Cotinine selectively activates a subpopulation of alpha3/alpha6beta2 nicotinic receptors in monkey striatum

The nicotine metabolite cotinine is an abundant long-lived bio-active compound that may contribute to the overall physiological effects of tobacco use. Although its mechanism of action in the central nervous system has not been extensively investigated, cotinine is known to evoke dopamine release in the nigrostriatal pathway through an interaction at nicotinic receptors (nAChRs). Because considerable evidence now demonstrates the presence of multiple nAChRs in the striatum, the present experiments were done to determine the subtypes through which cotinine exerts its effects in monkeys, a species that expresses similar densities of striatal alpha4beta2* (nAChR containing the alpha4 and beta2 subunits, but not alpha3 or alpha6) and alpha3/alpha6beta2* (nAChR composed of the alpha3 or alpha6 subunits and beta2) nAChRs. Competition binding studies showed that cotinine interacts with both alpha4beta2* and alpha3/alpha6beta2* nAChR subtypes in the caudate, with cotinine IC(50) values for inhibition of 5-[(125) I]iodo-3-[2(S)-azetinylmethoxy]pyridine-2HCl ([(125)I]A-85380) and (125)I-alpha-conotoxinMII binding in the micromolar range. This interaction at the receptor level is of functional significance because cotinine stimulated both alpha4beta2* and alpha3/alpha6beta2* nAChR [(3)H]dopamine release from caudate synaptosomes. Our results unexpectedly showed that nicotine evokes [(3)H]dopamine release from two alpha3/alpha6beta2* nAChR populations, one of which was sensitive to cotinine and the other was not. This cotinine-insensitive subtype was only present in the medial caudate and was preferentially lost with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced nigrostriatal damage. In contrast, cotinine and nicotine elicited equivalent levels of alpha4beta2* nAChR-mediated dopamine release. These data demonstrate that cotinine functionally discriminates between two alpha3/alpha6beta2* nAChRs in monkey striatum, with the cotinine-insensitive alpha3/alpha6beta2* nAChR preferentially vulnerable to nigrostriatal damage.     

Differential regulation of mesolimbic alpha 3/alpha 6 beta 2 and alpha 4 beta 2 nicotinic acetylcholine receptor sites and function after long-term oral nicotine to monkeys

Because the mesolimbic dopamine system plays a critical role in nicotine addiction/reinforcement and because nicotinic receptors regulate dopamine release, we initiated a study to evaluate the long-term effects of nicotine (>6 months at the final dose) on nicotinic acetylcholine receptor (nAChR) sites and function in the nucleus accumbens of nonhuman primates. Nicotine was given in the drinking water as this mode of administration is long-term but intermittent, thus resembling smoking in this aspect. We determined the effects of nicotine treatment on function and binding of the alpha3/alpha6beta2* and alpha4beta2* nAChRs subtypes in nucleus accumbens, a region directly implicated in the addictive effects of nicotine. To evaluate function, we measured nicotine and K+-evoked [3H]dopamine release from nucleus accumbens synaptosomes. Changes in alpha4beta2* and alpha3/alpha6beta2* nAChRs were measured using 125I-epibatidine, [125I]A85380 [5-[125I]iodo-3(2(S)-azetidinylmethoxy) pyridine] and 125I-alpha-conotoxin MII autoradiography. Chronic nicotine treatment, which led to plasma nicotine levels in the range of smokers, significantly increased nucleus accumbens alpha4beta2* nAChR sites and function compared with control. By contrast, this treatment did not significantly change alpha3/alpha6beta2* nAChR sites or evoked dopamine release in this region compared with control. Thus, these data are distinct from previous results in striatum in which the same nicotine treatment paradigm decreased striatal alpha3/alpha6beta2* nAChR sites and function. The finding that long-term nicotine treatment selectively modulates alpha4beta2* and not alpha3/alpha6beta2* nAChR expression in primate nucleus accumbens is consistent with the results of studies in nicotinic receptor mutant mice implicating the alpha4beta2* nAChR subtype in nicotine-mediated addiction.     

Declines in different beta2* nicotinic receptor populations in monkey striatum after nigrostriatal damage

In the present study we used the nicotinic ligand 5-iodo-A-85380 [5-iodo-3(2(S)-azetidinylmethoxy)pyridine], which selectively binds to beta2-containing nicotinic acetylcholine receptors, to elucidate the nicotinic receptor subtypes affected by nigrostriatal damage in the monkey. Autoradiographic studies in control monkeys showed that 5-[(125)I]A-85380 ([(125)I]A-85380) binds throughout the brain with the characteristics of a nicotinic receptor ligand. Competition experiments with cytisine and nicotine yielded K(i) values of approximately 1 and 10 nM, respectively, with complete inhibition of [(125)I]A-85380 binding at a 10(-6) M concentration of these ligands. In contrast, alpha-conotoxin MII blocked radioligand binding in the striatum by 30% at the highest concentrations, suggesting that a subset of striatal [(125)I]A-85380 sites are alpha-conotoxin MII-sensitive. Monkeys treated with the nigrostriatal neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine showed a selective decrease in striatal [(125)I]A-85380 sites, with a 42% reduction in the caudate and putamen of animals with moderate nigrostriatal lesioning and a 53% decline in the striatum of severely lesioned animals. Our previous work had demonstrated that there were two populations of nicotinic receptors eliminated after nigrostriatal damage, an alpha-conotoxin MII-sensitive and an alpha-conotoxin MII- resistant subtype. Analysis of both striatal [(125)I]A-85380 and [(125)I]epibatidine competition studies extend our earlier studies by demonstrating that the alpha-conotoxin MII-sensitive sites eliminated after moderate nigrostriatal lesioning appear to be composed of two nicotinic receptor subtypes. The data may be important for potential therapeutic approaches because they suggest that there are at least three populations of nicotinic receptors in monkey striatum, of which two are selectively vulnerable to nigrostriatal damage, while the third is more resistant.     

Chronic nicotine differentially regulates alpha6- and beta3-containing nicotinic cholinergic receptors in rat brain

We investigated the effects of chronic nicotine on alpha6- and beta3-containing nicotinic acetylcholine receptors (nAChRs) in two rat brain regions using three methodological approaches: radioligand binding, immunoprecipitation, and nicotine-stimulated synaptosomal release of dopamine. Nicotine was administered by osmotic minipumps for 2 weeks. Quantitative autoradiography with [(125)I]alpha-conotoxin MII to selectively label alpha6(*) nAChRs showed a 28% decrease in binding in the striatum but no change in the superior colliculus. Immunoprecipitation of nAChRs labeled by [(3)H]epibatidine in these two regions showed that chronic nicotine increased alpha4- and beta2-containing nAChRs by 39 to 67%. In contrast, chronic nicotine caused a 39% decrease in alpha6-containing nAChRs in striatum but no change in superior colliculus. No changes in beta3-containing nAChRs were seen in either region after chronic nicotine. The decreased expression of alpha6-containing nAChRs persisted for at least 3 days, recovering to baseline by 7 days after removal of the pumps. There was a small but significant decrease in total nicotine-stimulated dopamine release in striatal synaptosomes after nicotine exposure. However, the component of dopamine release that was resistant to alpha-conotoxin MII blockade was unaffected, whereas dopamine release that was sensitive to blockade by alpha-conotoxin MII was decreased by 56%. These findings indicate that the alpha6(*) nAChR is regulated differently from other nAChR subtypes, and they suggest that the inclusion of a beta3 subunit with alpha6 may serve to inhibit nicotine-induced down-regulation of these receptors.     

Increased nicotinic acetylcholine receptor protein underlies chronic nicotine-induced up-regulation of nicotinic agonist binding sites in mouse brain

Chronic nicotine treatment elicits a brain region-selective increase in the number of high-affinity agonist binding sites, a phenomenon termed up-regulation. Nicotine-induced up-regulation of α4β2-nicotinic acetylcholine receptors (nAChRs) in cell cultures results from increased assembly and/or decreased degradation of nAChRs, leading to increased nAChR protein levels. To evaluate whether the increased binding in mouse brain results from an increase in nAChR subunit proteins, C57BL/6 mice were treated with nicotine by chronic intravenous infusion. Tissue sections were prepared, and binding of [(125)I]3-((2S)-azetidinylmethoxy)-5-iodo-pyridine (A85380) to β2*-nAChR sites, [(125)I]monoclonal antibody (mAb) 299 to α4 nAChR subunits, and [(125)I]mAb 270 to β2 nAChR subunits was determined by quantitative autoradiography. Chronic nicotine treatment dose-dependently increased binding of all three ligands. In regions that express α4β2-nAChR almost exclusively, binding of all three ligands increased coordinately. However, in brain regions containing significant β2*-nAChR without α4 subunits, relatively less increase in mAb 270 binding to β2 subunits was observed. Signal intensity measured with the mAbs was lower than that with [(125)I]A85380, perhaps because the small ligand penetrated deeply into the sections, whereas the much larger mAbs encountered permeability barriers. Immunoprecipitation of [(125)I]epibatidine binding sites with mAb 270 in select regions of nicotine-treated mice was nearly quantitative, although somewhat less so with mAb 299, confirming that the mAbs effectively recognize their targets. The patterns of change measured using immunoprecipitation were comparable with those determined autoradiographically. Thus, increases in α4β2*-nAChR binding sites after chronic nicotine treatment reflect increased nAChR protein.     

Varenicline is a potent partial agonist at α6β2* nicotinic acetylcholine receptors in rat and monkey striatum

Extensive evidence indicates that varenicline reduces nicotine craving and withdrawal symptoms by modulating dopaminergic function at α4β2* nicotinic acetylcholine receptors (nAChRs) (the asterisk indicates the possible presence of other nicotinic subunits in the receptor complex). More recent data suggest that α6β2* nAChRs also regulate dopamine release and mediate nicotine reinforcement. The present experiments were therefore done to test the effect of varenicline on α6β2* nAChRs and their function, because its interaction with this subtype is currently unclear. Receptor competition studies showed that varenicline inhibited α6β2* nAChR binding (K(i) = 0.12 nM) as potently as α4β2* nAChR binding (K(i) = 0.14 nM) in rat striatal sections and with ～20-fold greater affinity than nicotine. Functionally, varenicline was more potent in stimulating α6β2* versus α4β2* nAChR-mediated [(3)H]dopamine release from rat striatal synaptosomes with EC(50) values of 0.007 and 0.086 μM, respectively. However, it acted as a partial agonist on α6β2* and α4β2* nAChR-mediated [(3)H]dopamine release with maximal efficacies of 49 and 24%, respectively, compared with nicotine. We also evaluated varenicline's action in striatum of monkeys, a useful animal model for comparison with humans. Varenicline again potently inhibited monkey striatal α6β2* (K(i) = 0.13 nM) and α4β2* (K(i) = 0.19 nM) nAChRs in competition studies. Functionally, it potently stimulated both α6β2* (EC(50) = 0.014 μM) and α4β2* (EC(50) = 0.029 μM) nAChR-mediated [(3)H]dopamine release from monkey striatal synaptosomes, again acting as a partial agonist relative to nicotine at both subtypes. These data suggest that the ability of varenicline to interact at α6β2* nAChRs may contribute to its efficacy as a smoking cessation aid.     

Quantification of alpha4beta2* nicotinic receptors in the rat brain with microPET and 2-[18F]F-A-85380

The radioligand 2-[(18)F]F-A-85380 has been used for PET studies of the alpha4beta2* subtype of nicotinic acetylcholine receptors (nAChRs) in the living brain of humans and nonhuman primates. In order to extend the capacity of microPET to quantify neuroreceptors in rat brain, we carried out studies of 2-[(18)F]F-A-85380 to measure the apparent binding potential BP* in individual rats, which were studied repeatedly over several months. Using a bolus-plus-infusion paradigm, 2-[(18)F]F-A-85380 (specific activity 20-1300 GBq/micromol) was administered intravenously over 8 to 9 h with K(bol) values of 350 to 440 min and a mean infusion rate of 0.03+/-0.01 nmol/kg/h. Studies included a 2-h nicotine infusion initiated 2 h before the end of scanning to displace specifically bound radioactivity. Steady state binding in brain was obtained within 5 h as defined by the occurrence of constant radioactivity concentrations in brain regions and constant, free arterial plasma levels of nonmetabolized radioligand. BP* averages (+/-SEM) for thalamus, forebrain, and cerebellum were 5.9+/-0.7, 2.6+/-0.4, and 1.0+/-0.1, respectively, which are consistent with the alpha4beta2* nAChR distribution in rat brain measured in vitro. Studies of receptor occupancy determined the ED(50) to be 0.29 nmol/kg/h. The demonstration that alpha4beta2* nAChRs are quantifiable in the rat brain using PET measurements, coupled with the ability to conduct longitudinal studies over several months in the same rats, suggests potential applications to studies of chronic nicotine use, its treatment, and abnormal functioning of alpha4beta2* receptors in a rat model.     

Paraquat exposure reduces nicotinic receptor-evoked dopamine release in monkey striatum

Paraquat, an herbicide widely used in the agricultural industry, has been associated with lung, liver, and kidney toxicity in humans. In addition, it is linked to an increased risk of Parkinson's disease. For this reason, we had previously investigated the effects of paraquat in mice and showed that it influenced striatal nicotinic receptor (nAChR) expression but not nAChR-mediated dopaminergic function. Because nonhuman primates are evolutionarily closer to humans and may better model the effects of pesticide exposure in man, we examined the effects of paraquat on striatal nAChR function and expression in monkeys. Monkeys were administered saline or paraquat once weekly for 6 weeks, after which nAChR levels and receptor-evoked [(3)H]dopamine ([(3)H]DA) release were measured in the striatum. The functional studies showed that paraquat exposure attenuated dopamine (DA) release evoked by alpha3/alpha6beta2(*) (nAChR that is composed of the alpha3 or alpha6 subunits, and beta2; the asterisk indicates the possible presence of additional subunits) nAChRs, a subtype present only on striatal dopaminergic terminals, with no decline in release mediated by alpha4beta2(*) (nAChR containing alpha4 and beta2 subunits, but not alpha3 or alpha6) nAChRs, present on both DA terminals and striatal neurons. Paraquat treatment decreased alpha4beta2(*) but not alpha3/alpha6beta2(*) nAChR expression. The differential effects of paraquat on nAChR expression and receptor-evoked [(3)H]DA release emphasize the importance of evaluating changes in functional measures. The finding that paraquat treatment has a negative impact on striatal nAChR-mediated dopaminergic activity in monkeys but not mice indicates the need for determining the effects of pesticides in higher species.     

Methyllycaconitine is a potent antagonist of alpha-conotoxin-MII-sensitive presynaptic nicotinic acetylcholine receptors in rat striatum

The plant alkaloid methyllycaconitine (MLA) is considered to be a selective antagonist of the alpha7 subtype of neuronal nicotinic acetylcholine receptor (nAChR). However, 50 nM MLA partially inhibited (by 16%) [(3)H]dopamine release from rat striatal synaptosomes stimulated with 10 microM nicotine. Other alpha7-selective antagonists had no effect. Similarly, MLA (50 nM) inhibited [(3)H]dopamine release evoked by the partial agonist (2-chloro-5-pyridyl)-9-azabicyclo[4.2.1]non-2-ene (UB-165) (0.2 microM) by 37%. In both cases, inhibition by MLA was surmountable with higher agonist concentrations, indicative of a competitive interaction. At least two subtypes of presynaptic nAChR can modulate dopamine release in the striatum, and these nAChR are distinguished by their differential sensitivity to alpha-conotoxin-MII (alpha-CTx-MII). MLA was not additive with a maximally effective concentration of alpha-CTx-MII (100 nM) in inhibiting [(3)H]dopamine release elicited by 10 microM nicotine or 0.2 microM UB-165, suggesting that both toxins act at the same site. This was confirmed in quantitative binding assays with (125)I-alpha-CTx-MII, which displayed saturable specific binding to rat striatum and nucleus accumbens with B(max) values of 9.8 and 16.5 fmol/mg of protein, and K(d) values of 0.63 and 0.83 nM, respectively. MLA fully inhibited (125)I-alpha-CTx-MII binding to striatum and nucleus accumbens with a K(i) value of 33 nM, consistent with the potency observed in the functional assays. We speculate that MLA and alpha-CTx-MII interact with a presynaptic nAChR of subunit composition alpha3/alpha6beta2beta3* on dopamine neurons. The use of MLA as an alpha7-selective antagonist should be exercised with caution, especially in studies of nAChR in basal ganglia.     

Effect of novel alpha-conotoxins on nicotine-stimulated [3H]dopamine release from rat striatal synaptosomes

Nicotine's action on the midbrain dopaminergic neurons is mediated by nicotinic acetylcholine receptors (nAChRs) that are present on the cell bodies and the terminals of these neurons. Previously, it was suggested that one of the nAChR subtypes located on striatal dopaminergic terminals may be an alpha3beta2 subtype, based on partial inhibition of nicotine-stimulated [(3)H]dopamine release by alpha-conotoxin MII, a potent inhibitor of heterologously expressed alpha3beta2 nAChRs. More recent studies indicated that alpha-conotoxin MII also potently blocks alpha6-containing nAChRs. In the present study, we have examined the nAChR subtype(s) modulating [(3)H]dopamine release from striatal terminals by using novel alpha-conotoxins that have 37- to 78-fold higher selectivity for alpha6-versus alpha3-containing nAChRs. All of the peptides partially (20-35%) inhibit nicotine-stimulated [(3)H]dopamine release with IC(50) values consistent with those obtained with heterologously expressed rat alpha6-containing nicotinic acetylcholine receptors. These results, together with previous studies by others, further support the idea that alpha6-containing nicotinic receptors modulate nicotine-stimulated dopamine release from rat striatal synaptosomes.     

N-n-alkylnicotinium analogs,a novel class of nicotinic receptor antagonists: interaction with alpha4beta2* and alpha7* neuronal nicotinic receptors

The current study demonstrates that N-n-alkylnicotinium analogs with increasing n-alkyl chain lengths from 1 to 12 carbons have varying affinity (Ki = 90 nM-20 microM) for S-(-)-[3H]nicotine binding sites in rat striatal membranes. A linear relationship was observed such that increasing n-alkyl chain length provided increased affinity for the alpha4beta2* nicotinic acetylcholine receptor (nAChR) subtype, with the exception of N-n-octylnicotinium iodide (NONI). The most potent analog was N-n-decylnicotinium iodide (NDNI; Ki = 90 nM). In contrast, none of the analogs in this series exhibited high affinity for the [3H]methyllycaconitine binding site, thus indicating low affinity for the alpha7* nAChR. The C8 analog, NONI, had low affinity for S-(-)-[3H]nicotine binding sites but was a potent inhibitor of S-(-)-nicotine-evoked [3H]dopamine (DA) overflow from superfused striatal slices (IC50 = 0.62 microM), thereby demonstrating selectivity for the nAChR subtype mediating S-(-)-nicotine-evoked [3H]DA overflow (alpha3alpha6beta2* nAChRs). Importantly, the N-n-alkylnicotinium analog with highest affinity for the alpha4beta2* subtype, NDNI, lacked the ability to inhibit S-(-)-nicotine-evoked [3H]DA overflow and, thus, appears to be selective for alpha4beta2* nAChRs. Furthermore, the present study demonstrates that the interaction of these analogs with the alpha4beta2* subtype is via a competitive mechanism. Thus, selectivity for the alpha4beta2* subtype combined with competitive interaction with the S-(-)-nicotine binding site indicates that NDNI is an excellent candidate for studying the structural topography of alpha4beta2* agonist recognition binding sites, for identifying the antagonist pharmacophore on the alpha4beta2* nAChR, and for defining the role of this subtype in physiological function and pathological disease states.     

Nicotine reduces L-DOPA-induced dyskinesias by acting at beta2* nicotinic receptors

L-DOPA-induced dyskinesias or abnormal involuntary movements (AIMs) are a debilitating adverse complication associated with prolonged L-DOPA administration for Parkinson's disease. Few treatments are currently available for dyskinesias. Our recent data showed that nicotine reduced L-DOPA-induced AIMs in parkinsonian animal models. An important question is the nicotinic acetylcholine receptor (nAChR) subtypes through which nicotine exerts this beneficial effect, because such knowledge would allow for the development of drugs that target the relevant receptor population(s). To address this, we used β2 nAChR subunit knockout [β2(-/-)] mice because β2-containing nAChRs are key regulators of nigrostriatal dopaminergic function. All of the mice were lesioned by intracranial injection of 6-hydroxydopamine into the right medial forebrain bundle. Lesioning resulted in a similar degree of nigrostriatal damage and parkinsonism in β2(-/-) and wild-type mice. All of the mice then were injected with L-DOPA (3 mg/kg) plus benserazide (15 mg/kg) once daily for 4 weeks until AIMs were fully developed. L-DOPA-induced AIMs were approximately 40% less in the β2(-/-) mice compared with the wild-type mice. It is interesting to note that nicotine (300 μg/ml in drinking water) reduced L-DOPA-induced AIMs by 40% in wild-type mice but had no effect in β2(-/-) mice with partial nigrostriatal damage. The nicotine-mediated decline in AIMs was much less pronounced in wild-type mice with near-complete degeneration, suggesting that presynaptic nAChRs on dopaminergic terminals have a major influence. These data demonstrate an essential role for β2* nAChRs in the antidyskinetic effect of nicotine and suggest that drugs targeting these subtypes may be useful for the management of L-DOPA-induced dyskinesias in Parkinson's disease.     

Modulation of inhibitory synaptic activity by a non-alpha4beta2, non-alpha7 subtype of nicotinic receptors in the substantia gelatinosa of adult rat spinal cord

The GABA/glycine-mediated inhibitory activity in the substantia gelatinosa (SG) of the spinal cord is critical in the control of nociceptive transmission. We examined whether and how SG inhibitory activity might be regulated by neuronal nicotinic receptors (nAChRs). Patch-clamp recordings were performed in SG neurons of spinal slice preparations from adult rats. We provided electrophysiological evidence that inhibitory presynaptic terminals in the SG expressed nAChRs and their activation resulted in large increases in the frequency of spontaneous and miniature inhibitory postsynaptic currents (sIPSCs and mIPSCs) in over 90% SG neurons tested. The enhancement of inhibitory activity was mediated by increases in the release of GABA/glycine, and direct Ca(2+) entry through SG presynaptic nAChRs appeared to be involved. Miniature IPSC frequency could be enhanced by the nAChR agonists nicotine or cytisine. Nicotine could still elicit large increases in mIPSC frequency in the presence of the alpha4beta2 nAChR antagonist dihydro-beta-erythroidine (5 microM) and the alpha7 nAChR-selective antagonist methyllycaconitine (40 nM). However, nicotine did not produce a significant enhancement of mIPSC frequency in the presence of the broad spectrum nAChR antagonist mecamylamine (5 microM). Nicotinic agonist-evoked whole-cell currents from SG neurons and the antagonist profiles also indicated the presence of a subtype of nAChRs, which were different from the major central nervous system nAChR subtypes, i.e. alpha4beta2* or alpha7 nAChRs. Together, our results suggest that a subtype of nAChR, possibly alpha3beta4* nAChR or a new nAChR type, is highly expressed at the inhibitory presynaptic terminals in SG of adult rats and play a role in the control of inhibitory activity in SG.     

Genetic approaches identify differential roles for alpha4beta2* nicotinic receptors in acute models of antinociception in mice

The effects of nicotine on the tail-flick and hot-plate tests were determined to identify nicotinic receptor subtypes responsible for spinally and supraspinally mediated nicotine analgesia in knockin mice expressing hypersensitive alpha(4) nicotinic receptors (L9'S), in seven inbred mouse strains (C57BL/6, DBA/2, A/2, CBA/2, BALB/cByJ, C3H/HeJ, and 129/SvEv), and in two F1 hybrids (B6CBAF1 and B6D2F1). L9'S heterozygotes were approximately 6-fold more sensitive to the antinociceptive effects of nicotine than the wild-type controls in the hot-plate test but not in the tail-flick assay. Large differences in the effects of nicotine were also observed with both tests for the seven mouse strains. A/J and 129 mice were 6- to 8-fold more sensitive than CBA and BALB mice. In addition, B6CBAF1 hybrid mice were even less sensitive than CBA mice. Nicotinic binding sites were measured in three spinal cord regions and the hindbrain of the inbred strains. Significant differences in cytisine-sensitive, high affinity [(125)I]epibatidine binding site levels (alpha(4)beta(2)(*) subtypes), but not in (125)I-alpha-bungarotoxin binding (alpha(7)(*) subtypes), were observed. Significant negative correlations between cytisine-sensitive [(125)I]epibatidine binding and nicotine ED(50) for both tests were noted. Our results indicate that alpha(4)beta(2)(*) acetylcholine nicotinic receptors (nAChR) are important in mediating nicotine analgesia in supraspinal responses, while also showing that alpha(4)beta(2)(*)-nAChR and at least one other nAChR subtype appear to modulate spinal actions.     

Bupropion inhibits nicotine-evoked [(3)H]overflow from rat striatal slices preloaded with [(3)H]dopamine and from rat hippocampal slices preloaded with [(3)H]norepinephrine

Bupropion, an efficacious antidepressant and smoking cessation agent, inhibits dopamine and norepinephrine transporters (DAT and NET, respectively). Recently, bupropion has been reported to noncompetitively inhibit alpha3beta2, alpha3beta4, and alpha4beta2 nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes or established cell lines. The present study evaluated bupropion-induced inhibition of native alpha3beta2* and alpha3beta4* nAChRs using functional neurotransmitter release assays, nicotine-evoked [(3)H]overflow from superfused rat striatal slices preloaded with [(3)H]dopamine ([(3)H]DA), and nicotine-evoked [(3)H]overflow from hippocampal slices preloaded with [(3)H]norepinephrine ([(3)H]NE). The mechanism of inhibition was evaluated using Schild analysis. To eliminate the interaction of bupropion with DAT or NET, nomifensine or desipramine, respectively, was included in the superfusion buffer. A high bupropion concentration (100 microM) elicited intrinsic activity in the [(3)H]DA release assay. However, none of the concentrations (1 nM-100 microM) examined evoked [(3)H]NE overflow and, thus, were without intrinsic activity in this assay. Moreover, bupropion inhibited both nicotine-evoked [(3)H]DA overflow (IC(50) = 1.27 microM) and nicotine-evoked [(3)H]NE overflow (IC(50) = 323 nM) at bupropion concentrations well below those eliciting intrinsic activity. Results from Schild analyses suggest that bupropion competitively inhibits nicotine-evoked [(3)H]DA overflow, whereas evidence for receptor reserve was obtained upon assessment of bupropion inhibition of nicotine-evoked [(3)H]NE overflow. Thus, bupropion acts as an antagonist at alpha3beta2* and alpha3beta4* nAChRs in rat striatum and hippocampus, respectively, across the same concentration range that inhibits DAT and NET function. The combination of nAChR and transporter inhibition produced by bupropion may contribute to its clinical efficacy as a smoking cessation agent.     

Subtype-selective up-regulation by chronic nicotine of high-affinity nicotinic receptors in rat brain demonstrated by receptor autoradiography

Subtypes of neuronal nicotinic acetylcholine receptors (nAChRs) are differentially sensitive to up-regulation by chronic nicotine exposure in vitro. To determine whether this occurs in animals, rats were implanted with minipumps containing saline +/- nicotine (6.0 mg/kg/rat/day) for 14 days. Autoradiography with [125I]epibatidine using 3-(2(S)-azetidinylmethoxy)pyridine dihydrochloride (A-85380) or cytisine as selective competitors allowed quantitative measurement in 33 regions of 3 families of nAChR binding, with properties of alpha4beta2, alpha3beta4, and alpha3/alpha6beta2. Chronic nicotine exposure caused increases of 20 to 100% for alpha4beta2-like binding in most regions surveyed. However, binding to this subtype was not increased in some regions, including habenulopeduncular structures, certain thalamic nuclei, and several brainstem regions. In 9 of 33 regions, including catecholaminergic areas and visual structures, alpha3/alpha6beta2-like binding represented >10% of total binding. Binding to this subtype was up-regulated by nicotine in only two of these nine regions: the nucleus accumbens and superior colliculus. alpha3beta4-Like binding represented >10% of total in 15 of the 33 regions surveyed. Binding to this subtype was increased by nicotine in only 1 of these 15 regions, and actually decreased in subiculum and cerebellum. These studies yielded two principal findings. First, chronic nicotine exposure selectively up-regulates alpha4beta2-like binding, with relatively little effect on alpha3/alpha6beta2-like and alpha3beta4-like binding in vivo. Second, up-regulation by chronic nicotine exposure shows considerable regional variation. Differential subtype sensitivity to chronic nicotine exposure may contribute to altered pharmacological response in individuals who smoke or use nicotine replacement therapy.     

Striatal alpha6* nicotinic acetylcholine receptors: potential targets for Parkinson's disease therapy

The presence of distinct nicotinic acetylcholine receptor (nAChR) subtypes in specific central nervous system (CNS) areas offers the possibility of developing targeted therapies for diseases involving the affected brain region. Parkinson's disease is a neurodegenerative movement disorder characterized by a progressive degeneration of the nigrostriatal system. alpha6-containing nAChRs (designated alpha6(*)1 nAChRs) have a relatively selective localization to the nigrostriatal pathway and a limited number of other CNS regions. In addition to a unique distribution, this subtype has a distinct pharmacology and specifically interacts with alpha-conotoxinMII, a toxin key in its identification and characterization. alpha6(*) nAChRs are also regulated in a novel manner, with a decrease in their number after nicotine treatment rather than the increase observed for alpha4(*) nAChRs. Striatal alpha6(*) receptors were functional and mediate dopamine release, suggesting that they have a presynaptic localization. This is further supported by lesion studies showing that both alpha6(*) nAChR sites and their functions are dramatically decreased with dopaminergic nerve terminal loss, in contrast to only small declines in alpha4(*) and no change in alpha7(*) receptors. Although the role of nigrostriatal alpha6(*) nAChRs is only beginning to be understood, an involvement in motor behavior is emerging. This latter observation coupled with the finding that nicotine protects against nigrostriatal damage suggest that alpha6(*) nAChRs may represent unique targets for neurodegenerative disorders linked to the nigrostriatal system such as Parkinson's disease.     

The novel alpha7 nicotinic acetylcholine receptor agonist N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-7-[2-(methoxy)phenyl]-1-benzofuran-2-carboxamide improves working and recognition memory in rodents

The relative contribution of alpha4beta2, alpha7 and other nicotinic acetylcholine receptor (nAChR) subtypes to the memory enhancing versus the addictive effects of nicotine is the subject of ongoing debate. In the present study, we characterized the pharmacological and behavioral properties of the alpha7 nAChR agonist N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-7-[2-(methoxy)phenyl]-1-benzofuran-2-carboxamide (ABBF). ABBF bound to alpha7 nAChR in rat brain membranes (Ki=62 nM) and to recombinant human 5-hydroxytryptamine (5-HT)3 receptors (Ki=60 nM). ABBF was a potent agonist at the recombinant rat and human alpha7 nAChR expressed in Xenopus oocytes, but it did not show agonist activity at other nAChR subtypes. ABBF acted as an antagonist of the 5-HT3 receptor and alpha3beta4, alpha4beta2, and muscle nAChRs (at higher concentrations). ABBF improved social recognition memory in rats (0.3-1 mg/kg p.o.). This improvement was blocked by intracerebroventricular administration of the alpha7 nAChR antagonist methyllycaconitine at 10 microg, indicating that it is mediated by alpha7 nAChR agonism. In addition, ABBF improved working memory of aged rats in a water maze repeated acquisition paradigm (1 mg/kg p.o.) and object recognition memory in mice (0.3-1 mg/kg p.o.). Rats trained to discriminate nicotine (0.4 mg/kg s.c.) from vehicle did not generalize to ABBF (0.3-30 mg/kg p.o.), suggesting that the nicotine cue is not mediated by the alpha7 nAChR and that selective alpha7 nAChR agonists may not share the abuse liability of nicotine. Our results support the hypothesis that alpha7 nAChR agonists may provide a novel therapeutic strategy for the treatment of cognitive deficits with low abuse potential.     

N-n-alkylpyridinium analogs,a novel class of nicotinic receptor antagonists: selective inhibition of nicotine-evoked [3H] dopamine overflow from superfused rat striatal slices

Structural simplification of N-n-alkylnicotinium analogs, antagonists at neuronal nicotinic acetylcholine receptors (nAChRs), was achieved by removal of the N-methylpyrrolidino moiety affording N-n-alkylpyridinium analogs with carbon chain lengths of C1 to C20. N-n-Alkylpyridinium analog inhibition of [3H]nicotine and [3H]methyllycaconitine binding to rat brain membranes assessed interaction with alpha4beta2* and alpha7* nAChRs, respectively, whereas inhibition of nicotine-evoked 3H overflow from [3H]dopamine ([3H]DA)-preloaded rat striatal slices assessed antagonist action at nAChR subtypes mediating nicotine-evoked DA release. No inhibition of [3H]methyllycaconitine binding was observed, although N-n-alkylpyridinium analogs had low affinity for [3H]nicotine binding sites, i.e., 1 to 3 orders of magnitude lower than that of the respective N-n-alkylnicotinium analogs. These results indicate that the N-methylpyrrolidino moiety in the N-n-alkylnicotinium analogs is a structural requirement for potent inhibition of alpha4beta2* nAChRs. Importantly, N-n-alkylpyridinium analogs with n-alkyl chains < C10 did not inhibit nicotine-evoked [3H]DA overflow, whereas analogs with n-alkyl chains ranging from C10 to C20 potently and completely inhibited nicotine-evoked [3H]DA overflow (IC50 = 0.12-0.49 microM), with the exceptions of N-n-pentadecylpyridinium bromide (C15) and N-n-eicosylpyridinium bromide (C20), which exhibited maximal inhibition of approximately 50%. The mechanism of inhibition of a representative analog of this structural series, N-n-dodecylpyridinium iodide, was determined by Schild analysis. Linear Schild regression with slope not different from unity indicated competitive antagonism at nAChRs mediating nicotine-evoked [3H]DA overflow and a KB value of 0.17 microM. Thus, the simplified N-n-alkylpyridinium analogs are potent, selective, and competitive antagonists of nAChRs mediating nicotine-evoked [3H]DA overflow, indicating that the N-methylpyrrolidino moiety is not a structural requirement for interaction with nAChR subtypes mediating nicotine-evoked DA release.     

Nicotinic receptor subtypes in rat hippocampal slices are differentially sensitive to desensitization and early in vivo functional up-regulation by nicotine and to block by bupropion

To identify the brain nicotinic acetylcholine receptor (nAChR) subtypes that may be involved in nicotine addiction, we investigated the actions of bupropion, a drug used in cigarette smoking cessation programs, and nicotine on three pharmacologically identified nAChRs in rat hippocampal slices, namely, type IA, type II, and type III nAChRs, likely representing alpha7, alpha4beta2, and alpha3beta4 subunits, respectively. Using whole-cell patch-clamp recordings from interneurons of acute hippocampal slices prepared from male rat pups, we studied the effect of nicotine on in vivo up-regulation and in vitro desensitization of nAChRs. Two subcutaneous injections of nicotine (0.586 mg/kg free base, in less than a day) to rats at postnatal days 14 to 15 significantly enhanced the magnitude of functional responses arising from type III and type II, but not type IA nAChRs. This treatment did not increase the functional affinity for acetylcholine at type II nAChRs. A single injection of nicotine also produced a significant increase in type III nAChR response. In addition, type III and type II, but not type IA nAChRs, are desensitized by in vitro exposure to nicotine at concentrations found in the venous blood of cigarette smokers. Bupropion at 1 muM produced 56, 15, and 0% inhibition of type III, type II, and type IA nAChR responses, respectively, in the slices. Our results suggest that in vivo-nicotine-induced nAChR up-regulation observed in neurons of intact brain tissue is a physiologically relevant phenomenon and that early up-regulation of type III and type II nAChRs could be an important biological signal in nicotine addiction.