChRs) cells was 14 times lower than in M10 cells (a4b2 nAChRs), thus, the lower anity of nicotine for the a3 subunit results in the higher concentration of nicotine necessary to produce an upregulation of nAChRs in these cells. The mechanism by which nAChR number is increased following nicotine exposure is thought to involve reduced turnover of cell surface receptors; it is proposed that conformational changes in the receptor structure occur, preventing it from being removed from the cell surface (Peng et al., 1994). This view is supported by observations from a number of studies. Chronic nicotine administration in mice increases the number of ‰3 HŠ nicotine sites observed in brain but does not increase a2, a3, a4, b5 or b2 mRNA levels indicating that increased receptor number results from post transciptional mechanisms (Marks et al., 1992). Exposure of receptors, both to nicotine and to the non-competitive channel blocker mecamylamine, have been observed to increase nAChR number, but not exposure to the non-competitive antagonist chlorisondamine (el-Bizri and Clarke, 1994; Pauly et al., 1996) suggesting that, receptor blockade is not sucient to induce conformational changes necessary for receptor upregulation. The desensitization of nAChRs is thought to involve phosphorylation of the receptor mediated by protein kinase A and protein kinase C, both forsklin and phorbol esters have been observed to increase nAChR binding sites and to synergistically enhance nicotine induced receptor upregulation, with evidence suggesting that a4 nAChR subunits may be speci®c targets of PKA phosphorylation (Gopalakrishnan et al., 1997; Hsu et al., 1997). Following incubation of oocyte expressed a4b2 and a3b2 nAChRs with nicotine, human a4b2 nAChRs expressed in HEK 293 cells are upregulated by chronic nicotine exposure, as measured by an increase in ‰3 HŠ epibatidine binding and functionally deactivated, measured as an abolition of Ca2+ in¯ux (Eilers et al., 1997). Similar deactivation of receptors could be produced by decreasing PKC activity by application of the PKC inhibitor NPC-15437 suggesting that, phosphorylation of the receptor protein by PKC activity is necessary for ion-channel receptor function. Following deactivation, functional recovery of receptors occurs

82

D. Paterson, A. Nordberg / Progress in Neurobiology 61 (2000) 75±111

slowly (4±6 h) but may be accelerated by inhibition of PKA activity. From these observations it is hypothesised that receptor deactivation involves dephosphorylation of the receptor protein at PKC sites, putatively by PKA action and that reactivation involves re-phosphorylation of these same sites. It is likely that the phosphorylation state of the receptor will be in¯uential on turnover and removal from the cell surface which will thus in¯uence receptor number. Evidence to support PKA involvement in deactivation and upregulation of receptors comes from studies involving mutant PC12 cells expressing nAChRs which are de®cient in cAMP dependent PKA I and II (Madhok et al., 1995). Following nicotine treatment, a dose dependent increase in ‰3 HŠ nicotine sites is observed in wild type PC12 cells but not in mutant PC12 cells; furthermore, a3 mRNA levels were observed to decrease and b2 mRNA levels to increase in wild type PC 12 cells but no change in subunit mRNA levels was observed in mutant PC12 cells. Therefore, upregulation of nAChRs would appear to involve a PKA mediated mechanism and that, b2 containing receptors constitute the major subtype upregulated, following chronic agonist exposure.

2. Neuronal nicotinic receptor subtypes in the human brain Classically, much of the knowledge pertaining to neuronal nAChRs in human brain has been obtained from in vitro binding studies on postmortem tissue and on neuroblastoma cell lines expressing nAChRs, with evidence suggesting that nAChRs can be broadly divided into two main subtypes. More recently, the cloning of nAChR subunits and the expression of recombinant nAChRs in oocytes and additional techniques such as in situ hybridisation and immunohistochemistry has furthered understanding of the structure, subunit composition and pharmacological pro®le of individual nAChRs. Studies have revealed that multiple subtypes of functional neuronal nAChRs can be formed from various combinations of nAChR subunits, with evidence to suggest that as many as four functionally distinct subtypes of nAChR exist (Zoli et al., 1998). However, it appears that the majority of high anity nAChRs in the brain comprise the a4b2 subtype (Whiting et al., 1987; Flores et al., 1992; Zoli et al., 1998). Heteromeric a3 and homomeric a7 subtypes constitute the other major neuronal nAChRs present, with the remainder made up of various combinations of a2, a3, a5, a6 and b2 and b4 subunits coexpressed to form heteromeric receptors.

2.1. Ligand binding studies on human brain tissue In human brain, neuronal nAChRs can be divided by radioligand binding into at least two classes: (1) Alpha bungarotoxin sites (BTXRs) that bind aBTX with high anity, and the nicotinic agonists nicotine, ACh and cytisine with low anity and (2) high anity nicotine sites (AChRs) that bind nicotine, ACh and cytisine with high anity and aBTX with low anity (Clarke et al., 1985; Sugaya et al., 1990). Original homogenate and autoradiographic ligand binding studies on postmortem human brain tissue (Shimohama et al., 1985; Flynn and Mash, 1986; Nordberg and Winblad, 1986; Nordberg et al., 1987; Adem et al., 1988; Perry et al., 1992) identi®ed the presence of high and low anity ‰3 HŠ nicotine and ‰3 HŠ ACh sites in the cortex, hippocampus and thalamus of human brain, which are hypothesised to correspond to AChRs and BTXRs, respectively. Additional investigation revealed the presence of super high, high and low anity agonist binding sites in the brain, indicating that more than two receptor subtypes exist (Nordberg et al., 1988, 1989a; Nordberg and Winblad, 1986). Homogenate binding studies utilising ‰3 HŠ cytisine have also been performed on human brain tissue, where a single high anity site (Kd = 0.147±0.245 nM) was identi®ed in the hippocampus, cingulate gyrus and cortex with highest levels of binding observed in the thalamus, Bmax = 48 fmol/mg protein (Hall et al., 1993). Binding studies with the high anity AChR agonist ‰3 HŠ epibatidine have also been performed on human postmortem brain tissue identifying the presence of two high anity sites in cortex with Kd values of 0.3 and 28.4 pM, respectively, observations consistent with ‰3 HŠ ACh studies (Houghtling et al., 1995). Recent autoradiographic studies comparing the distribution of ‰3 HŠ nicotine and ‰3 HŠ epibatidine binding sites in human brain have con®rmed this observation and have identi®ed two high anity binding sites in the temporal cortex, thalamus and cerebellum (Marutle et al., 1998; Sihver et al., 1998a). The binding of each of the nicotinic agonists ‰3 HŠ nicotine, ‰3 HŠ cytisine and ‰3 HŠ epibatidine to AChRs is una€ected by the presence of aBTX or MLA, indicating that, they bind at the classical ACh recognition site on a receptor subtype, distinct to that at which ‰125 IŠ aBTX binds. In comparison to the large number of studies mapping high anity AChR sites, relatively few ligand binding studies with ‰125 IŠ aBTX have been performed on human postmortem brain. Studies reveal, however, a single high anity site with a distribution distinct to that of ‰3 HŠ nicotine (Davies and Feisullin, 1981; Lang and Henke, 1983; Sugaya et al., 1990; Rubboli et al., 1994b; Spurden et al., 1997; Sabbagh et al., 1998). ‰125 IŠ aBTX binding is highest in the hippocampal formation, particularly the dentate gyrus and CA1±CA3

D. Paterson, A. Nordberg / Progress in Neurobiology 61 (2000) 75±111

regions, and in the thalamus with relatively little binding observed in the cortex. These studies describe the presence of three probable nAChR sites in human postmortem brain distinguishable by their relative anities for nicotinic agonist ligands and aBTX. 2.2. Binding studies in human neuroblastoma cell lines A number of ligand binding experiments have been performed on human neuroblastoma cell lines such as IMR32, SH-SY5Y and SK-N-BE. These cell lines express various cell surface nAChR subtypes and are therefore extremely useful tools for examining native human nAChRs. Binding studies with ‰125 IŠ aBTX and ‰3 HŠ ACh on IMR 32 and SH-SY5Y neuroblastoma cell lines (Lang and Henke, 1983; Gotti et al., 1986; Lukas, 1993; Galzi and Changeux, 1995; Gotti et al., 1995) have identi®ed the presence of a single site for ‰125 IŠ aBTX binding (Kd, 1±10 nM) and two sites for ‰3 HŠ ACh. The low anity (Kd, 100 nM) ACh site is present in high abundance and the second high anity (Kd, 1±2 nM) site is present in low abundance. Binding of ‰3 HŠ ACh to both of these sites is una€ected by the presence of aBTX indicating that the ‰125 IŠ aBTX and ‰3 HŠ ACh binding sites are distinct from one another. Binding of ‰3 HŠ nicotine and ‰3 HŠ epibatidine to nAChRs expressed by SH-SY5Y cells has also been investigated (Warpman et al., 1998). The level of nonspeci®c binding of ‰3 HŠ nicotine was very high (>90%) in this cell line, while in contrast ‰3 HŠ epibatidine bound with very high speci®city (non-speci®c binding <10%). ‰3 HŠ epibatidine identi®ed a single binding site in SH-SY5Y cells (Kd = 0.12 20.02 nM, Bmax = 322.5 2 4.1 fmol/mg). The low level of speci®c binding observed with ‰3 HŠ nicotine is consistent with the fact that SH-SY5Y cells predominantly express a3 but no a4b2 nAChRs. Additionally, the single site identi®ed with ‰3 HŠ epibatidine is consistent with its high anity for a3 nAChRs, while the lack of a second binding site is explained by the absence of a4b2 nAChRs in this cell line, for which epibatidine also displays high anity. The interaction of the nAChR agonist (R,S)-3pyridyl-1-methyl-2-3(3-pyridyl)-azetidine (MPA) with nAChRs expressed in SH-SY5Y cells has also been investigated (Zhang et al., 1998). In this study, MPA displaced ‰3 HŠ epibatidine from a single binding site with a Ki = 35.9 2 4.1 nM, a value similar to that observed for displacement of ‰3 HŠ epibatidine from rat cortical membranes, Ki = 23.78 20.36 nM. In general, these observations are consistent with results from human postmortem binding studies and provide evidence to support the presence of at least two (probably three) classes of nAChR in human brain, distinguishable by their relative anities for ‰3 HŠ nicotine, ‰3 HŠ epibatidine and ‰125 IŠ aBTX.

83

2.3. Nicotinic receptor subunit expression in oocytes and transfected cell lines The high degree of sequence homology existing between rat and human nAChR subunits (82±95%) has allowed the use of rat cDNAs encoding di€erent subunits to be used as molecular probes for the identi®cation and cloning of human nAChR subunits. Nine receptor subunits have thus far been identi®ed and cloned in human brain and include a2, a3, a4, a5, a6, a7, b2, b3, and b4 subunits. The isolation and cloning of the individual human nAChR subunits combined with the use of appropriate expression systems such as Xenopus oocytes has allowed investigation into the biochemical structure, subunit composition and pharmacological pro®le of functional human nAChRs. A large number of such studies have been performed involving most of the human nAChR subunits and have resulted in a number of common conclusions, including the identi®cation of two main structural subtypes of nAChR. 2.3.1. Heteromeric nicotinic receptors Following individual injection into an appropriate expression system, a2, a3, or a4 and b2 or b4 subunits are not capable of forming functional receptor-ion channel complexes. However, when any of the a2±a4 subunits are expressed pairwise with b2 or b4 subunits, functional receptors are produced (i.e. a2b2, a2b4, a3b2, a3b4, a4b2, a4b4) that have distinct electrophysiological and pharmacological pro®les (Sargent, 1993; McGehee and Role, 1995). These observations indicate that this group of subunits can only form heteromeric receptors. Both a and b subunits contribute to the pharmacological and functional pro®le of each receptor. For example, when expressed with b2 subunits, each of a2, a3 and a4 subunits form functional nAChRs with di€erent single channel conductance, average channel open times and antagonist sensitivity. More speci®cally, b subunits appear to have a profound in¯uence on the dissociation rate of agonists and antagonists from the receptor as well as the rate at which agonist bound receptors open (Papke and Heinemann, 1991; Papke et al., 1993). Functional nAChRs may also be formed from combinations of three or more individual subunit types (Role and Berg, 1996; Wang et al., 1996). For example, the a5 subunit when expressed alone or in combination with any of the b subunits is unable to form a functional ion channel, however, when expressed with a4 and b2 subunits, it gives rise to a functional nAChR subtype a4b2a5. Furthermore, a receptor composed of four di€erent subunits is formed when a5 is co-expressed with a4, b2 and b4 subunits. Until recently, the a6 subunit was thought to be an ``orphan'' subunit (as is the b3 subunit) in that it had been unable to form a functional ion

5.4620.60a,g

b

Receptors expressed in oocytes. Receptors stably expressed in HEK 293 cells. c Peng et al., 1994. d Gopalakrishnan et al., 1995. e Gerzanich et al., 1995. f Gopalakrishnan et al., 1996. g Wang et al., 1996. h Buisson et al., 1996. i Chavez-Noriega et al., 1997. j Gerzanich et al., 1998. k Stauderman et al., 1998.

a

a7

a4b4

a3b4a5 a4b2

a3b2a5 a3b4

0.18720.029b,k

0.2520.02a,g 4.9020.60a,g 0.23020.012b,k 2.8020.30a,g

0.1220.04a,g

a3b2

b,k

[3 HŠ epibatidine Kd (nM) 0.04220.01

[3 HŠ nicotine Kd (nM)

a2b2 a2b4

Receptor composition

0.21 20.04b,f

[3 HŠ cytisine Kd (nM)

Table 2 Anity of radioligands and agonist potency at recombinant human nicotinic receptorse,i

0.81a,c 0.7120.11b,d

[125 IŠ aBTX Kd (nM)

DMPP > Nicotine > Cytisine > ACha,i Nicotine > DMPP > Cytisine > ACha,i Epibatidine > Cytisine > Suberycholine=Nicotine=DMPPb,k DMPP > Cytisine > ACha,i Epibatidine > Nicotine > ACha,i DMPP > Cytisine > Nicotine > ACha,i,j DMPP > Cytisine > Nicotine > ACha,i Epibatidine > DMPP=Cytisine=Nicotine=Suberycholineb,k DMPP > Cytisine > Nicotine > ACha,i Cytisine > Nicotine > DMPP > ACha,i Nicotine > ACh > Cytisineb,h Cytisine > Nicotine > DMPP > ACha,i Epibatidine > Cytisine=Suberycholine > nicotine > DMPPb,k

Rank order of agonist potency

84 D. Paterson, A. Nordberg / Progress in Neurobiology 61 (2000) 75±111

D. Paterson, A. Nordberg / Progress in Neurobiology 61 (2000) 75±111

channel individually or in combination with any of the other subunits. However, recent studies have demonstrated the assembly of a functional a3b4a6 receptorion channel (composed of chick embryo cDNAs expressed in human BOSC 23 cells) with an anity for ACh threefold lower than that of a3b4 (Fucile et al., 1998). It is likely therefore that a similar functional receptor may be formed from the comparable human nAChR subunits. 2.3.2. Homomeric nicotinic receptors In contrast to a2±a4 and b2±b4 subunits, a7 and a8 subunits when expressed in oocytes with any of the other a or b subunits or multiple combinations thereof, fail to form functional ion channels. However, when expressed individually, they form functional homoligomeric receptors that are consistent in size with the pentameric structure displayed by their heteroligomeric counterparts but have distinct pharmacological and physiological pro®les (Anand et al., 1993). The multiple combinations of nAChR subunit possible, therefore, give rise to a multitude of nAChR subtypes. Studies in which recombinant human nAChRs were expressed in oocytes or human cell lines have identi®ed numerous combinations of subunits that form functional nAChRs. Although no de®nitive conclusion can be drawn from these studies, it is likely that the subunit compositions and structural conformations of these recombinant nAChRs will re¯ect the structure of nAChRs expressed in human brain. Human nAChRs comprising of a3b2, a4b2, a3b4 and a7 subunits have been stably transfected into oocytes (Peng et al., 1994; Gerzanich et al., 1995; Wang et al., 1996) and a4b2, a4b4 and a7 subtypes have also been expressed in HEK293 cells (Gopalakrishnan et al., 1995, 1996; Stauderman et al., 1998), where they have been characterised with ligand binding experiments. Results indicate that the heteromeric nAChRs containing combinations of a and b subunits show greatest anity for nicotinic agonists such as epibatidine, nicotine and cytisine (e.g. a3b3 Kd = 0.12 20.0 nM, a3b2a5 Kd = 0.25 20.02 nM, for ‰3 HŠ epibatidine and a4b2 Kd = 0.21 2 0.04 nM for ‰3 HŠ cytisine), whereas, homomeric a7 receptors displayed highest anity for ‰125 IŠ aBTX (Kd = 0.71 2 0.11 nM) Table 2. It is therefore believed that the high anity nicotine binding sites are composed of the heteromeric ab receptors, while the homomeric a7 (and a8) receptors represent the high anity aBTX sites in human brain. Support for these observations comes from combined immunoprecipitation and ligand binding studies on IMR 32 cells using subunit speci®c antibodies directed against human a3, a5, a7, b2 and b4 subunits and ‰125 IŠ aBTX (Gotti et al., 1995). The antibodies directed against the individual subunits were used to immunoprecipitate ‰125 IŠ aBTX labelled nAChRs expressed by IMR32

85

cells. Of the antibodies, only the anti-a7 was capable of precipitating almost all of the radiolabelled receptors. Anti-a3, a4, a5, b2 and b4 antibodies were incapable of precipitating any of the labelled receptors. Thus, it appears that aBTX sites contain the a7 subunit but not any of the a3, a4, a5, b2 or b4 subunits. From this observation it is likely that a7 is the sole constituent of aBTX receptor but it is possible that as yet unidenti®ed subunits are also involved. Few similar such immunoprecipitation studies have been performed with the high anity nicotinic receptor sites expressed in neuroblastoma cell lines, mainly due to the lack of subunit speci®c antibodies. However, native nAChRs expressing b2 subunits have recently been characterised using a monoclonal antibody (mAb290), which is speci®c for this receptor subunit (Wang et al., 1996). Following immunoprecipitation and western blotting, b2 containing nAChRs expressed by SH-SY5Y cells were also found to contain a3 and a5 subunits. In the same study ‰3 HŠ epibatidine labelled a3 containing receptors in the SH-SY5Y cells were immunoprecipitated with mAb290 revealing that at least 56% of a3 containing ‰3 HŠ epibatidine sites also contain b2 subunits. The sedimentation properties of these a3 containing nAChRs in SH-SY5Y cells were additionally analysed and compared to those of recombinant a3b2 and a3b2a5 subtypes expressed in oocytes. The receptors were found to have the same sedimentation coecient of 11S, which is consistent with that of a pentameric nAChR. There are as yet no b4 subunit speci®c antibodies available and thus, nAChRs containing this subtype have not been characterised. However, the above observations generally support the view that in human brain the high anity aBTX sites are composed of homomeric a7 (and probably also homomeric a8) receptors and that the high anity nicotine binding sites consist of heteromeric receptors composed of multiple combinations of a and b subunits but not of a7 and a8. 2.4. Electrophysiological studies The structural diversity displayed by the numerous nAChR subtypes is re¯ected in their functional pro®les, with individual receptors having distinct functional characteristics which are determined by their subunit composition. The pharmacologic and functional properties of a range of recombinant human nAChRs (e.g a2b2, a2b4, a3b2, a3b4, a4b2, a4b4, a7) expressed in oocytes and HEK 293 cells have been characterised (Luetje and Patrick, 1991; Galzi and Changeux, 1995; Gerzanich et al., 1995, 1998; Zoli et al., 1995; Gopalakrishnan et al., 1996; Chavez-Noriega et al., 1997; Olale et al., 1997; Stauderman et al., 1998; Zoli et al., 1998). Electrophysiological techniques such as patch clamp were used to measure currents pro-

86

D. Paterson, A. Nordberg / Progress in Neurobiology 61 (2000) 75±111

duced by a range of nicotinic agonists including ACh, nicotine, cytisine, DMPP and epibatidine with each subtype displaying di€erent kinetics of activation and inactivation (Table 2). ACh was found to be highly ecacious in producing currents at all subtypes except for a3b2 where DMPP was markedly more ecacious. Cytisine was the least e€ective agonist at b2 containing receptors but was ecacious in producing currents in a2b4, a3b4 and a4b4, as well as a7 receptors (ChavezNoriega et al., 1997). Human a4b2 receptors stably expressed in HEK293 cells diplayed agonist induced cation currents consistent with native neuronal a4b2 nAChRs, while also showing high anity for ‰3 HŠ cytisine (Kd = 0.2 nM) with a good correlation observed between anity in transfected cells and native human nAChRs (Gopalakrishnan et al., 1995). Epibatidine was found to be the most potent of a range of agonists in eliciting currents in a2b4, a3b4 and a4b4 receptors expressed in HEK 293 cells (Stauderman et al., 1998). a7 receptor activation in the same cell line following nicotine or ACh application evoked whole cell currents with fast activation and inactivation kinetics that were sensitive to blockade with aBTX (Gopalakrishnan et al., 1995). Speci®c binding of ‰125 IŠ aBTX was also observed in these cells with a Kd = 0.7 nM. In oocytes expressing a3b2 and a3b4 receptors, the addition of an a5 subunit (i.e. producing a3b2a5 and a3b4a5 receptors) had variable e€ects on receptor function. In a3b2 receptors, a5 produced a 50 fold increase in the sensitivity of the receptor to ACh, but had little e€ect on a3b4 nAChR function (Gerzanich et al., 1998). Ion channel function in all receptor subtypes was observed to be sensitive to nicotinic antagonists such as DHbE, mecamylamine and d-tubocurarine. Human a4b2 and a4b4 were more sensitive to blockade by DHbE than d-tubocurarine, whereas, a7 and a3b4 were more sensitive to d-tubocurarine than DHbE (Chavez-Noriega et al., 1997). The non-competitive antagonist mecamylamine (3 mM) produced greater than 80% block of a2b2 and a4b4 nAChRs compared to approximately 50% block of a4b2, a2b2 and a7 receptors. The di€erent receptor subtypes also display individual rates of inactivation and desensitisation following agonist exposure. Following exposure of a7, a4b2, a3b2a5 and a3b4a5 receptors expressed in oocytes to submicromolar concentrations of nicotine, the majority of a4b2 and a7 receptors were irreversibly deactivated, while a3b2a5 and a3b4a5 subtypes were much less a€ected (Olale et al., 1997). 2.5. Evidence for four functional subtypes of nicotinic receptor The studies outlined in the previous sections provide evidence for numerous nAChR subtypes with distinct pharmacological and functional pro®les. However,

despite the individual functional pro®les of nAChRs characterized in expression systems, evidence exists to suggest that nAChRs expressed in vivo may be functionally characterized into three or four main subtypes. From studies on cultured rat hippocampal neurons Alkondon and Albuquerque (1993) provided pharmacological and functional evidence for at least three distinct nAChR subtypes. Using whole cell patch clamp techniques and the decay kinetics of the currents elicited by the application of 3 mM ACh, they described four current types termed IA, IB, II and III in rat hippocampal neurons. The order of potency of agonists in activating the currents varied between current types, as did the sensitivity of each current to blockade by a number of nicotinic antagonists. Type IA currents, the most common observed (present in 83% of neurons), were rapidly decaying in nature and could be blocked by aBTX (10 nM), kBTX (10 nM) and MLA (1 nM). This suggests that this current type may result from a7 nAChR activation, as only homomeric a7 receptors are sensitive to aBTX (Couturier et al., 1990). Type II currents (present in 5% of neurons) were blocked by DHbE (10 nM) and by high concentrations of MLA and kBTX (100 nM each), but not by aBTX. Type III currents (present in 2% of neurons), slowly decaying in nature were blocked by mecamylamine (1 mM) but not by aBTX, kBTX or MLA in concentrations of up to 100 nM. Approximately, 10% of the neurons investigated displayed mixed responses to agonist activation (Type IB). This type of current was partially blocked by MLA (1 nM) or DHbE (10 nM) alone and completely blocked by a combination of the two antagonists. A number of the agonists used were useful in discriminating between the currents elicited. ACh, carbachol, nicotine and suberylcholine were particularly e€ective in producing Type II currents, while cytisine appeared to be speci®c for Type III. This functional classi®cation of three nAChR subtypes has recently been expanded to four receptor subtypes by Zoli et al. (1998) from autoradiographic and patch clamp studies of nAChRs in brain slices of b2 knockout mice . . . Table 3. Autoradiographic studies mapping the distribution of ‰3 HŠ ACh, ‰3 HŠ nicotine, ‰3 HŠ cytisine, ‰3 HŠ epibatidine and ‰125 IŠ aBTX in b2 knockout mice and wild type mice were performed. Distribution of all ligands in brain sections of wild type mice was consistent with distribution of these ligands in previous studies in the mouse and with distribution in rat brain. In brain sections of b2 knockout mice, however, high anity ‰3 HŠ nicotine binding sites could no longer be detected, with the binding of ‰3 HŠ ACh, ‰3 HŠ cytisine and ‰3 HŠ epibatidine signi®cantly reduced. In contrast, the pattern of ‰125 IŠ aBTX binding was not substantially di€erent from that of wild type mice. Electrophysiological analysis of nAChRs in wild type and b2 knockout brain sections was then correlated to

a7 b2a4(a5?), b2(a2?), b2(a3?), b2(a6b3?) b4a3(a5?) (b4a4?) (b4a2?) Zoli et al. (1998) aBTX and MLA sensitive MLA insensitive Nic > Cyt, DHbE = MCA MLA insensitive, Cyt = Nic, DHbE < MCA MLA insensitive, Cyt = Nic, DHbE < MCA V. fast desensitisation Mixed Slowly decaying when Nic. > 100 mM Fast decay when Nic. > 100 mM

87

NA NA NA NA

regional binding pattern of the radioligands. The currents elicited by application of a range of nicotinic agonists allowed assignment of responses to the activation of four subtypes of nAChR. Type 1 receptors are aBTX sensitive with low anity for nicotinic agonists and their distribution as mapped by ‰125 IŠ aBTX is not signi®cantly di€erent in wild type and b2 knockout mice. Their sensitivity to aBTX and the fast activation and decay kinetics displayed by these receptors indicate that they are a7 homomeric receptors and that, they correspond to the receptors producing Type IA currents as described by Alkondon and Albuquerque (1993). This is supported by disappearance of receptors with a similar pharmacological and functional pro®le in a7 knockout mice (Orr-Urtreger et al., 1997). Type 2 receptors contain the b2 subunit and represent the vast majority of nAChRs in rodent brain as evinced by their disappearance in b2 knockout mice. All of the nicotinic agonists used, bound to this receptor type with high anity (nanomolar to subnanomolar range). The rank order of agonist potency for these receptors is consistent with that of recombinant a4b2 receptors expressed in oocytes (Luetje and Patrick, 1991), and it is proposed that the composition of the major isoform forming Type 2 receptors is a4b2. It is likely however, that other subunits (e.g. a2, a3, a5 and b4) also co-assemble with b2 to form a range of functional receptors that contribute to Type 2 high anity nAChR sites. Type 3 receptors do not contain b2 and bind only ‰3 HŠ epibatidine with high anity. Electrophysiological studies indicate that the rank order of agonist potency for this receptor is consistent with a3b4 containing receptors (Luetje and Patrick, 1991). The distribution of these receptors is also consistent with the distribution of a3 and b4 subunit mRNA, suggesting an a3b4 composition for this subtype. Type 4 receptors bind ‰3 HŠ epibatidine and ‰3 HŠ cytisine with high anity and display agonist potency consistent with b4 containing receptors (Luetje and Patrick, 1991). In contrast to Type 2 receptors, they display a signi®cantly faster desensitization rate suggesting a subunit composition of a2 and or a4 with b4. Although the data from these studies is derived from rodent brain, it is feasible that a similar hierarchy of functional nAChRs will exist within human brain, although further investigation will (of course) be necessary. Therefore, while multiple subtypes of neuronal nAChR exist in vivo, it appears that they be functionally categorized into four groups. 3. Distribution of nicotinic receptors in the human brain

1 2 3 4

aBTX EPI > Nic = Cyt = MCC = ACh EPI EPI > Cyt > MCC = ACh

a7 a7, a4b2 a4b2 a3b4 Alkondon and Albuquerque (1993) MLA, aBTX, kBTX sensitive NA MLA+DHbE sensitive NA DHbE sensitive NA MLA sensitive NA Rapidly decaying Fast and slow decay Slowly decaying Slowly decaying 83% 10% 5% 2% IA IB II III

Receptor type

Table 3 Four functional subtypes of nicotinic receptor

Pharmacology

High anity binding

Subunit composition Major current attributes Relative frequency

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In comparison to muscarinic receptors, neuronal nAChRs are expressed in relatively low density in the human brain. In addition, their pattern of distribution

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Table 4 Distribution of nicotinic receptors in human brain Region [3 HŠ nicotinea High density Moderate density Low density [3 HŠ epibatidineb High density Moderate density Low density [125 IŠ aBTXc High density Moderate density Low density

Thalamus, caudate nucleus, substantia nigra Frontal cortex, parietal cortex Occipital cortex, temporal cortex, hippocampus, cereÂbellum Thalamus Caudate-putamen, parietal cortex, cerebellum Frontal cortex, occipital cortex, temporal cortex, hippocampus Nucleus reticularis, lateral and medial geniculate bodies, pontine nucleus, horizontal limb of the diagonal band of Broca, NBM, inferior olivary nucleus Hippocampus, hypothalamus, pons, medulla Cortex, cerebellum

a

Data from Adem et al. (1987), Nordberg et al. (1988) and Perry et al. (1992). Data from Marutle et al. (1998). c Data from Rubboli et al. (1994a), Breese et al., 1997 and Spurden et al. (1997).

b

is relatively homogenous and is not restricted to the well de®ned brain cholinergic pathways. The neuroanatomical distribution of various nAChR subtypes and subunit mRNA has been fairly extensively characterised in rodent and chick brain but has been less well characterised in human brain. The majority of studies performed to date, have mapped the distribution of high anity AChR sites utilising ‰3 HŠ nicotine, ‰3 HŠ epibatidine and ‰3 HŠ cytisine with some additional studies mapping ‰125 IŠ aBTX binding sites. A few studies mapping the distribution of nAChR subunit mRNA in human brain have also been performed, predominantly using isotopic in situ hybridisation. However, despite the incomplete nature of the studies performed to date, it is possible to draw a map of nAChR distribution in

the human brain Table 4 and 5). Nicotinic receptors are present in a variety of brain structures, in particular the thalamus, cortex and the striatum. This distribution of receptors is consistent with that described in the human brain with PET, a non-invasive imaging technique (see Section 4.1). 3.1. Ligand binding studies High anity nicotinic AChR sites as mapped by the binding of ‰3 HŠ nicotine in both homogenate and autoradiographic studies displays a distinct pattern in the human brain. Highest levels of binding are observed in the thalamus and nucleus basalis of Meynert (NBM) with relatively lower levels in the hippocampus, cortex

Table 5 Distribution of nicotinic receptor subunit mRNA in the human braina Brain region Cortex Prefrontal Motor Entorhinal Cingular Temporal Thalamus Dorsomedial Lateroposterior Reticular Ventro-posterolateral Geniculate bodies Hippocampus Dentate gyrus Caudate putamen Cerebellum

b2 + + + + + +

b3

b4

+

+

+

+ +++ ++

+ +(+) +(+) +(+) +

+ +

+ +

a3

a4

++ ++ ++ + +

+ + + + +(+)

+++

+

+

a7 ++ +++ +

++

+(+)

+++

++

+ + +

a5

+(+)

+ +

++ ++ ++

a Data from Breese et al. (1997), Court and Clementi (1995), HellstroÈm-Lindahl et al. (1998, 1999), Rubboli et al. (1994a), SchroÈder et al. (1995) and Wevers et al. (1994).

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89

Fig. 3. Autoradiographic distribution of: (a) ‰3 HŠ (-) nicotine; (b) ‰3 HŠ cytisine and (c) ‰3 HŠ epibatidine binding in the cerebral cortex of a human hemisphere. Pseudocolour images are not standardized to each other, as the series of autoradiograms for each ligand was created individually. The density of binding sites increase in the colour sequence black, blue, yellow, red. Abbreviations: cs, central sulcus (BA3b; BA4); ifrs, inferior frontal sulcus (BA6); mfrs, medial frontal sulcus (BA8); sfrs, superior frontal sulcus (BA9); oc, occipital cortex (BA18); sts, superior temporal sulcus (BA39); pc, parietal cortex (BA40). Reprinted from Neuroscience, Sihver et al., 1998a Copyright (1998), with permission from Elsevier Science.

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and caudate putamen (Shimohama et al., 1985; Adem et al., 1988, 1989; Perry et al., 1992; Court et al., 1995). High levels of binding are observed in the majority of thalamic nuclei with greatest density observed in the lateral dorsal nuclei, medial and lateral geniculate nuclei and anterior thalamic nuclei (Spurden et al., 1997; Adem et al., 1988). Cortical AChRs are concentrated in the entorhinal cortex and the subicular complex (Court and Clementi, 1995). However, regional di€erences in ‰3 HŠ nicotine binding are observed throughout the cortex, with distinct laminar distribution of ligand in individual regions. In the somatosensory cortex, highest levels are observed in the uppermost and innermost layers with signi®cantly less binding observed in layer IV. In contrast, in the primary motor and temporal cortices, ligand binding was observed to be considerably denser in the outer layers than in the inner layers with a distinct high density band observed in layer III of the temporal cortex (Perry et al., 1992). Greatest levels of cortical ‰3 HŠ nicotine binding are observed in the subicular complex, in particular, the presubiculum and entorhinal cortex (Perry et al., 1992). In the hippocampus ‰3 H], nicotine binding is generally low in the CA1-4 and dentate gyrus, but is greater in the lacunosum moleculare in CA2-3 (Perry et al., 1992). This distribution of high anity nicotinic AChR sites in human brain has recently been con®rmed by autoradiographic studies comparing ‰3 HŠ nicotine and ‰3 HŠ epibatidine (Marutle et al., 1998; Sihver et al., 1998a). Two high anity binding sites in the temporal cortex, thalamus and cerebellum of human brain have been identi®ed with ‰3 HŠ epibatidine (Houghtling et al., 1995; Marutle et al., 1998). These sites are likely to represent binding to a4b2 and a3 nAChRs. However, di€erences in regional binding between ‰3 HŠ nicotine and ‰3 HŠ epibatidine were observed with a proportionally higher level of ‰3 HŠ epibatidine binding in the thalamus and cerebellum, possibly re¯ecting selectivity for di€erent nAChR subtypes between nicotine and epibatidine Ð i.e. greater selectivity of ‰3 HŠ epibatidine for a3 nAChRs. Thalamic nAChRs have also been mapped in the human brain with ‰3 HŠ nicotine and ‰3 HŠ ACh (Adem et al., 1988) with high number of binding sites observed in the antero-ventral and dorsomedial thalamic nuclei and low numbers of binding sites observed in the postero-lateral and postero-lateral-ventral nuclei. The pattern of nAChR sites in the cortex of human brain with regard to their laminar distribution has also recently been determined with the use of ‰3 HŠ nicotine, ‰3 HŠ cytisine and ‰3 HŠ epibatidine and autoradiographic analysis of whole human brain hemispheres (Fig. 3 . . . (Sihver et al., 1998a). The laminar distribution of all three ligands was generally similar to the highest levels of binding observed in layers I, III and V, and particularly high levels observed in layer III of the primary

sensory motor cortex and inferior frontal sulcus. However, examination of the regional distribution of the three ligands suggests the presence of three di€erent binding sites within the human cortex. The ®rst site is thought to be a common site for ‰3 HŠ nicotine, ‰3 HŠ cytisine and ‰3 HŠ epibatidine, and is likely to represent binding to a4 subunits in the brain. The morphological distribution of ‰3 HŠ nicotine and ‰3 HŠ epibatidine indicate that they bind to an additional site speci®cally noticeable in the primary motor cortex, layer IIIb of the occipital corex and layer V of the superior temporal sulcus, as their binding in these regions is signi®cantly greater than that of ‰3 HŠ cytisine. The high levels of ‰3 HŠ nicotine binding observed in layers I and VI of the primary motor cortex, deeper layer V of the primary sensory cortex, layer III of the superior temporal sulcus and layer VI of the parietal cortex suggest the presence of a third site (Sihver et al., 1998a). Although more detailed, these observations are generally consistent with the regional laminar distribution of ‰3 HŠ nicotine binding sites as described by Perry et al. (1992). Although relatively few ‰125 IŠ aBTX binding studies have been performed on human brain, comparison of ‰125 IŠ aBTX and ‰3 HŠ nicotine autoradiography reveals a distinct pattern of binding for the two ligands (Table 4). A single high anity ‰125 IŠ aBTX site is identi®ed in human brain, with the highest density of binding observed in the hippocampus contrasting to the relatively sparse concentration of ‰3 HŠ nicotine binding sites in this region (Rubboli et al., 1994a; Court and Clementi, 1995; Breese et al., 1997; HellstroÈm-Lindahl et al., 1999). ‰125 IŠ aBTX and ‰3 HŠ nicotine also show a distinct pattern of distribution in the thalamus. A recent study has compared the relative distribution of the two ligands in this brain region (Spurden et al., 1997). Consistent with previous reports ‰3 HŠ nicotine binding was high in the majority of thalamic nuclei, particularly the lateral dorsal, medial geniculate, lateral geniculate and anterior nuclei. In contrast, the relative level of ‰125 IŠ aBTX binding is lower in all of these nuclei, with the highest level of binding observed in the reticular nucleus, NBM and the horizontal limb of the diagonal band of Broca (Breese et al., 1997). 3.2. Subunit mRNA distribution A small number of studies mapping nAChR subunit mRNA distribution in human brain (Table 5) have been performed mainly utilising in situ hybridisation (Rubboli et al., 1994a, 1994b; Wevers et al., 1994; SchroÈder et al., 1995; Court and Clementi, 1995; Breese et al., 1997; HellstroÈm-Lindahl et al., 1998, 1999). The majority of investigations thus far have concentrated on the distribution of a7, a4, a3 and b2 mRNA, with apparently only one study additionally

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examining a5 and b3 and b4 distribution in the post mortem human brain (HellstroÈm-Lindahl et al., 1998). The majority of studies compared subunit distribution to the pattern of ‰3 HŠ nicotine and ‰125 IŠ aBTX binding sites and have generally reached similar conclusions. Consistent with the theory that a4b2 nAChRs constitute the predominant subtype present in the brain, the distribution of b2 mRNA is fairly homogeneous. Moderate to low amounts are observed in most brain regions including the cortex, thalamus, caudate-putamen and hippocampus. Distribution of subunit mRNA in di€erent cortical layers has been examined to a limited extent (SchroÈder et al., 1995) and is generally similar to the pattern of ‰3 HŠ nicotine binding. Observations indicate that a3 mRNA is most abundant in the thalamus (e.g. dorsomedial, lateroposterior, reticular, and ventroposterolateral nuclei), is present in low to moderate amounts in most cortical regions (e.g. prefrontal, motor, entorhinal, cingular), and hippocampus, and absent in the caudate-putamen (Rubboli et al., 1994a; Court and Clementi, 1995). The pattern of a3 mRNA in the thalamus corresponds to that of ‰3 HŠ nicotine and ‰3 HŠ cytisine (which is almost identical) but is distinct from binding in the hippocampus (Court and Clementi, 1995). In the cortex, a3 mRNA is most predominantly expressed in pyramidal neurons layers III±VI, moderately expressed in layer II and minimally expressed in layer IV (Wevers et al., 1994; SchroÈder et al., 1995). In another study, a3 mRNA was observed to be evenly distributed in the parietal cortex, frontal cortex and hippocampus, but signi®cantly lower in the temporal cortex and cerebellum (HellstroÈm-Lindahl et al., 1999). In contrast, the expression of a4 mRNA observed in the same study, was signi®cantly higher in the temporal cortex and cerebellum compared to the other brain regions. The distribution of a4 mRNA in the neocortex is more widespread than that of a3, but both are associated with pyramidal neurons (Court and Clementi, 1995). In the frontal cortex a4, mRNA is abundant in all layers except I and IV (SchroÈder et al., 1995). b2 mRNA shows a strong signal in the insular cortex, the granular layer of the dentate gyrus and the CA2/3 region of the hippocampus, with a signal of lower intensity observed in the subicular and entorhinal cortex (Rubboli et al., 1994b). High levels of b2 mRNA have also been reported in the cortex and cerebellum of prenatal and aged brain (HellstroÈm-Lindahl et al., 1998). In contrast, Court and Clementi, (1995), report only moderate levels of b2 mRNA in the dentate gyrus and the CA2/3 of the hippocampus, with a pattern distinct to that of ‰3 HŠ nicotine binding. b2 mRNA is also present in the striatum but is not so predominant in the thalamus. In a study comparing the regional expression of a7 mRNA and ‰125 IŠ aBTX binding in human postmortem brain, Breese et al. (1997)

91

observed the reticular nucleus of the thalamus, the lateral and medial geniculate bodies, the horizontal limb of the diagonal band of Broca, and the NBM as regions with high levels of both a7 mRNA and ‰125 IŠ aBTX binding. Moderate levels of a7 mRNA were observed in the cortex and hippocampus where ‰125 IŠ aBTX binding did not correlate so well to probe signal. However, in the majority of brain regions ‰125 IŠ aBTX binding and a7 mRNA were localized to the same cell bodies. These observations are supported by Rubboli et al. (1994b), who report high levels of a7 mRNA in the dentate gyrus, CA2/3 of the hipocampus, certain thalamic nuclei and the caudate nucleus, a distribution which was matched by the pattern of ‰125 IŠ aBTX binding. In the frontal cortex, a7 mRNA is high in layers II and III, moderate in layers V and VI and low in layers I and IV (SchroÈder et al., 1995). a5, b3 and b4 subunit mRNA distribution has been studied in prenatal and aged human post mortem brain (HellstroÈm-Lindahl et al., 1998). These subunits can be detected in the spinal cord, medulla oblongata, pons, cerebellum, mesencephalon, subcortical forebrain and cortex and thus, have a fairly widespread distribution. a5 was most abundant in the cortex, whilst b3 was highest in the cerebellum. In comparison, mRNA levels for a5 and b4 subunits were signi®cantly lower in aged cortex and cerebellum. 4. Imaging of nicotinic receptors with PET and SPECT Quantitative imaging of functional and pathological processes in the living mammalian brain has recently become feasible through the development of PET (positron emission tomography) and SPECT (single photon emission computed tomography) imaging techniques. PET and SPECT are non-invasive tomographic methods for imaging the regional distribution of radioactive tracers. PET utilises tracers labelled with positron emitting radionuclides such as ‰11 C], ‰13 NŠ and ‰18 F], while SPECT utilises g or photon embodying radioisotopes such as ‰123 IŠ and ‰99m Tc]. Following injection of radiotracer, brain distribution of radioactivity is detected by means of externally placed banks of paired detectors that register co-incidental energy (in the form of 2g rays) from the annihilation of emitted positrons with electrons in the case of PET and by use of a rotating g camera or multiple detector rings in the case of SPECT. These techniques have been successfully used to map cerebral metabolic function and blood ¯ow, as well as a number of receptor systems in the living human brain. With the use of these techniques, the distribution and binding of nicotinic receptors in the human brain can be studied in vivo. This is of particular interest when considering the involvement of these receptors in the pathology of neu-

92

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rodegenerative conditions such as Alzheimer's disease, Parkinson's disease, numerous pathological conditions such as epilepsy, schizophrenia and depression, and their implication in neurological processes such as learning and memory. Brain imaging of nicotinic receptors therefore o€ers further understanding of the involvement of these receptors in pathological conditions as well as insight into their role in the normal functioning of the brain. 4.1. PET The development of methods for the synthesis of radiolabelled nicotine (Maziere et al., 1976; LaÊngstroÈm et al., 1982; Halldin et al., 1992) has allowed the uptake and distribution of nicotine in the brain of animals and man to be studied. Following studies in mice, in which injection of ‰3 HŠ nicotine produced brain uptake and distribution of radioactivity consistent with nAChR density (Broussolle et al., 1989), ‰11 CŠ nicotine was developed and used in rhesus monkeys and then in humans to map the distribution of nicotinic receptors in the living brain. (Nordberg et al., 1989b, 1990; NybaÈck et al., 1989). The distribution of ‰11 CŠ radioactivity measured with PET was generally consistent with the known pattern of nAChRs measured by in vitro binding in autopsy brain tissue (Nordberg et al., 1989a). High levels of radioactivity were observed in the thalamus and caudate nucleus, moderate levels in the frontal and temporal parietal cortices and in the cerebellum with low levels in white matter tracts. ‰11 CŠ nicotine has been used to image nAChRs in Alzheimer patients (Fig. 4) and has revealed signi®cant reductions in uptake of ‰11 CŠ nicotine in the frontal and temporal cortices of these patients when compared to healthy, aged, matched volunteers (Nordberg et al., 1990, 1995). These observations con®rmined earlier postmortem ®ndings (Nordberg et al., 1989a). Additionally, a positive correlation was observed between uptake of tracer into temporal cortex and cognitive performance in Alzheimer's patients. These observations indicate the viability of ‰11 CŠ nicotine and PET as a diagnostic tool in Alzheimer's disease. However, a number of factors make ‰11 CŠ nicotine a less than ideal ligand for the imaging of nicotinic receptors in vivo, in man. It displays high levels of non speci®c binding, rapid metabolism and rapid washout of the brain (Grunwald et al., 1996). The heterogeneity of ‰11 CŠ nicotine binding in the brain also precludes the identi®cation of a reference region which may be used to accurately determine non speci®c binding. Furthermore, the study of (NybaÈck et al., 1994) suggests that brain uptake and retention of ‰11 CŠ nicotine may not be entirely mediated by speci®c binding to nAChRs, with the suggestion that brain distribution of the tracer may be

in¯uenced to a signi®cant degree by cerebral blood ¯ow. However, kinetic studies of ‰11 CŠ nicotine uptake into the brain of Alzheimer's patients involving compartmental modelling in which the e€ect of blood ¯ow was considered allowed calculation of a kinetic rate constant k2 expressing ‰11 CŠ nicotine binding (Nordberg et al., 1995, 1997). A study performed in monkeys con®rmed that the calculated k2 constant was blood ¯ow independent (Lundqvist et al., 1998). A signi®cant reduction in k2 was observed in the temporal and frontal cortices and the hippocampus of AD patients compared to age matched controls (Nordberg et al., 1995, 1997). Additionally, a signi®cant correlation was observed between cognitive status and ‰11 CŠ nicotine binding (expressed as k2 † in the temporal cortex in AD patients (Nordberg et al., 1997). However, the drawbacks involved in the use of ‰11 CŠ nicotine as a tracer for the imaging of nicotinic receptors has led in the last few years to the search for new ligands (see Fig. 5) with more suitable pro®les i.e. high speci®c to non speci®c binding ratio, longer retention (slower washout) from brain and less rapid metabolism. In addition, ligands with nicotinic receptor subtype speci®city are also of interest, speci®cally, a ligand with selectivity for a4b2 receptors which are recognised to be the predominant subtype lost in Alzheimer's disease. Initially, interest centred upon the development of the nAChR agonist cytisine as a PET radiotracer, but in vivo studies in rodents with ‰3 HŠ cytisine indicated that although brain retention was longer than that of ‰11 CŠ nicotine, it displayed low blood brain barrier penetration making it a poor candidate ligand. PET studies with ‰11 CŠ ABT 418 and N-[11 CŠ methylcytisine (high anity nAChR agonists) have also been attempted in baboons (Valette et al., 1997). However both ligands displayed low levels of brain uptake, rapid washout from brain and rapid metabolism. More favourable results were forthcoming with a series of ‰3 HŠ labelled nicotine analogues, including ‰3 H](R,S)-5-isothiocyanonicotine and ‰3 H]-(R,S)-5-aminonicotine, which have been evaluated in vitro and in vivo and appear to possess appropriate attributes for potential PET ligands (Kim et al., 1994). The recent discovery of the potent high anity nicotinic receptor agonist epibatidine provided a potentially excellent ligand for the in vivo imaging of nAChRs. In vivo in rodents ‰3 HŠ epibatidine and the epibatidine analogue ‰3 HŠ norchloroepibatidine display regional brain localization consistent with the known pattern of high anity nAChR sites (i.e. thalamus > cortex > cerebellum), high speci®c to non speci®c binding and slow clearance from the brain (London et al., 1995; Sche€el et al., 1995). This favourable pro®le in rodents has led to the development of positorn labelled analogues of epibatidine potentially suitable for use as

D. Paterson, A. Nordberg / Progress in Neurobiology 61 (2000) 75±111

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Fig. 4. (S)(-)[11 CŠ Nicotine uptake in the brain of two Alzheimer's patients. Horizontal PET images show the distribution of (S)(-)[11 CŠ nicotine in the brain of two AD patients at: (A) the level of the basal ganglia and (B) the frontal association cortex±parietal cortex. The ®gures represent a summation picture of the brain uptake of ‰11 CŠ radioactivity following an intravenous injection of a tracer dose of (S)(-)[11 CŠ nicotine. ``Hot'' colours represent areas with high tracer uptake and ``cold'' colours represent areas with low uptake. One patient shows right side de®cits (upper sections), while the other shows left side de®cits (lower sections) in (S)(-)[11 CŠ nicotine brain uptake (indicated by the arrows). Reduced (S)(-)[11 CŠ nicotine uptake in these areas is likely to correspond to a loss of nAChRs.

PET ligands in man. A number of these compounds and other novel nAChR ligands will now be discussed. 4.1.1. [18 FŠ NFEP or ‰18 FŠ FPH [18 FŠ ¯uoronorchloroepibatidine ((2)exo-(2-[18 FŠ ¯uoro-5-pyridyl)-7-azabicyclo[2.2.1] heptane) is a ‰18 FŠ 2 ¯uoro pyridyl analog of epibatidine that has been named as both ‰18 FŠ FPH and ‰18 FŠ NFEP, and has been assessed as a nAChR PET ligand via ex-vivo autoradiography in mouse brain, in vitro autoradiography in human brain tissue and with PET in the living baboon brain (Horti et al., 1997; Villemagne et al., 1997; Gatley et al., 1998). Its potential as a possible PET ligand for use in man was initially indicated by promising preliminary studies performed with ‰3 HŠ NFEP (Sche€el et al., 1995). Ligand distribution in both mouse and human brain indicates binding

consistent with nAChR distribution as mapped by ‰3 HŠ nicotine, ‰3 HŠ cytisine and ‰3 HŠ epibatidine (Gatley et al., 1998). Following injection in mice ‰18 FŠ FPH=‰18 F NFEPŠ displays rapid uptake and a heterogeneous distribution in the brain with preinjection of a range of nAChR ligands including nicotine, cytisine and lobelline inhibiting tracer binding in all brain regions except the cerebellum, where no e€ect was observed. In contrast, the preinjection of the non competitive nAChR antagonist mecamylamine, the muscarinic agonist scopolamine, the dopamine receptor agonist apomorphine, and the 5-HT receptor antagonist ketanserin had no e€ect on radioligand binding in any brain region, con®rming that speci®c binding to nAChRs accounts for radioligand distribution in the brain (Horti et al., 1997). PET analysis in baboon indicated that ‰18 FŠ FPH=‰18 FŠ NFEP displays

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Fig. 5. Structure of (S)[11 CŠ nicotine and a selection of ‰11 C], ‰18 FŠ and ‰123=125 IŠ radiolabelled nAChR ligands developed for PET and SPECT.

rapid uptake and heterogeneous distribution in the brain which correlates well with that observed in mouse (Villemagne et al., 1997). Furthermore, administration of unlabelled cytisine signi®cantly reduced ‰18 FŠ FPH=‰18 FŠ NFEP levels in the brain indicating that brain retention of tracer is mediated by speci®c binding to nAChRs, possibly of the a4b2 subtype. ‰18 FŠ FPH=‰18 FŠ NFEP therefore has excellent potential for use as a PET ligand in man. However, norchloroepibatidine exhibits relatively high toxicity which may preclude extensive use of ‰18 FŠ FPH=‰18 FŠ NFEP as a PET agent in man (Gatley et al., 1998). 4.1.2. [18 FŠ A-85380 and ‰11 CŠ A-8548 Recently, a number of novel compounds with subnanomolar anity for nAChRs have been synthesized for the treatment of Alzheimer's disease (Abreo et al., 1996). Among these compounds are A-85380 (3-(2-(S)azetidinylemthoxy)pyridine) and A-85453 (3-[(1methyl-2(S)-pyrrolidinyl)methoxy]pyridine). Both of these compounds display an anity for a4b2 receptors

equal to that of epibatidine, but signi®cantly lower anity than epibatidine for a7 receptors. Additionally, A853580 is respectively 40 and 100 times less potent than epibatidine in activating a4b2 and a3b4 subtypes. (Sullivan et al., 1996). It has therefore been hypothesised that these compounds would be suitable PET ligands as they may selectively label a4b2 over a7 receptors and have a greater margin of safety between adequate tracer dose and dose producing biological e€ect compared to ligands such as ‰18 FŠ FPH=‰18 FŠ NFEP]. ‰18 FŠ A-85380 and ‰11 CŠ A-85453 have recently been synthesized and their brain uptake and distribution assessed in mice via ex vivo autoradiography (Horti et al., 1998; Kassiou et al., 1998). Following administration, both ligands displayed high uptake into the brain with radioactivity peaking after approximately 5 min with a slow washout observed thereafter. A high speci®c to non-speci®c binding ratio was observed with regional distribution of radioactivity consistent with nAChR density. Pre-administration of non-radiolabelled nicotinic ligands such as

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nicotine, cytisine and epibatidine signi®cantly reduced binding of both radioligands in all brain areas with the exception of the cerebellum where observed e€ects were minimal. In contrast, administration of the noncompetitive ligand mecamylamine had no e€ect on brain tracer level, nor did the administration of a number of non-nicotinic ligands, thus indicating that brain retention of these ligands is mediated by speci®c binding to nAChRs (Horti et al., 1998; Kassiou et al., 1998). These results suggest that both ‰18 FŠ A-85380 and ‰11 CŠ A-85453 deserve further investigation as possible PET radiotracers.

4.1.3. [11 CŠ MPA A further potential PET radiotracer is ‰11 CŠ MPA ((R,S)-1-methyl-2-(3-pyridyl) azetidine). Initial in vitro evaluation of this compound in rodent brain indicated that it displayed characteristics suitable for a PET ligand (Sihver et al., 1998b) and recent preliminary PET studies comparing it to ‰11 CŠ ABT 418 and ‰11 CŠ nicotine in rhesus monkeys appear to con®rm this (Sihver et al., 1999b). Uptake of ‰11 CŠ MPA into the brain was rapid following injection, similar in extent to ‰11 CŠ ABT 418 and ‰11 CŠ nicotine. Pre-injection of animals with nicotine resulted in 25% reduction ‰11 CŠ MPA uptake in the thalamus, a 19% reduction in the temporal cortex and an 11% reduction in the cerebellum, indicating that brain retention of ‰11 CŠ MPA in nAChR mediated. In contrast, nicotine pre-treatment was observed to produce increases in ‰11 CŠ ABT 418 and ‰11 CŠ nicotine brain uptake, suggesting that ‰11 CŠ MPA may be a more suitable PET ligand for nAChRs.

4.1.4. [76 BrŠ BAP In a similar manner to ‰11 CŠ MPA, ‰76 BrŠ BAP (576 [ Br]-bromo-3-((2(s )-azetidinyl) methoxy)pyridine) has been evaluated in vitro and in vivo in rats with preliminary studies additionally performed in rhesus monkeys (Sihver et al., 1999b). With in vitro autoradiographic analysis, highest levels of binding were observed in the thalamus and presubiculum with moderate levels observed in the cortex and striatum and low levels observed in the hippocampus and cerebellum. Ex-vivo autoradiographic analysis following injection into rats revealed a similar regional binding pattern, with brain uptake blocked by preinjection of nicotine. In rhesus monkeys, PET evaluation revealed high levels of tracer retention in the thalamus, up to 60% of which could be displaced by preinjection with cytisine and 50% by preinjection of nicotine. The high levels of speci®c uptake observed for this ligand in rat and monkey brain suggest a promising PET ligand.

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4.2. SPECT Development of SPECT agents for the imaging of nicotinic receptors holds a number of advantages over PET. The greater availability of SPECT (mainly due to its lower cost), the longer half-life of SPECT ligands (hours as opposed to minutes) and the commercial availability of the appropriate radioisotopes make SPECT a cheaper and easier imaging alternative. However, despite the availability of ‰11 C]-nicotine and the development of a number of epibatidine analogs and synthetic compounds for use with PET, the number of SPECT ligands currently available and in development is relatively few. A radioiodinated form of nicotine ([125 I]-(s)-nicotine) has been developed (Kampfer et al., 1996) and evaluated in rats (Saji et al., 1995). But as with its ‰11 CŠ niciotine PET counterpart, it su€ers from substantial non-speci®c binding. Only recently the radioiodinated compounds ‰125=123 IŠ IPH ((2)-exo-2-iodo-5-pyridyl)-7-azabicyclo[2.2.1] heptane) and ‰125 IŠ 5-I-A-85380 have been developed and evaluated (Musachio et al., 1997, 1998; Vaupel et al., 1998). Following intravenous injection in mice ‰125 IŠ IPH displays similar qualities to its PET counterpart ‰18 FŠ FPH, with high levels of brain uptake observed, high speci®c to non-speci®c binding and regional distribution appropriate for a nicotinic receptor ligand. Additionally, pre-administration of unlabelled IPH, nicotine and cytisine blocked receptor binding, an e€ect not produced by pre-administration of scopolamine, ketaserin or mecamylamine. SPECT analysis of ‰123 IŠ IPH in baboon produced similar observations. Tracer uptake was rapid, reaching a plateau after approximately 45 min with cortical, subcortical and cerebellar structures all identi®able. Observed activity was highest in the thalamus, moderate in the cortex and low in the cerebellum. Subsequent injection of 1 mg/kg cystine signi®cantly reduced brain levels of tracer with the thalamus showing the most profound e€ect, indicating that the ligand binds speci®cally to nAChRs (Musachio et al., 1997). ‰125 IŠ 5-I-A-85380 has been evaluated in mice via ex vivo autoradiography (Vaupel et al., 1998) and preliminary SPECT studies with ‰123 IŠ 5-I-A-85380 have also been performed in baboons (Musachio et al., 1999). ‰125=123 IŠ 5-I-A-85380 shows similar characteristics to its IPH and its PET counterpart. High brain uptake is observed with regional distribution of radioactivity consistent with nAChR density. Although further characterisation is necessary, both of these compounds have excellent potential for use as SPECT agents in man and with the advantages of SPECT over PET will perhaps provide the most convenient method of imaging nicotinic receptors in pathological states such as Alzheimer's diseases.

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5. Nicotinic receptor function in the CNS 5.1. Functional and behavioural e€ects of nicotine Nicotine is a potent modulator of CNS function. It enhances ion ¯ux and neurotransmitter release, augments or gates of a number of neuronal systems and elicits a variety of behavioural states. Nicotine administration produces a number of physiological e€ects including increased heart rate and blood pressure and dose dependent increase in the secretion of prolactin and ACTH, resulting in a subsequent increase in corticosterone secretion (Newhouse et al., 1990; Benowitz, 1996). Speci®c CNS e€ects of nicotine include EEG desynchronisation, producing a shift in the direction of higher frequency (Edwards and Warburton, 1982), increased cerebral blood ¯ow and increased cerebral glucose utilization through stimulation of nAChRs in the basal forebrain; e€ects which can be blocked by the nicotinic antagonist mecamylamine (Pickworth et al., 1988; London, 1990; Linville and Arneric, 1991; Linville et al., 1993). In humans, nicotine increases arousal, visual attention and perception (Jones et al., 1992), but also decreases reaction time, prevents a decline in eciency over time and improves the ability to withold inappropriate responses (Jones et al., 1992; Wesnes and Warburton, 1983). In both smokers and non-smokers, nicotine produces an improvement in the speed and accuracy of motor function (Hindmarch et al., 1990; West and Jarvis, 1986) and improves performance in complex psychomotor tasks such as car driving. However, its withdrawal worsens performance and other vigilance tasks (Heimstra et al., 1967, 1989). This e€ect may be dose dependent with large doses of nicotine worsening car driving (Sherwood, 1995). Despite the complex e€ects elicited by nicotine, the exact role of brain nAChRs remains unclear. A signi®cant body of evidence suggests that presynaptic nAChRs exist on several cell populations in cortical, hippocampal and cerebellar brain regions (Wonnacott, 1997). Nicotine interacts with a variety of presynaptic nAChRs to facilitate the release of a number of neurotransmitters including ACh, DA, NA, 5-HT, GABA and glutamate, many of which have been implicated in mediating/modulating a number of behavioural tasks (de Sarno and Giacobini, 1989; Wonnacott et al., 1990; McGehee and Role, 1995). Therefore, it has been proposed that the role of nAChRs in the brain is to modify the excitability of neurons, that is to produce the optimal performance of neurons by adjuting their excitability, an action which is likely to be of importance in a number of behavioural responses and particularly in cognitive processes (McGehee and Role, 1995). The most obvious behavioural action mediated by nAChRs is the addiction to nicotine in tobacco smoke. The mesolimbic DA pathway is thought to

mediate the addictive e€ects of nicotine and other substances of abuse, with nicotine known to stimulate release of DA in this pathway (Pich et al., 1997). Addiction to nicotine is motivated by both positive and negative reinforcing factors (Benowitz, 1996). Positive factors are relaxation, reduced stress, increased vigilance, improved cognition and reduced body weight (LindstroÈm, 1997). Enhanced release of mesolimbic DA through activation of presynaptic nAChRs may feasibly mediate these rewarding e€ects. Negative reinforcing factors are unpleasant withdrawal symptoms including nervousness, restlessness, irritability, anxiety, impaired concentration and cognition and weight gain, and may result from reduced stimulation of mesolimbic DA neurons (LindstroÈm, 1997). In a recent functional MRI study (Stein et al., 1998), nicotine administration was observed to produce a dose dependent increase in several rewarding behavioural parameters and to increase neuronal activity in limbiccortical structures including the amygdala, nucleus accumbens, and cingulate and frontal cortices. These structures are consistent with nicotines behaviour arousing and behaviour reinforcing properties in humans and have previously been identi®ed to participate in the reinforcing, mood elevating and cognitive properties of other abused drugs such as cocaine, amphetamines and opiates (Stein et al., 1998). The mechanisms by which nicotine produces tolerance and addiction and identi®cation of the receptor subtype(s) which mediate these e€ects is complicated by a number of factors including the ability of nAChRs to be activated by acute exposure to agonists, to be reversibly desensitized on longer exposure, and to be permanently deactivated following prolonged exposure with the additional result of increasing nAChR number (LindstroÈm, 1997). Furthermore, identifying which e€ects are the result of activation and which of inactivation of nAChRs is unclear. However, the rewarding e€ects of nicotine can be blocked by pre-administration of the nAChR channel blocker mecamylamine, indicating that activation of nAChRs is important in positive reinforcement and self administration (Henning®eld, 1984). The development of tolerance to nicotine exhibited by smokers and the adverse e€ects of nicotine on naive users is more obviously explained by the reversible desensitization of nAChRs following exposure to nicotine and the permanent inactivation of nAChRs, despite upregulation of numbers following prolonged exposure to nicotine (LindstroÈm, 1997). 5.2. Role of nicotinic receptors in cognitive and memory functions A considerable body of evidence exists to suggest that nicotine and nicotinic agonsits have cognitive and memory enhancing properties in animals and humans,

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while antagonists such as mecamylamine impair memory function (Levin and Simon, 1998). In rodents and non-human primates, short and long term treatment with nicotinic agonists improve performance on a variety of memory tasks. Short term nicotine treatment has been shown to improve working memory performance in a number of experimental studies on rats (Levin et al., 1993; Decker, 1995; Decker et al., 1995) and to improve memory in delayed matching to sample tasks in monkeys (Buccafusco and Jackson, 1991). This mnemonic improvement is also observed following administration of the nAChR agonists lobelline, dimethylethsanolamine (DMEA), ABT-418 and GTS-21 (Decker et al., 1993, 1994; Levin et al., 1995). However, contradictory evidence indicating that nicotine has no e€ect or even a detrimental e€ect on memory performance in animals has been reported (Dunnett and Martel, 1990). Acute nicotine induced memory improvements can be blocked by the concurrent administration of either nicotinic or muscarinic antagonists suggesting a possible role for muscarinic receptors in mediating the mnemonic e€ects of nicotine (Newhouse et al., 1997). Long term nicotine treatment has also been shown to improve memory in animal studies with surprisingly no development of tolerance (Levin et al., 1990, 1993; Levin and Torry, 1996). Interestingly, improvements in memory persisted for up to 2 weeks after drug withdrawal, although the mechanisms by which this e€ect occurs are unclear. Long term nicotine induced memory facilitation is inhibited by concurrent administration of mecamylamine but not by short term mecamylamine injection during nicotine infusion (Newhouse et al., 1997). When administered alone, mecamylamine causes working memory impairments (Andrews et al., 1994; Oliverio, 1966). Nicotine and nicotinic agonsits also reverse memory de®cits in brain lesion studies (Decker et al., 1992, 1994; Levin et al., 1993; Muir et al., 1995; Grigoryan et al., 1996) and age related memory de®cits are improved by bolus nicotine administration (Arendash et al., 1995; Socci et al., 1995; Levin and Torry, 1996). Despite the large number of studies performed, the cognitive e€ects of nicotine on humans remain to be fully elucidated. Di€erent means of administration, di€erent doses and the participation of smoking and non-smoking subjects in studies have resulted in diculties in interpretation of results. Many studies have used tobacco smoking as a means of nicotine administration, ignoring the fact that nicotine is unlikely to be the only active substance in tobacco smoke. It should also be noted that the test subjects participating in these studies were often tobacco smokers who had been deprived of tobacco for some time. Under these conditions, nicotine has been observed to improve performance in a variety of cognitive and mnemonic tasks. However, it is unclear

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whether the improvements observed represents a primary action of nicotine or merely a return to predeprivation level of performance. Interpretation of observations from this type of deprivation study is further complicated by the fact that chronic tobacco smoking increases the number of high anity nAChRs in various brain areas (Hindmarch and Sherwood, 1995; Stolerman and Jarvis, 1995). In addition, nicotine produces prolonged behavioural e€ects in animals including a signi®cant period following drug withdrawal, and it is likely that tobacco smokers will be subject to similar prolonged behavioural e€ects following cessation to smoking. Therefore, the cogntive improvements produced by nicotine in this type of deprivation study may be misleading. Nicotine induced improvements in memory and cognitive tasks in nonsmokers and non-deprived smokers have been more dicult to demonstrate. For example, reports have suggested that nicotine has no variable and even negative e€ects on memory and learning (Levin, 1992). However, in general it has been accepted that nicotine has a number of cognitive enhancing actions in humans. Nicotine increases arousal, visual attention and perception, and may prevent fatigue induced de®cits in vigilance and long term performance (Jones et al., 1992; Newhouse et al., 1992). It shortens information processing time and improves reactions (Le Houezec et al., 1994) and in non-deprived smokers it enhances recognition memory (Rusted et al., 1994). Nicotine is thought to improve short term memory by facilitating the storage of information received (Warburton et al., 1986; Levin, 1992; Levin et al., 1992) and have a consolidating e€ect on memory (Warburton et al., 1986; Colrain et al., 1992; Newhouse et al., 1995). In complimentary fashion, the non-competitive nAChR antagonist mecamylamine produces detrimental e€ects on learning and memory. The studies described above support the theory that nAChRs play an important role in cognition and memory. This theory becomes more compelling when considering the signi®cant loss of nAChRs from the hippocampus and frontal cortex observed in AD (two brain regions important in cognitive function) and the cognitive decline associated with the disease. However, further work is necessary to determine which nAChR subtypes are involved in cognitive processes and to elucidate the interactions of these receptors with other neurotransmitter systems which are critical in cognitive function. 6. Pathology of neuronal nicotinic receptors 6.1. Epilepsy Epilepsy is a heterogeneous group of disorders that

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a€ects about 2% of the population. Autosomal dominant frontal lobe epilepsy (ADNFLE) is a partial epilepsy that causes brief seizures that occur during light sleep that are often mis-diagnosed as nightmares, although most patients also su€er to some degree from violent generalized seizures (Sche€er et al., 1995). It has recently been recognized that ADNFLE results from a missense mutation in the a4 subunit gene, replacing a highly conserved serine at position 247 of the M2 channel lining domain of the subunit with a phenylalanine (Steinlein et al., 1995). The mutation is thought to impair channel function and when expressed in Xenopus oocytes with normal a b2 subunit, a mutant receptor is formed that displays reduced Ca2+ permeability, reduced channel opening (probably due to a faster desensitization rate) and slower recovery from the desensitized state (Weiland et al., 1996). A second gene mutation involving the insertion of a leucine after position 259 in the extracellular C-terminal end of the M2 domain of the a4 subunit also causes ADNFLE (Steinlein et al., 1995). This mutation is thought to be relatively well tolerated however, having less impact on receptor function, but as with the 247 serine mutation Ca2+ channel permeability is reduced. Studies on reconstituted a4b2 nAChRs expressed in Xenopus oocytes with the serine 247 mutation indicate that these receptors display a decrease in apparent anity of ACh of about 7 fold and currents 5 times smaller than controls at saturating concentrations of ACh. Additionally, these receptors desensitize to an agonist concentration 3000 times lower than controls (Bertrand et al., 1998). As epilepsy results from excessive neuronal activation, it is somewhat contradictory that mutations resulting in reduced nAChR function should cause seizures. A possible explanation for this lies in the action of presynaptic a4b2 receptors to promote the release of many neurotransmitters including inhibitory GABA and glycine (Wonnacott et al., 1990; Wonnacott, 1997). Facilitation of GABA or glycine release or activation of such inhibitory neurons by a4b2 receptors may prevent the onset of seizures between sleep and wakefulness, and thus, reduced receptor function resulting from the above mutations may trigger ADFLNE. A further link of nAChRs to epilepsy comes from the observation that mice with unusually high numbers of ‰125 IŠ aBTX binding sites are more susceptible to seizures in response to nicotine administration (Marks et al., 1989) suggesting that the a7 nAChR may also play a role in epilepsy. 6.2. Alzheimer's disease Alzheimer's disease (AD) is a progressive neurodegenerative condition a€ecting almost 1 in 10 individuals over the age of 65 (Evans et al., 1989). It accounts

for over 50% of senile dementia and the majority of pre-senile dementia cases, and is characterised by progressive deterioration of higher cognitive functions including the loss of memory (Octave, 1995). Postmortem AD brains display two distinctive neuropathological features which constitute conclusive diagnostic markers for AD: intracellular neuro®briallary tangles and extracellular neuritic senile plaques. Intracellular neuro®brillary tangles accumulate in neuronal perikarya and consist of paired helical ®laments containing the microtubule associated protein tau (Delacourte and Defossez, 1986). The presence of tau in neuronal cell bodies represents a highly aberrant localisation of the protein, as compared to the axonal localisation observed in normal neurons (Kowall and Kosik, 1987). In AD brains, tau is present in tangles in a hyperphosphorylated form (Grundke-Iqbal et al., 1986). The amount of phosphorylated tau in AD brain is several 100 fold greater to that in normal brains, making tau an excellent disease marker (Vandermeeren et al., 1993). The neuritic plaques observed extracellularly in AD brains contain amyloid peptide ®brils in their core. These ®brils consist of the amyloid b or A4 (bA4) peptide (Glenner et al., 1984). The bA4 peptide is derived from a larger precursor peptide termed the amyloid precursor peptide/protein (APP), a glycosylated transmembrane protein with a single membrane spanning domain (Kang et al., 1987). APP is normally cleaved within its transmembrane domain yielding a secretory form, the exact physiological role of which is not entirely understood. The primary neurodegenerative e€ects of AD appear to be closely linked to amyloid production. In addition to these speci®c neuropathological features, AD brains exhibit extensive cellular atrophy and cell loss, shrinkage of cortical thickness, enlargement of sulci and ventricles and changes in multiple neurochemical systems including ACh, glutamate, GABA and 5-HT. However, the most consistent and severe neurochemical abnormality associated with AD is the loss of cholinergic innervation of the cerebral cortex and hippocampus (Coyle et al., 1983). ChAT activity is signi®cantly reduced in the cortex and hippocampus of AD brains (Reisine et al., 1978). Post mortem analysis of AD brains reveals that the NBM, the major source of cortical and hippocampal cholinergic innervation, is degenerated in AD (Whitehouse et al., 1982). In addition, a linear correlation between reduced cortical ChAT activity and degree of dementia has been observed (Perry et al., 1978). Observations such as these prompted (Bartus et al., 1982) to propose the cholinergic hypothesis of AD, which speci®cally attributed the cognitive deterioration associated with the disease to the degeneration of the cholinergic pathways from the basal forebrain (nucleus basalis of Meynert) to the cortex and hippocampus. In addition to measurement of ChAT levels, numerous

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studies characterising changes in cholinergic receptor density in AD brains have been performed. Marked reductions in high anity cortical nAChRs have been observed in the brains of AD patients compared to age matched controls (Whitehouse et al., 1986; Flynn and Mash, 1986; Nordberg and Winblad, 1986; Aubert et al., 1992; Warpman and Nordberg, 1995). Interestingly, a signi®cant correlation cannot be observed between histopathological score and number of nAChRs in autopsy brain cortical tissue (Svensson et al., 1997), although a signi®cant correlation is observed between cognition and nAChRs measured in vivo by PET (Nordberg et al., 1995). The in¯uence of bA4 on cholinergic neurotransmission has recently been studied in autopsy brain tissue from subjects carrying the Swedish APP 670/671 mutation, and in brain tissue from sporadic AD cases (Marutle et al., 1999). Signi®cant reductions in nAChR numbers were observed in the cortex of the Swedish APP 670/671 mutation cases (73±87%) and in the sporadic cases (37±57%) as measured by ‰3 HŠ epibatidine and ‰3 HŠ nicotine binding. ‰3 HŠ epibatidine saturation analysis revealed two binding sites in the cortex of Swedish APP 670/671 brains, with a signi®cant decrease (82%) in the number of high anity sites and no change in Kd observed, compared to control subjects. Besides a signi®cant positive correlation between the number of neuronal plaques and ‰3 HŠ nicotine binding sites in the parietal cortex, no strict correlation between nAChR de®cits and neuropathological markers could be observed in the cortex of Swedish APP 670/671 brains, suggesting that, although these processes may be related, they are not strictly dependent upon one another (Marutle et al., 1999). These observations support the assumption that the nAChR might be impaired very early in the course of AD. The most signi®cant changes are observed in the temporal, parietal and occipital cortices (Nordberg, 1994). A recent study comparing ‰3 HŠ nicotine, ‰3 HŠ epibatidine, ‰3 HŠ cytisine and ‰3 HŠ vesamicol (representing vesicular ACh transporter sites) binding observed signi®cant reductions in the temporal cortex of Alzheimer's patients compared to aged matched controls for all ligands (Fig. 6), although the reduction in ‰3 HŠ vesamicol binding was only half as much as the reduction observed with the nAChR ligands (Sihver et al., 1999c). Evidence from postmortem binding studies and transfected human nAChRs indicates that a4b2 nAChRs constitute the major subtype of nAChRs lost in AD (Warpman and Nordberg, 1995). This view is supported by the observation that a4 and b2 subunit mRNA levels decrease with age in the frontal cortex of human brain, with b2 levels additionally reduced in the hippocampus (Tohgi et al., 1998). In a similar study, the level of a3 subunit mRNA in the entorhinal cortex of human brain was observed to decrease with age, however no signi®cant

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Fig. 6. Average binding densities of ‰3 HŠ nicotine, ‰3 HŠ epibatidine, ‰3 HŠ cytisine and ‰3 HŠ vesamicol in the temporal cortex of control (n = 7) and Alzheimer brains (n = 9). Data are presented as mean 2SEM fmols binding per mg of brain. P < 0.05 Student's t-test. (Sihver et al., 1999c).

di€erence between mRNA levels in entorhinal cortex, hippocampus and thalamus of AD and age matched controls was measured (Terzano et al., 1998). Furthermore, the mRNA levels of a4, a5, a7, b2, and b4 are signi®cantly higher in prenatal cortex and cerebellum compared to aged brain (HellstroÈm-Lindahl et al., 1998). A signi®cant correlation between reduced ‰3 HŠ epibatidine sites and reduced levels of the presynaptic marker, synaptophysin in the frontal cortex of AD brains has also been identi®ed, suggesting that the majority of nAChRs lost are presynaptic. Interestingly, in the same study reductions in ChAT activity were observed but did not correlate with the reductions in ‰3 HŠ epibatidine binding measured, suggesting that the nAChRs lost in AD are not exclusive to cholinergic neurons (Sabbagh et al., 1998). This view is supported by a recent study where basal forebrain lesions with the selective cholinergic immunotoxin 192 IgG saporin produced signi®cant reductions in ChAT activity in the parietal cortex of rats but had no e€ect on nAChR numbers as measured by ‰3 HŠ epibatidine and ‰3 HŠ cystisine (Bednar et al., 1998). 6.2.1. Alzheimer's disease therapy Cholinergic transmitter replacement therapy forms the mainstay of AD treatment and is based on the theory that low levels of ACh are responsible for the cognitive decline associated with the disease. Classically, replacement therapy has involved the use of cholin-

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esterase inhibitors such as tacrine, donepezil and rivastigmine, which prevent the breakdown of ACh released from cholinergic neurons, thereby increasing the concentration of transmitter available to interact with receptors. These drugs have moderate palliative e€ects on symptoms as well as having some ability to slow disease progression (Amberla et al., 1993; Maltby et al., 1994; Nordberg et al., 1998; Nordberg and Svensson, 1998). Although, only moderately e€ective cholinesterase therapy currently constitutes the best treatment available for AD (Nordberg and Svensson, 1998). It is likely that the therapeutic bene®t of cholinesterase inhibitor treatment occurs at least in part, through activation of neuronal nAChRs Ð by direct action of the increased levels of ACh on these receptors and through allosteric activation of the receptors by the drugs (e.g. tacrine, galanthamine) themselves (Maelicke et al., 1995; Svensson and Nordberg, 1996). Neuronal nAChR activation is therefore currently being investigated as a strategy for AD therapy (SjoÈberg et al., 1998). The potential therapeutic bene®t in AD from nAChR stimulation is based on three main observations. Firstly, stimulation of nAChRs by nicotine improves mnemonic function. In animal experiments, nicotine treatment has been observed to improve performance in memory related tasks (Levin, 1992), furthermore, b2 knockout mice show abnormal behaviour in avoidance learning indicating the involvement of nAChRs (Picciotto et al., 1995). In humans, the nicotinic antagonist mecamylamine produces impairments in short term memory (Newhouse et al., 1992), while nicotine improves performance of human subjects in memory related tasks (Colrain et al., 1992; Rusted and Warburton, 1992; Rusted et al., 1994). Nicotine has also been observed to produce similar cognitive improvements in AD patients (Jones et al., 1992; Valenzuela et al., 1994; Vidal, 1996; Newhouse et al., 1997). Secondly, nAChR activation modulates the release of a number of neurotransmitters such as ACh, DA, GABA and glutamate, and will enhance the release of ACh (Beani et al., 1985; McGehee and Role, 1995; Pontieri et al., 1996; Marshall et al., 1997). Thirdly, there is evidence to suggest that nAChR activation provides protection against b-amyloid neurotoxicity. Nicotine protects cultured cortical neurons against b-amyloid induced neuronal death, an e€ect blocked by the a4b2 selective antagonist DHbE. Furthermore, cytisine, an a4b2 selective agonist, also inhibits b-amyloid toxicity indicating that the a4b2 nAChR is important in neuroprotection (Kihara et al., 1998). These observations are consistent with the loss of a4b2 nAChRs in AD, an occurrence which may thus potentiate the neurotoxic action of b-amyloid. Interestingly however, there is evidence to suggest that stimulation of a7 nAChRs may also be neuroprotective. Nicotine and cholinesterase inhibitors have been

observed to attenuate b-amyloid toxicity, an action proposed to occur through a7 activation (Kihara et al., 1997; Zamani et al., 1997; Svensson and Nordberg, 1998). Nicotinic receptor activation would therefore appear to be a promising strategy for treatment of AD. The adverse side e€ects produced by the nonspeci®c action of nicotine render it unsuitable as a therapy for AD. There is however, tremendous scope for the development of a4b2 and a7 selective agonists as potential therapeutic agents for AD. 6.3. Parkinson's disease Parkinson's disease (PD) is a progressive neurodegenerative condition involving the dopaminergic neurons of the substantial nigra. It is characterised by diculty in initiating and smoothly sustaining movement. In a manner similar to AD there is a loss of cholinergic cells in the basal forebrain accompanied by a signi®cant reduction in the number of high anity nicotine binding sites in the brain (Whitehouse et al., 1983; Aubert et al., 1992; Lange et al., 1993). In addition to motor dysfunction, PD patients often have accompanying cognitive impairments or dementia with a greater loss of cholinergic markers and nAChRs in demented patients than non-demented patients (Perry et al., 1995). The reduction in cortical nAChR number in PD patients parallels the degree of dementia observed with progression of the disease (Whitehouse et al., 1988a; Aubert et al., 1992) and as with AD may result from degeneration of cholinergic projection neurons in the basal forebrain. The most potent environmental factor a€ecting susceptibility to PD is tobacco use, with smokers having a lower than expected incidence of PD (Morens et al., 1995). Although there are many constituents of tobacco smoke other than nicotine, it appears that it is the best candidate as the protective agent. Chronic nicotine treatment in rodents protects against mechanical and neurotoxin induced nigrostriatal lesions, preventing DA neuronal degeneration, increasing DA levels and counteracting DA D2 receptor upregulation (Janson et al., 1989, 1994; Fuxe et al., 1990; Janson and Moller, 1993). In 1-methyl-4-phenyl-1., 2,3,6-terahydropyridine (MPTP) lesioned mice nicotine is partially neuroprotective, with acute administration of nicotine prior to or combined with MPTP treatment reducing DA neuronal degeneration in the neostriatum and substantial nigra (Janson et al., 1988, 1992). However, chronic nicotine administration via minipums enhances MPTP neurotoxicity in the striatum (Janson et al., 1992). The protective e€ect of nicotine is thought to be related to reduced MPP+ (neurotoxic metabolite of MPTP) uptake into DA neurons via nicotine induced increases in DA release. Conversely, enhanced MPTP neurotoxicity is thought to result from failure of nAChRs to

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desensitize following chronic nicotine administration, leading to chronic increased Na+/Ca2+ ion in¯ux via nAChRs located on DA neurons, with associated Ca+ ion toxicity and increased energy demands (Janson et al., 1992). However, nicotine has been observed to counteract the locomotor e€ects MPTP in animal models of PD (Sershen et al., 1987; Maggio et al., 1998). In addition the novel a4b2, nAChR selective agonist SIB-1508Y ((S)-(-)-5-ethynyl-3-(1-methyl-2-pyrrodinyl)-pyridine) has been observed to improve cognitive and motor performance in monkeys in the same MPTP model of PD (Cosford et al., 1996; Schneider et al., 1998a, 1998b). When administered alone, SIB1508Y (1 mg/kg) did not signi®cantly improve cognitive or motor function, but when combined with levodopa signi®cant improvements were observed in both cognitive and motor task performance and at doses of levodopa between one third and one sixth of that necessary to improve motor performance alone (Schneider et al., 1998b). 6.4. Schizophrenia Schizophrenia is a chronically deteriorating heterogeneous psychosis beginning in late adolescence or early adulthood and is characterized by hallucinations, delusions, bizzare behaviour, apathy and blunted a€ect (Arnold and Trojanowski, 1996; Tsuang, 1993). The etiology of this condition or group of conditions is unclear but studies indicate that schizophrenia has a strong genetic component, although the inheritance pattern appears to be complex involving an uncertain mode of transmission, incomplete penetrance and probable genetic heterogeneity (Risch, 1990; Tsuang, 1993). Possible loci for schizophrenia have been identi®ed at a number of chromosomal sites (Pulver et al., 1994; Wang et al., 1995; Silverman et al., 1996). However, these loci do not account for all cases of schizophrenia and do not delineate which aspects of this multifactorial illness might be in¯uenced by a speci®c locus. A dopamine hypothesis for schizophrenia has been proposed, suggesting that the symptomology results from an excess of dopamine, although similar symptoms can be produced by administration of drugs like phencyclidine (PCP) an NMDA and nAChR channel blocker. The possible involvement of nAChRs in schizophrenia was suggested by the high percentage of smokers present in the schizophrenic population compared to the general population, 90% compared to 33% (Lohr and Flynn, 1992). Furthermore, the number of ‰3 HŠ cytisine and ‰125 IŠ BTX binding sites in CA3 region of the hippocampus in postmortem schizophrenic brains was signi®cantly reduced compared to control brains, indicating a de®cit in nAChR number in schizophrenia (Freedman et al., 1995). Schizophrenic patients have also been

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observed to have high levels of nAChR antibodies, which may be a contributing factor in the reduced number of nAChRs observed in schizophrenia (Mukherjee et al., 1994). From these observations it was postulated that the high incidence of smoking in schizophrenics is an attempt on their part to self-medicate nicotine to overcome a de®cit in nicotinic neurotransmission. In this regard, nicotine has been observed to normalize two psychophysiological de®cits in schizophrenic patients (Adler et al., 1992). De®cits in the regulation of response to sensory stimuli are likely to be a major feature underlying the overt symptoms of schizophrenia, such as hallucinations and delusions. Attention to apparently extraneous stimuli in their surroundings, which are generally ignored by normal subjects, is characteristic of schizophrenics and suggests that neuronal mechanisms responsible for ®ltering or gating of sensory input are impaired. Increased sensitivity to auditory stimuli in schizophrenics and their relatives involves diminished gating of P50 brain waves upon repeated auditory stimulation. In normal subjects, response to an initial auditory stimuli elicits an excitatory response that also activates inhibitory mechanisms, which then diminsh the excitatory response to subsequent auditory stimuli. The ability of schizophrenics and their relatives to diminish the excitatory response following the second of paired auditory stimuli is reduced compared to normal subjects but is transiently normalized by nicotine administration or following smoking (Adler et al., 1985, 1992, 1993). The inheritance of this neuronal defect has been linked to a dinucleotide polymorphism at chromosome 15 which is also the locus for the a7 nAChR (Chini et al., 1994; Freedman et al., 1997). The a7 nAChR is further implicated in schizophrenia by the observation that the protein level of this subunit is signi®cantly reduced in the frontal cortex of schizophrenic brain compared to age matched controls (Guan et al., 1999). These observations suggest that a7 nAChRs may be extremely important in schizophrenia. 6.5. Tourette's syndrome Gilles de la Tourette syndrome (TS) is a neuropsychiatric disorder of unknown etiology which starts in childhood and is characterized by persistent motor and verbal tics as well as by the frequent occurrence of hyperactivity, anxieties, phobias or obsessive compulsive disorders. Classical neuroleptics such as haloperidol are used to treat TS but are not always e€ective and produce side e€ects such as sedation and the possible development of tardive dyskinesia. A number of studies have reported that administration of nicotine by means of gum or transdermal patches potentiate the action of neuroleptics and is e€ective in ameliorating the symptoms of TS (Sanberg et al., 1988;

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McConville et al., 1992; Dursun and Reveley, 1997; Sanberg et al., 1997). Up to 4 weeks of bene®t have been reported in TS patients treated for only 2 days with 10 mg nicotine patches (Dursun and Reveley, 1997). The mechanism by which the bene®cial e€ects of nicotine are produced has yet to be elucidated but may possibly involve modulation of dopamine release. Prolonged exposure to nicotine produces reversible desensitization and eventually permanent inactivation of nAChRs, especially a4b2 and a7 subtypes (Hsu et al., 1996; Olale et al., 1997). Permanent inactivation of these receptors followed by a slow rate of resynthesis might account for the weeks of bene®t displayed following nicotine administration. The development of nicotinic agonists without the side e€ect pro®le of nicotine would therefore appear to be desirable for the treatment of TS. As yet, there is no direct evidence of nAChR involvement in the condition but the positive e€ects of nicotine suggests that they may play a role, further investigation into the role of nAChRs in this condition is therefore necessary. 6.6. Anxiety and depression nAChRs are apparently involved in the pathophysiology of both anxiety disorders and depression. Nicotine administration has been observed to have anxiolytic e€ects in humans and in animal models of anxiety (Pomerleau, 1986; Decker et al., 1994). This action can be blocked by administration of the noncompetitive nAChR antagonist mecamylamine and by the benzodiazepine inverse agonist ¯umazenil. These observations suggest that the anxiolytic action of nicotine is produced by enhanced release of the inhibitory neurotransmitter GABA, which then acts on central benzodiazepine±GABAA receptor complex. Both retrospective and prospective clinical studies have demonstrated a relationship between smoking and major depression; persons with major depression are more likely to smoke, to have greater diculty in stopping, and are at increased risk of su€ering mild to severe depression having succeeded in stopping (Glassman et al., 1988, 1990; Breslau et al., 1993; Dalack et al., 1995; Stage et al., 1996; Covey et al., 1997, 1998). This results from shared predispositions involving genetic or environmental factors, although separate causal mechanisms may exist including self medication of depressed mood and neuropharmacologic e€ects of nicotine and other smoke substances on neurotransmitters linked to depression (Breslau, 1995; Breslau et al., 1998). Depression increases the likelihood of smoking, as well as nicotine and other dependencies. Transdermal nicotine patches improve the mood of non-smoking depressed patients and increase the duration of REM sleep (Salin-Pascual et al., 1996; SalinPascual and Drucker-Colin, 1998). Furthermore, nic-

otine has been observed to act as an antidepressant in animal models of depression (Semba et al., 1998). There is also a considerable body of evidence linking the action of classical tricyclic antidepressants (eg. imipramine, desipramine) and the newer serotonin uptake inhibitors (e.g. ¯uoxteine, paroxetine) to nAChRs. The tricyclic antidepressants imipramine, desipramine, amytriptyline and nortriptyline, all produce a non-competitive inhibition of nAChRs with a reversible inhibition of agonist induced currents (Scho®eld et al., 1981; Arita et al., 1987; Rana et al., 1993). The serotonin uptake inhibitors ¯uoxetine, paroxetine, sertaline, venlafaxine and nefazodone also produce a similar reversible non-competitive inhibition of nAChRs (Fairclough et al., 1993; Dalack et al., 1995; Garcia-Colunga et al., 1997; Hennings et al., 1997; Maggio et al., 1998; Fryer and Lukas, 1999). Fluoxetine (Prozac) has been most extensively studied, blocking nAChR currents in a voltage dependent manner, while also increasing the rate of desensitization of the receptor (Garcia-Colunga et al., 1997; Maggio et al., 1998). Furthermore, in rat hippocampal slices, ¯uoxetine inhibits nicotine induced release of noradrenaline in a dose dependent manner (Hennings et al., 1997). Despite the well characterised connection between depression, antidepressants and nicotine the role of nAChRs as targets for antidepressant treatment remains under appreciated. 7. Conclusions A considerable amount is now known about nAChRs in the human brain. A number of structural and functional subtypes have been identi®ed with individual pharmacological pro®les and distinct patterns of distribution. To date, nine individual receptor subunits have been identi®ed and cloned in human brain which combine in various conformations to form individual receptor subtypes. The distribution nAChRs has been mapped using various radioligands possessing a modest degree of subtype selectivity and the distribution of individual subunit mRNA has also been mapped to a limited degree with in situ hybridisation and other techniques. Nicotinic receptors apparently play a pivotal role in a number of functional processes including learning and memory and are implicated in several CNS disorders including Alzheimer's disease, Parkinson's disease, schizophrenia and epilepsy, as well as mediating the addiction to nicotine presented in chronic tobacco users. However, a great deal of information still remains to be elucidated concerning human neuronal nAChRs. The structure of individual receptors present in human brain and their subunit composition remains to be fully investigated. The pattern of distribution in human brain of the subtypes thus far identi®ed is far from comprehensive and

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would be aided by the development of subtype selective radioligands. In this vein, subtype selective radioligands would have important application for PET and SPECT and would extend the potential of these techniques as diagnostic and mapping tools for nAChRs in the living human. Loss of nAChRs might be an early presymptomatic diagnostic marker for AD. Additionally, the expression pattern of each of the individual nAChR subunits is incomplete. The functional roles of nAChRs and subunits in human brain has also yet to be fully elucidated, with roles for a5 and a6 subunits beginning to emerge. Similarly, the exact role of nAChRs in various pathologies remains unclear as in Alzheimer's disease where it is debatable whether the loss of nAChRs observed is symptomatic or causative. The apparent bene®t of nAChR stimulation in a number of these conditions identi®es neuronal nAChRs as potential therapeutic targets. Identi®cation of the speci®c receptor subtypes involved in each of the conditions is therefore desirable as is the development of subtype selective nAChR agonists which would potentially provide selective therapies for individual conditions. A considerable amount of research is therefore necessary to fully elucidate the structure, function, physiological and pathological involvement and therapeutic potential of nAChRs, which remain an exciting research prospect. Acknowledgements This study was supported by grants from the Swedish Medical Research Council (project number 05817), Loo and Hans Ostermans Foundation, Stiftelsen foÈr Gamla TjaÈnarinnor, Stohnes Foundation, and KI Foundations. References Abreo, M.A., Lin, N.H., Garvey, D.S., Gunn, D.E., Hettinger, A.M., Wasicak, J.T., Pavlik, P.A., Martin, Y.C., Donnellyroberts, D.L., Anderson, D.J., Sullivan, J.P., Williams, M., Arneric, S.P., Holladay, M.W., 1996. Novel 3-Pyridyl ethers with subnanomolar anity for central neuronal nicotinic acetylcholine receptors. J. Med. Chem 39, 817±825. Adem, A., Synnergren, B., Botros, M., Ohman, B., Winblad, B., Nordberg, A., 1987. [3H] acetylcholine nicotinic recognition sites in human brain: characterization of agonist binding. Neurosci. Lett. 83, 298±302. Adem, A., Jossan, S.S., d'Argy, R., Brandt, I., Winblad, B., Nordberg, A., 1988. Distribution of nicotinic receptors in human thalamus as visualized by 3H-nicotine and 3H-acetylcholine receptor autoradiography. J. Neural. Transm 73, 77±83. Adem, A., Nordberg, A., Jossan, S.S., Sara, V., Gillberg, P.G., 1989. Quantitative autoradiography of nicotinic receptors in large cryosections of human brain hemispheres. Neurosci. Lett 101, 247± 252. Adler, L.E., Ho€er, L.D., Wiser, A., Freedman, R., 1993.

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