Nigrostriatal damage preferentially decreases a subpopulation of alpha6beta2* nAChRs in mouse, monkey, and Parkinson's disease striatum

Parkinson's disease is a neurodegenerative movement disorder characterized by a loss of substantia nigra dopamine neurons, and corresponding declines in molecular components present on striatal dopaminergic nerve terminals. These include the alpha6beta2(*) nicotinic acetylcholine receptors (nAChRs), which are localized exclusively on dopamine terminals in striatum ((*)denotes the presence of possible additional subunits). In this study, we used a novel alpha-conotoxin MII (alpha-CtxMII) analog E11A to further investigate alpha6beta2(*) nAChR subtypes in mouse, monkey, and human striatum. Receptor competition studies with (125)I-alpha-CtxMII showed that E11A inhibition curves were biphasic, suggesting the presence of two distinct alpha6beta2(*) nAChR subtypes. These include a very high (femtomolar) and a high (picomolar) affinity site, with approximately 40% of the sites in the very high affinity form. It is noteworthy that only the high-affinity form was detected in alpha4 nAChR-null mutant mice. Because (125)I-alpha-CtxMII binds primarily to alpha6alpha4beta2beta3 and alpha6beta2beta3 nAChR subtypes in mouse striatum, these data suggest that the population lost in the alpha4 knockout mice was the alpha6alpha4beta2beta3 subtype. We next investigated the effect of nigrostriatal lesioning on these two striatal alpha6beta2(*) populations in two animal models and in Parkinson's disease. There was a preferential loss of the very high affinity subtype in striatum of mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), monkeys treated with MPTP, and patients with Parkinson's disease. These data suggest that dopaminergic terminals expressing the alpha6alpha4beta2beta3 population are selectively vulnerable to nigrostriatal damage. This latter nAChR subtype, identified with alpha-CtxMII E11A, may therefore provide a unique marker for dopaminergic terminals particularly sensitive to nigrostriatal degeneration in Parkinson's disease.     

Decrease in alpha3*/alpha6* nicotinic receptors but not nicotine-evoked dopamine release in monkey brain after nigrostriatal damage

Nicotinic acetylcholine receptors (nAChRs) are decreased in the striata of patients with Parkinson's disease (PD) or in experimental models after nigrostriatal damage. Because presynaptic nAChRs on striatal dopamine terminals mediate dopamine release, receptor loss may contribute to behavioral deficits in PD. The present experiments were done to determine whether nAChR function is affected by nigrostriatal damage in nonhuman primates, because this model shares many features with PD. Initial characterization of nicotine-evoked [3H]dopamine release from monkey striatal synaptosomes revealed that release was calcium-dependent and inhibited by selective nAChR antagonists. It is noteworthy that a greater proportion (approximately 70%) of release was inhibited by the alpha3*/alpha6* antagonist alpha-conotoxinMII (alpha-CtxMII) compared with rodents. Monkeys were lesioned with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and [3H]dopamine release, dopamine transporter, and nAChRs were measured. As anticipated, lesioning decreased the transporter and alpha3*/alpha6* nAChRs in caudate and putamen. In contrast, alpha3*/alpha6* nAChR-evoked [3H]dopamine release was reduced in caudate but not putamen, demonstrating a dissociation between nAChR sites and function. A different pattern was observed in the mesolimbic dopamine system. Dopamine transporter levels in nucleus accumbens were not reduced after MPTP, as expected; however, there was a 50% decline in alpha3*/alpha6* nAChR sites with no decrease in alpha3*/alpha6* receptor-evoked dopamine release. No declines in alpha-CtxMII-resistant nAChR (alpha4*) binding or nicotine-evoked release were observed in any region. These results show a selective preservation of alpha3*/alpha6* nAChR-mediated function in the nigrostriatal and mesolimbic dopamine systems after nigrostriatal damage. Maintenance of function in putamen, a region with a selective loss of dopaminergic terminals, may be important in PD.     

Pharmacology of alpha-conotoxin MII-sensitive subtypes of nicotinic acetylcholine receptors isolated by breeding of null mutant mice

Subtypes of nicotinic acetylcholine receptors (nAChR) containing alpha6 subunits comprise 25 to 30% of the presynaptic nAChRs expressed in striatal dopaminergic terminals in rodents and 70% in monkeys. This class of receptors, potentially important in nicotine addiction, binds alpha-conotoxin MII (alpha-CtxMII) with high affinity and is heterogeneous, consisting of several subtypes in mice, possibly an important consideration for the design of compounds that selectively activate or antagonize the alpha6 subclass of nAChRs. Selected-null mutant mice were bred to generate isolated subtypes of alpha6beta2* nAChRs expressed in vivo for assessing pharmacology of alpha6beta2* nAChRs. Binding to striatal membranes and function in synaptosomes from (alpha4-/-)(beta3+/+) and (alpha4-/-)(beta3-/-) mice were measured and compared with wild-type (alpha4+/+)(beta3+/+) mice. Gene deletions (alpha4 and beta3) decreased binding of (125)I-alpha-CtxMII without affecting affinity for alpha-CtxMII or inhibition of alpha-CtxMII binding by epibatidine or nicotine. Deletion of the alpha4 subunit substantially increased EC(50) values for both nicotine- and cytisine-stimulated alpha-CtxMII-sensitive dopamine release from striatal synaptosomes. A further increase in EC(50) values was seen upon the additional deletion of the beta3 subunit. The data indicate that one alpha-CtxMII-sensitive nAChR subtype, prevalent on wild-type dopaminergic terminals, has the lowest EC(50) for a nicotine-mediated function so far measured in mice. In conclusion, the gene deletion strategy enabled isolation of alpha6* subtypes, and these nAChR subtypes exhibited differential activation by nicotine and cytisine.     

Expression of nigrostriatal alpha 6-containing nicotinic acetylcholine receptors is selectively reduced, but not eliminated, by beta 3 subunit gene deletion

mRNAs for the neuronal nicotinic acetylcholine receptor (nAChR) alpha6 and beta3 subunits are abundantly expressed and colocalized in dopaminergic cells of the substantia nigra and ventral tegmental area. Studies using subunit-null mutant mice have shown that alpha6- or beta3-dependent nAChRs bind alpha-conotoxin MII (alpha-CtxMII) with high affinity and modulate striatal dopamine release. This study explores the effects of beta3 subunit-null mutation on striatal and midbrain nAChR expression, composition, and pharmacology. Ligand binding and immunoprecipitation experiments using subunit-specific antibodies indicated that beta3-null mutation selectively reduced striatal alpha6* nAChR expression by 76% versus beta3(+/+) control. Parallel experiments showed a smaller reduction in both midbrain alpha3* and alpha6* nAChRs (34 and 42% versus beta3(+/+) control, respectively). Sedimentation coefficient determinations indicated that residual alpha6* nAChRs in beta3(-/-) striatum were pentameric, like their wild-type counterparts. Immunoprecipitation experiments on immunopurified beta3* nAChRs demonstrated that almost all wild-type striatal beta3* nAChRs also contain alpha4, alpha6, and beta2 subunits, although a small population of non-beta3 alpha6* nAChRs is also expressed. beta3 subunit incorporation seemed to increase alpha4 participation in alpha6beta2* complexes. (125)I-Epibatidine competition binding studies showed that the alpha-CtxMII affinity of alpha6* nAChRs from the striata of beta3(-/-) mice was similar to those isolated from beta3(+/+) animals. Together, the results of these experiments show that the beta3 subunit is important for the correct assembly, stability and/or transport of alpha6* nAChRs in dopaminergic neurons and influences their subunit composition. However, beta3 subunit expression is not essential for the expression of alpha6*, high-affinity alpha-CtxMII binding nAChRs.     

Sazetidine-A is a potent and selective agonist at native and recombinant alpha 4 beta 2 nicotinic acetylcholine receptors

Sazetidine-A has been recently proposed to be a "silent desensitizer" of alpha4beta2 nicotinic acetylcholine receptors (nAChRs), implying that it desensitizes alpha4beta2 nAChRs without first activating them. This unusual pharmacological property of sazetidine-A makes it, potentially, an excellent research tool to distinguish between the role of activation and desensitization of alpha4beta2 nAChRs in mediating the central nervous system effects of nicotine itself, as well as those of new nicotinic drugs. We were surprised to find that sazetidine-A potently and efficaciously stimulated nAChR-mediated dopamine release from rat striatal slices, which is mediated by alpha4beta2(*) and alpha6beta2(*) subtypes of nAChR. The agonist effects on native striatal nAChRs prompted us to re-examine the effects of sazetidine-A on recombinant alpha4beta2 nAChRs in more detail. We expressed the two alternative stoichiometries of alpha4beta2 nAChR in Xenopus laevis oocytes and investigated the agonist properties of sazetidine-A on both alpha4(2)beta2(3) and alpha4(3)beta2(2) nAChRs. We found that sazetidine-A potently activated both stoichiometries of alpha4beta2 nAChR: it was a full agonist on alpha4(2)beta2(3) nAChRs, whereas it had an efficacy of only 6% on alpha4(3)beta2(2) nAChRs. In contrast to what has been published before, we therefore conclude that sazetidine-A is an agonist of native and recombinant alpha4beta2 nAChRs but shows differential efficacy on alpha4beta2 nAChRs subtypes.     

Long-term nicotine treatment decreases striatal alpha 6* nicotinic acetylcholine receptor sites and function in mice

Alpha-conotoxin MII-sensitive nicotinic acetylcholine receptors (nAChRs) are distinct from other subtypes in their relatively restricted localization to the striatum and some other brain regions. The effect of nicotine treatment on nAChR subtypes has been extensively investigated, with the exception of changes in alpha-conotoxin MII-sensitive receptor expression. We therefore determined the consequence of long-term nicotine administration on this subtype and its function. Nicotine was given in drinking water to provide a long-term yet intermittent treatment. Consistent with previous studies, nicotine exposure increased 125I-epibatidine and 125I-A85380 (3-[2-(S)-azetidinylmethoxy]pyridine), but not 125I-alpha-bungarotoxin, receptors in cortex and striatum. We observed an unexpected reduction (30%) in striatal 125I-alpha-conotoxin MII sites, which occurred because of a decrease in B(max). This decline was more robust in older (>8-month-old) compared with younger (2-4-month-old) mice, suggesting age is important for nicotine-induced disruption of nAChR phenotype. Immunoprecipitation experiments using nAChR subunit-directed antibodies indicate that alterations in subunit-immunoreactivity with nicotine treatment agree with those in the receptor binding studies. To determine the relationship between striatal nAChR sites and function, we measured nicotine-evoked [3H]dopamine release. A decline was obtained with nicotine treatment that was caused by a selective decrease in alpha-conotoxin MII-sensitive but not alpha-conotoxin MII-resistant dopamine release. These results may explain previous findings that nicotine treatment decreased striatal nAChR-mediated dopamine function, despite an increase in [3H]nicotine (alpha4*) sites. The present data suggest that the alpha6* nAChR subtype represents a key factor in the control of dopamine release from striatum, which adapts to long-term nicotine treatment by down-regulation of alpha6* receptor sites and function.     

Heterogeneity and selective targeting of neuronal nicotinic acetylcholine receptor (nAChR) subtypes expressed on retinal afferents of the superior colliculus and lateral geniculate nucleus: identification of a new native nAChR subtype alpha3beta2(alpha5 or beta3) enriched in retinocollicular afferents

The activation of neuronal nicotinic acetylcholine receptors (nAChRs) has been implicated in the activity-dependent development and plasticity of retina and the refinement of retinal projections. Pharmacological and functional studies have also indicated that different presynaptic nAChRs can have a modulatory function in retinotectal synapses. We biochemically and pharmacologically identified the multiple nAChR subtypes expressed on retinal afferents of the superior colliculus (SC) and lateral geniculate nucleus (LGN). We found that the alpha6beta2(*) and alpha4(nonalpha6)beta2(*) nAChRs are the major receptor populations expressed in both SC and LGN. In addition, the LGN contains two minor populations of alpha2alpha6beta2(*) and alpha3beta2(*) subtypes, whereas the SC contains a relatively large population of a new native subtype, the alpha3beta2(alpha5/beta3) nAChR. This subtype binds the alpha-conotoxin MII with an affinity 50 times lower than that of the native alpha6beta2(*) subtype. Studies of tissues obtained from eye-enucleated animals allowed the identification of nAChRs expressed by retinal afferents: in SC alpha6beta2(*), alpha4alpha6beta2(*), and alpha3beta2(*) (approximately 45, 35, and 20%, respectively), in LGN, alpha4alpha6beta2(*), alpha6beta2(*), alpha4beta2(*), alpha2alpha6beta2(*), and alpha3beta2(*) (approximately 40, 30, 20, 5, and 5%, respectively). In both regions, more than 50% of nAChRs were not expressed by retinal afferents and belonged to the alpha4beta2(*) (90%) or alpha4alpha5beta2(*) (10%) subtypes. Moreover, studies of the SC tissues obtained from wild-type and alpha4, alpha6, and beta3 knockout mice confirmed and extended the data obtained in rat tissue and allowed a comprehensive dissection of the composition of nAChR subtypes present in this retinorecipient area.     

Subunit composition and pharmacology of two classes of striatal presynaptic nicotinic acetylcholine receptors mediating dopamine release in mice

Pharmacological evaluation of nicotine-stimulated dopamine release from striatum has yielded data consistent with activation of a single population of nicotinic acetylcholine receptors (nAChR). However, discovery that alpha-conotoxin MII (alpha-CtxMII) partially inhibits the response indicates that two classes of presynaptic nAChRs mediate dopamine release. We have investigated the pharmacology and subunit composition of these two classes of nAChR. Inhibition of nicotine-stimulated dopamine release from mouse striatal synaptosomes by alpha-CtxMII occurs within minutes; recovery is slow. The IC50 is 1 to 3 nM. alpha-CtxMII-sensitive and -resistant components have significant differences in pharmacology. The five agonists tested were more potent at activating the alpha-CtxMII-sensitive nAChRs; indeed, this receptor is the highest affinity functional nAChR found, so far, in mouse brain. In addition, cytisine was more efficacious at the alpha-CtxMII-sensitive sites. Methyllycaconitine was 9-fold more potent at inhibiting the alpha-CtxMII-sensitive sites, whereas dihydro-beta-erythroidine was a 7-fold more potent inhibitor of the alpha-CtxMII-resistant response. Both the transient and persistent phases of nicotine-stimulated dopamine release were partially inhibited by alpha-CtxMII with equal potency. The subunit composition of functional nAChRs, was assessed in mice with null mutations for individual nAChR subunits. The beta2 subunit is an absolute requirement for both classes. In contrast, deletion of beta4 or alpha7 subunits had no effect. The alpha-CtxMII-sensitive response requires beta3 and is partially dependent upon alpha4 subunits, probably alpha6beta3beta2 and alpha4alpha6beta3beta2, whereas the alpha-CtxMII-resistant release requires alpha4 and is partially dependent upon alpha5 subunits, probably alpha4beta2 and alpha4alpha5beta2.     

The subtypes of nicotinic acetylcholine receptors on dopaminergic terminals of mouse striatum

This review summarizes studies that attempted to determine the subtypes of nicotinic acetylcholine receptors (nAChR) expressed in the dopaminergic nerve terminals in the mouse. A variety of experimental approaches has been necessary to reach current knowledge of these subtypes, including in situ hybridization, agonist and antagonist binding, function measured by neurotransmitter release from synaptosomal preparations, and immunoprecipitation by selective antibodies. Early developments that facilitated this effort include the radioactive labeling of selective binding agents, such as [(125)I]-alpha-bungarotoxin and [(3)H]-nicotine, advances in cloning the subunits, and expression and evaluation of function of combinations of subunits in Xenopus oocytes. The discovery of epibatidine and alpha-conotoxin MII (alpha-CtxMII), and the development of nAChR subunit null mutant mice have been invaluable in determining which nAChR subunits are important for expression and function in mice, as well as allowing validation of the specificity of subunit specific antibodies. These approaches have identified five nAChR subtypes of nAChR that are expressed on dopaminergic nerve terminals. Three of these contain the alpha6 subunit (alpha4alpha6beta2beta3, alpha6beta2beta3, alpha6beta2) and bind alpha-CtxMII with high affinity. One of these three subtypes (alpha4alpha6beta2beta3) also has the highest sensitivity to nicotine of any native nAChR that has been studied, to date. The two subtypes that do not have high affinity for alpha-CtxMII (alpha4beta2, alpha4alpha5beta2) are somewhat more numerous than the alpha6* subtypes, but do bind nicotine with high affinity. Given that our first studies detected readily measured differences in sensitivity to agonists and antagonists among these five nAChR subtypes, it seems likely that subtype selective compounds could be developed that would allow therapeutic manipulation of diverse nAChRs that have been implicated in a number of human conditions.     

Rationale, pharmacology and clinical efficacy of partial agonists of alpha4beta2 nACh receptors for smoking cessation

Most smokers repeatedly fail in their attempts to stop smoking because of the addictive nature of the nicotine in tobacco products. Nicotine dependence is probably mediated through the activation of multiple subtypes of neuronal nicotinic acetylcholine receptor (nAChR), among which the mesolimbic alpha(4)beta(2) subtype has a pivotal role. Here, we discuss the rationale for and the design of alpha(4)beta(2) nAChR partial agonists as novel treatments for tobacco addiction. Such agents are expected to exhibit a dual action by sufficiently stimulating alpha(4)beta(2)-nAChR-mediated dopamine release to reduce craving when quitting and by inhibiting nicotine reinforcement when smoking. Potent and selective alpha(4)beta(2) nAChR partial agonists that exhibit dual agonist and antagonist activity in preclinical models can be identified. The validity of this approach is demonstrated by the clinical efficacy of the alpha(4)beta(2) nAChR partial agonist varenicline, which has significantly better quit rates than do other treatments and offers a new option for smoking cessation pharmacotherapy.     

Alpha6-containing nicotinic acetylcholine receptors dominate the nicotine control of dopamine neurotransmission in nucleus accumbens.

Modulation of striatal dopamine (DA) neurotransmission plays a fundamental role in the reinforcing and ultimately addictive effects of nicotine. Nicotine, by desensitizing beta2 subunit-containing (beta2*) nicotinic acetylcholine receptors (nAChRs) on striatal DA axons, significantly enhances how DA is released by reward-related burst activity compared to nonreward-related tonic activity. This action provides a synaptic mechanism for nicotine to facilitate the DA-dependent reinforcement. The subfamily of beta2*-nAChRs responsible for these potent synaptic effects could offer a molecular target for therapeutic strategies in nicotine addiction. We explored the role of alpha6beta2*-nAChRs in the nucleus accumbens (NAc) and caudate-putamen (CPu) by observing action potential-dependent DA release from synapses in real-time using fast-scan cyclic voltammetry at carbon-fiber microelectrodes in mouse striatal slices. The alpha6-specific antagonist alpha-conotoxin-MII suppressed DA release evoked by single and low-frequency action potentials and concurrently enhanced release by high-frequency bursts in a manner similar to the beta2*-selective antagonist dihydro-beta-erythroidine (DHbetaE) in NAc, but less so in CPu. The greater role for alpha6*-nAChRs in NAc was not due to any confounding regional difference in ACh tone since elevated ACh levels (after the acetylcholinesterase inhibitor ambenonium) had similar outcomes in NAc and CPu. Rather, there appear to be underlying differences in nAChR subtype function in NAc and CPu. In summary, we reveal that alpha6beta2*-nAChRs dominate the effects of nicotine on DA release in NAc, whereas in CPu their role is minor alongside other beta2*-nAChRs (eg alpha4*), These data offer new insights to suggest striatal alpha6*-nAChRs as a molecular target for a therapeutic strategy for nicotine addiction.     

Excitation of rat striatal large neurons by dopamine and/or glutamate released from nerve terminals via presynaptic nicotinic receptor (A4beta2 type) stimulation

Previous in vivo experiments using rats anesthetized with chloral hydrate have revealed that nicotine applied iontophoretically increased firing of striatal neurons receiving excitatory dopaminergic input from the substantia nigra, and nicotine-induced firing was inhibited by domperidone, a dopamine D2 antagonist. The results suggest that nicotine increases release of dopamine from the terminals of dopaminergic neurons. Therefore, we performed the present patch clamp study using slice and acutely dissociated preparations of the rat striatum to elucidate the mechanisms underlying the nicotine-induced excitation of striatal neurons. Application of nicotine (100 microM) to large striatal neurons in slice preparations did not produce any effect on the resting membrane potential, but did increase the frequency of miniature postsynaptic potentials (mpps) and action potentials in all 15 neurons tested. The nicotine-induced increase in mpps and action potentials were inhibited during simultaneous application of domperidone; L-glutamic acid diethyl ester hydrochloride, a non-selective glutamate receptor antagonist; and/or dihydro-beta-erythroidine, a central nicotinic acetylcholine receptor (alpha4beta2 type) antagonist. Postsynaptic current was not induced by nicotine applied by U-tube in 96% of acutely dissociated striatal neurons. The present findings suggest that nicotine mainly acts on the presynaptic nicotinic receptors in the nerve terminals to release neurotransmitters such as dopamine and/or glutamate, thereby activating the striatal large neurons.     

Presynaptic alpha 7- and beta 2-containing nicotinic acetylcholine receptors modulate excitatory amino acid release from rat prefrontal cortex nerve terminals via distinct cellular mechanisms

Nicotine can enhance working memory and attention. Activation of both alpha7 and beta2(*) nicotinic acetylcholine receptors (nAChRs) in the prefrontal cortex (PFC) has been implicated in these processes. The ability of presynaptic nAChRs to modulate neurotransmitter release, notably glutamate release, is postulated to contribute to nicotine's effects. We have examined the cellular mechanisms underlying alpha7 and beta2(*) nAChR-mediated [(3)H]d-aspartate release from the PFC in vitro. Using the alpha7 and beta2(*) nAChR-selective agonists (R)-N-(1-azabicyclo[2.2.2]-oct-3-yl)(5-(2-pyridyl)thiophene-2-carboxamide) (compound A) and 5-iodo-3-(2(S)-azetidinylmethoxy)pyridine (5-iodo-A-85380), respectively, in conjunction with inhibitors of voltage-operated Ca(2+) channels (VOCCs) and intracellular Ca(2+) stores, we show that [(3)H]d-aspartate release evoked by activation of beta2(*) nAChRs occurs via VOCCs. In contrast, alpha7 nAChR-evoked release was unaffected by VOCC blockers but was abolished by modulators of Ca(2+) stores, including ryanodine. The alpha7 nAChR ligand alpha-bungarotoxin and ryanodine receptors were colocalized to a subpopulation of PFC synaptosomes. Compound A-evoked [(3)H]d-aspartate release was also blocked by mitogen-activated protein kinase kinase 1 inhibitors, implicating extracellular signal-regulated kinase (ERK)1/2 in alpha7 nAChR-evoked exocytosis. Western blotting confirmed that compound A, but not 5-iodo-A-85380, application increased ERK2 phosphorylation in PFC synaptosomes, and this was dependent on ryanodine-sensitive stores. Compound A also promoted synapsin-1 phosphorylation at ERK1/2-dependent sites, in a ryanodine-sensitive manner. Thus, beta2(*) and alpha7 nAChR subtypes in the PFC mediate [(3)H]d-aspartate release via distinct mechanisms as a result of their differential coupling to VOCCs and Ca(2+)-induced Ca(2+) release (CICR), respectively. The ability of alpha7 nAChRs to promote the phosphorylation of presynaptic ERK2 and synapsin-1, downstream of CICR, provides a potential mechanism for presynaptic facilitation in the PFC.     

Pharmacological profile of the alpha4beta2 nicotinic acetylcholine receptor partial agonist varenicline, an effective smoking cessation aid

The preclinical pharmacology of the alpha4beta2 nicotinic acetylcholine receptor (nAChR) partial agonist varenicline, a novel smoking cessation agent is described. Varenicline binds with subnanomolar affinity only to alpha4beta2 nAChRs and in vitro functional patch clamp studies in HEK cells expressing nAChRs show that varenicline is a partial agonist with 45% of nicotine's maximal efficacy at alpha4beta2 nAChRs. In neurochemical models varenicline has significantly lower (40-60%) efficacy than nicotine in stimulating [(3)H]-dopamine release from rat brain slices in vitro and in increasing dopamine release from rat nucleus accumbens in vivo, while it is more potent than nicotine. In addition, when combined with nicotine, varenicline effectively attenuates the nicotine-induced dopamine release to the level of the effect of varenicline alone, consistent with partial agonism. Finally, varenicline reduces nicotine self-administration in rats and supports lower self-administration break points than nicotine. These data suggest that varenicline can reproduce to some extent the subjective effects of smoking by partially activating alpha4beta2 nAChRs, while preventing full activation of these receptors by nicotine. Based on these findings, varenicline was advanced into clinical development and recently shown to be an effective and safe aid for smoking cessation treatment.     

Subunit composition of nicotinic receptors in monkey striatum: effect of treatments with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or L-DOPA

Nicotinic acetylcholine receptors (nAChRs) represent an important modulator of striatal function both under normal conditions and in pathological states such as Parkinson's disease. Because different nAChR subtypes may have unique functions, immunoprecipitation and ligand binding studies were done to identify their subunit composition. As in the rodent, alpha2, alpha4, alpha6, beta2, and beta3 nAChR subunit immunoreactivity was identified in monkey striatum. However, distinct from the rodent, the present results also revealed the novel presence of alpha3 nAChR subunit-immunoreactivity in this same region, but not that for alpha5 and beta4. Relatively high levels of alpha2 and alpha3 subunits were also identified in monkey cortex, in addition to alpha4 and beta2. Experiments were next done to determine whether striatal subunit expression was changed with nigrostriatal damage. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment decreased alpha6 and beta3 subunit immunoreactivity by approximately 80% in parallel with the dopamine transporter, suggesting that they are predominantly expressed on nigrostriatal dopaminergic projections. In contrast, alpha3, alpha4, and beta2 subunit immunoreactivity was decreased approximately 50%, whereas alpha2 was not changed. These data, together with those from dual immunoprecipitation and radioligand binding studies ([(3)H]cytisine, (125)I-alpha-bungarotoxin, and (125)I-alpha-conotoxin MII) suggest the following: that alpha6beta2beta3, alpha6alpha4beta2beta3, and alpha3beta2* nAChR subtypes are present on dopaminergic terminals and that the alpha4beta2 subtype is localized on both dopaminergic and nondopaminergic neurons, whereas alpha2beta2* and alpha7 receptors are localized on nondopaminergic cells in monkey striatum. Overall, these results suggest that drugs targeting non-alpha7 nicotinic receptors may be useful in the treatment of disorders characterized by nigrostriatal dopaminergic damage, such as Parkinson's disease.     

L-DOPA treatment modulates nicotinic receptors in monkey striatum

Nicotinic acetylcholine receptor (nAChR) activation is well known to stimulate dopamine release in the striatum. This phenomenon may be physiologically significant in the control of motor function, as well as in pathological conditions such as Parkinson's disease. An understanding of the mechanisms that influence nAChR expression and function is therefore important. Because the dopamine precursor l-DOPA is the most commonly used therapeutic agent for Parkinson's disease, we investigated the effects of l-DOPA treatment on striatal nAChR expression in unlesioned and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned monkeys. In unlesioned animals, l-DOPA (15 mg/kg) administered twice daily for 2 weeks decreased both 125I-epibatidine and [125I]iodo-3-[2(S)-azetidinylmethoxy]pyridine (A-85380) binding sites in the caudate and putamen, but did not affect 125I-alpha-CtxMII sites. alpha-CtxMII inhibition of striatal 125I-epibatidine and [125I]A-85380 binding with alpha-CtxMII suggest that there are both high- (Ki < 0.2 nM) and low-affinity (Ki > 100 nM) alpha-CtxMII-sensitive sites, as well as alpha-CtxMII-resistant sites, and that l-DOPA treatment influences only the low-affinity alpha-CtxMII-sensitive subtype. The l-DOPA effect was selective for striatal nAChRs with no change in cortical sites. Monkeys with severe nigrostriatal damage did not exhibit l-DOPA-induced declines in striatal nAChRs, suggesting that l-DOPA primarily affects nAChRs associated with dopaminergic terminals. In summary, these data show that l-DOPA treatment decreases nAChR expression, in contrast with the well established up-regulation of these sites by chronic nicotine exposure. Furthermore, they demonstrate preferential l-DOPA regulation of a novel low-affinity alpha-CtxMII-sensitive site. These declines in nAChRs with l-DOPA may be relevant to both the therapeutic and side effect profiles of l-DOPA therapy in Parkinson's disease.     

Dose-related neuroprotective effects of chronic nicotine in 6-hydroxydopamine treated rats, and loss of neuroprotection in alpha4 nicotinic receptor subunit knockout mice

The present study examined the effect of a range of doses of chronic nicotine (0.75, 1.5, 3.0 and 30.0 mg kg(-1) day(-1), s.c., 14 days) upon striatal dopaminergic nerve terminal survival following 6-hydroxydopamine (6-OHDA; 10 microg intrastriatal unilaterally) in rats; and the effects of acute nicotine (1 mg kg(-1), s.c.) pretreatment upon striatal neurodegeneration induced by methamphetamine (5 mg kg(-1), i.p., three doses at 2 h intervals) in wild-type and alpha4 nicotinic receptor (nAChR) subunit knockout mice. In both models of Parkinsonian-like damage, loss of striatal dopaminergic nerve terminals was assessed by [(3)H]-mazindol autoradiography. In rats, chronic nicotine infusion delivered by osmotic minipump implanted subcutaneously 7 days prior to intrastriatal 6-OHDA injection produced significant and dose-related protection against 6-OHDA-induced neurodegeneration. Low (0.75 and 1.5 mg kg(-1) day(-1)) but not high (3.0 and 30.0 mg kg(-1) day(-1)) nicotine doses significantly inhibited 6-OHDA-induced degeneration. In wild-type mice, acute nicotine treatment produced significant inhibition of methamphetamine-induced neurodegeneration. In alpha4 nAChR subunit knockout mice, acute nicotine treatment failed to inhibit methamphetamine-induced neurodegeneration. Nicotine is capable of protecting dopaminergic neurons against Parkinsonian-like neurodegeneration in vivo. In rats, this neuroprotective effect is critically dependent upon nicotine dose and is consistent with the activation of nAChRs, as high, desensitizing doses of nicotine fail to be neuroprotective. Further, neuroprotection is absent in alpha4 nAChR subunit knockout mice. The current results therefore suggest that activation of alpha4 subunit containing nAChRs constitutes a major component of the neuroprotective effect of nicotine upon Parkinsonian-like damage in vivo.     

Chronic pre-treatment with nicotine enhances nicotine-evoked striatal dopamine release and alpha6 and beta3 nicotinic acetylcholine receptor subunit mRNA in the substantia nigra pars compacta of the rat

Whilst local intrastriatal infusion of nicotine consistently elicits striatal dopamine release, systemic administration often fails to do so. Since chronic nicotine administration is known to result in desensitisation-induced upregulation of nicotinic acetylcholine receptors (nAChRs), the present study investigated whether chronic pre-treatment could enhance the response to systemic nicotine and, if so, whether increases in specific nAChR subunit mRNA levels in the substantia nigra pars compacta (SNc) may underlie this effect. In vivo microdialysis studies in male Sprague-Dawley rats revealed that following 4 days pre-treatment with nicotine (0.8 mg kg(-1)s.c.), local intrastriatal nicotine infusion (3 mM) elicited significantly higher dopamine efflux compared to vehicle pre-treated controls (peak release: 1273 +/- 199% basal versus 731 +/- 113% basal), whereas systemic nicotine challenge (0.8 mg kg(-1)s.c.) elicited no response. In contrast, following 8 days pre-treatment with nicotine (0.8 mg kg(-1)s.c.), systemic nicotine challenge (0.8 mg kg(-1)s.c.) now produced significantly higher dopamine efflux than that of vehicle pre-treated controls (147 +/- 30% basal versus 91 +/- 5% basal). Eight days pre-treatment with nicotine also significantly elevated the levels of alpha6 (approximately 55%) and beta3 (approximately 43%) nAChR subunit mRNA in the SNc, suggesting that up-regulation of these nAChR subunit genes in the nigrostriatal tract may contribute to the enhanced nicotine-evoked striatal dopamine release.     

Nicotine, but neither the alpha4beta2 ligand RJR2403 nor an alpha7 nAChR subtype selective agonist, protects against a partial 6-hydroxydopamine lesion of the rat median forebrain bundle

Although previous studies suggest nicotine protects against a 6-hydroxydopamine (6-OHDA)-induced lesion of the nigrostriatal tract in rats, it is not known whether functional motor recovery occurs or which nicotinic acetylcholine receptor (nAChR) subtypes mediate this effect. These issues were investigated by comparing the effects of the subtype-specific nAChR agonists, RJR2403 (alpha4beta2 preferring) and (R)-N-(1-azabicyclo[2.2.2.]oct-3-yl)(5-(2-pyridyl)thiopene-2-carboxamide (Compound A; alpha7-selective) and nicotine given 30 min prior to and daily for 14 days after a partial 6-OHDA lesion. In vehicle treated animals, 6-OHDA (6 microg) produced a 65 +/- 1.8% loss of striatal tyrosine hydroxylase (TH) immunoreactivity in the lesion versus intact hemisphere. This loss was reduced in animals treated with nicotine (0.6 and 0.8 mg kg(-1)), reaching significance at the higher dose (36.6 +/- 3.7% loss; P < 0.01 versus vehicle). Treatment with nicotine (0.6 and 0.8 mg kg(-1)) also significantly reduced the number of amphetamine-induced rotations compared to vehicle treatment. In contrast, treatment with RJR2403 (0.2 and 0.4 mg kg(-1)) or Compound A (10 and 20 mg kg(-1)) reduced neither the degree of amphetamine-induced rotations nor the loss of striatal TH immunoreactivity. These data suggest that whilst nicotine is neuroprotective in this partial lesion model, activation of neither the alpha4beta2 nor alpha7 subtypes alone is sufficient to provide protection.     

Varenicline: an alpha4beta2 nicotinic receptor partial agonist for smoking cessation

Herein we describe a novel series of compounds from which varenicline (1, 6,7,8,9-tetrahydro-6,10-methano-6H-pyrazino[2,3-h][3]benzazepine) has been identified for smoking cessation. Neuronal nicotinic acetylcholine receptors (nAChRs) mediate the dependence-producing effects of nicotine. We have pursued alpha4beta2 nicotinic receptor partial agonists to inhibit dopaminergic activation produced by smoking while simultaneously providing relief from the craving and withdrawal syndrome that accompanies cessation attempts. Varenicline displays high alpha4beta2 nAChR affinity and the desired in vivo dopaminergic profile.