Chronic nicotine treatment increases nAChRs and microglial expression in monkey substantia nigra after nigrostriatal damage.

Our previous work had shown that long-term nicotine administration improved dopaminergic markers and nicotinic receptors (nAChRs) in the striatum of monkeys with nigrostriatal damage. The present experiments were done to determine whether nicotine treatment also led to changes in the substantia nigra, the region containing dopaminergic cell bodies. Monkeys were chronically treated with nicotine in the drinking water for 6 months after which they were injected with low dose of 1-methyl-4-phenyl-1,2,3,6-tetrahydrophridine (MPTP) for a further 6-month period. Nicotine was administered until the monkeys were euthanized 2 months after the last MPTP injection. Nicotine treatment did not affect the dopamine transporter or the number of tyrosine hydroxylase positive cells in the substantia nigra of lesioned monkeys. However, nicotine administration did lead to a greater increase in alpha3/alpha6beta2* and alpha4beta2* nAChRs in lesioned monkeys compared to controls. Nicotine also significantly elevated microglia and reduced the number of extracellular neuromelanin deposits in the substantia nigra of MPTP-lesioned monkeys. These findings indicate that long-term nicotine treatment modulates expression of several molecular measures in monkey substantia nigra that may result in an improvement in nigral integrity and/or function. These observations may have therapeutic implications for Parkinson's disease.

Multiple roles for nicotine in Parkinson's disease.

There exists a remarkable diversity of neurotransmitter compounds in the striatum, a pivotal brain region in the pathology of Parkinson's disease, a movement disorder characterized by rigidity, tremor and bradykinesia. The striatal dopaminergic system, which is particularly vulnerable to neurodegeneration in this disorder, appears to be the major contributor to these motor problems. However, numerous other neurotransmitter systems in the striatum most likely also play a significant role, including the nicotinic cholinergic system. Indeed, there is an extensive anatomical overlap between dopaminergic and cholinergic neurons, and acetylcholine is well known to modulate striatal dopamine release both in vitro and in vivo. Nicotine, a drug that stimulates nicotinic acetylcholine receptors (nAChRs), influences several functions relevant to Parkinson's disease. Extensive studies in parkinsonian animals show that nicotine protects against nigrostriatal damage, findings that may explain the well-established decline in Parkinson's disease incidence with tobacco use. In addition, recent work shows that nicotine reduces l-dopa-induced abnormal involuntary movements, a debilitating complication of l-dopa therapy for Parkinson's disease. These combined observations suggest that nAChR stimulation may represent a useful treatment strategy for Parkinson's disease for neuroprotection and symptomatic treatment. Importantly, only selective nAChR subtypes are present in the striatum including the alpha4beta2*, alpha6beta2* and alpha7 nAChR populations. Treatment with nAChR ligands directed to these subtypes may thus yield optimal therapeutic benefit for Parkinson's disease, with a minimum of adverse side effects.

Nicotine is neuroprotective when administered before but not after nigrostriatal damage in rats and monkeys.

Nicotine reduces dopaminergic deficits in parkinsonian animals when administered before nigrostriatal damage. Here we tested whether nicotine is also beneficial when given to rats and monkeys with pre-existing nigrostriatal damage. Rats were administered nicotine before and after a unilateral 6-hydroxydopamine lesion of the medial forebrain bundle, and the results compared with those in which rats received nicotine only after lesioning. Nicotine pre-treatment attenuated behavioral deficits and lessened lesion-induced losses of the striatal dopamine transporter, and alpha6beta2* and alpha4beta2* nicotinic receptors (nAChRs). By contrast, nicotine administered 2 weeks after lesioning, when 6-hydroxydopamine-induced neurodegenerative effects are essentially complete, did not improve these same measures. Similar results were observed in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned monkeys. Nicotine did not enhance striatal markers when administered to monkeys with pre-existing nigrostriatal damage, in contrast to previous data that showed improvements when nicotine was given to monkeys before lesioning. These combined findings in two animal models suggest that nicotine is neuroprotective rather than neurorestorative against nigrostriatal damage. Receptor studies with (125)I-alpha-conotoxinMII and the alpha-conotoxinMII analog E11A were next performed to determine whether nicotine treatment pre- or post-lesioning differentially affected expression of alpha6alpha4beta2* and alpha6(nonalpha4)beta2* nAChR subtypes in striatum. The observations suggest that protection against nigrostriatal damage may be linked to striatal alpha6alpha4beta2* nAChRs.

Prominent role of alpha3/alpha6beta2* nAChRs in regulating evoked dopamine release in primate putamen: effect of long-term nicotine treatment.

Brain dopaminergic systems are critical in motor control as evidenced by findings that their disruption results in movement disorders such as Parkinson's disease. Nicotinic acetylcholine receptor (nAChR) activation plays an important role in regulating striatal dopaminergic function. Rodent studies show that short-term nicotine exposure influences stimulated striatal dopamine release with responsiveness dependent on neuronal activity. However, studies have not yet been done in nonhuman primates, nor has work been done to evaluate the effect of long-term nicotine exposure, which is relevant for therapies for chronic neurological disorders. Here, we used voltammetry to assess the role of nAChRs on evoked dopamine release from monkey putamen slices. In both ventral and dorsal putamen, alpha3/alpha6beta2(*) nAChRs regulated > or =80% of non-burst- (single pulse) nAChR-modulated dopamine release, and alpha4beta2(*) nAChRs regulated the remainder. Similar results were observed with burst-firing in ventral but not dorsal putamen, indicating that nAChR-modulated effects on release depend on the subregion and firing frequency. Next, we investigated the consequence of long-term nicotine exposure via the drinking water on nAChR-modulated responsiveness. Nicotine treatment altered both non-burst- and burst-stimulated dopamine release in ventral but not dorsal putamen. Altogether, these data support a predominant role for alpha3/alpha6beta2(*) nAChRs in the regulation of evoked dopamine release in nonhuman primate putamen. They also show that long-term nicotine treatment selectively modifies nAChR-modulated release in distinct striatal subregions. These findings have implications for the development of treatments for addiction and neurological disorders with nAChR dysfunction.

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.

Pre-synaptic dopaminergic compensation after moderate nigrostriatal damage in non-human primates.

Despite a dramatic loss of nigrostriatal dopaminergic neurons in Parkinson's disease, clinical symptoms only arise with 70-80% reduction of striatal dopamine. The mechanisms responsible for this functional compensation are currently under debate. Although initial studies showed an enhanced pre-synaptic dopaminergic function with nigrostriatal degeneration, more recent work suggests that functional compensation is not dopamine-mediated. To address this issue, we used cyclic voltammetry to directly measure endogenous dopamine release from striatal slices of control monkeys and animals with a moderate or severe MPTP-induced dopaminergic lesion. The moderately lesioned monkeys were asymptomatic, while the severely lesioned animals were parkinsonian. In monkeys with a moderate lesion, a 300% increase was obtained in endogenous striatal dopamine release. In contrast, in striatal slices from severely lesioned animals, a small % of evoked dopamine signals were similar in amplitude to control while the greater majority were undetectable. These findings suggest that pre-synaptic dopaminergic compensation develops in residual dopaminergic terminals with moderate lesioning, but that this response is lost with severe nigrostriatal damage. Such an interpretation is supported by the results of dopamine turnover studies. This enhanced pre-synaptic dopaminergic activity may be important in maintaining normal motor function during the initial stages of Parkinson's disease.

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.

Nicotine reduces levodopa-induced dyskinesias in lesioned monkeys.

OBJECTIVE: Levodopa, the gold standard for Parkinson's disease treatment, is associated with debilitating abnormal involuntary movements or dyskinesias, for which few treatments are currently available. Studies have implicated numerous neurotransmitters in the development of levodopa-induced dyskinesias. However, the cholinergic system has received little attention despite an extensive overlap between dopaminergic terminals and cholinergic interneurons in the striatum and the well-known ability of nicotine to stimulate striatal dopamine release. Our objective, therefore, was to determine whether nicotine treatment reduced levodopa-induced dyskinesias. METHODS: The effect of nicotine (provided in the drinking water) was determined on dyskinesias induced by levodopa (5 mg/kg twice daily by oral gavage) in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated monkeys. RESULTS: Nicotine pretreatment reduced peak and total levodopa-induced dyskinesias in levodopa-naive monkeys over an 8-week period, with a decrease in total dyskinesias of about 50%. A crossover study was then done in which levodopa-treated monkeys originally receiving vehicle were administered nicotine, whereas the levodopa-treated animals initially receiving nicotine were placed on vehicle. Nicotine treatment to levodopa-primed monkeys led to an approximately 35% reduction in total dyskinesias that lasted for at least 8 weeks, at which time the study was ended. In contrast, a significant increase in levodopa-induced dyskinesias was observed in the group of animals that had previously received nicotine and were then switched to vehicle. Nicotine treatment did not appear to affect the antiparkinsonian action of levodopa. INTERPRETATION: These data suggest that nicotine or selective nicotinic agonists may represent a useful treatment strategy to reduce levodopa-induced dyskinesias.

Guidelines on nicotine dose selection for in vivo research.

RATIONALE: This review provides insight for the judicious selection of nicotine dose ranges and routes of administration for in vivo studies. The literature is replete with reports in which a dosaging regimen chosen for a specific nicotine-mediated response was suboptimal for the species used. In many cases, such discrepancies could be attributed to the complex variables comprising species-specific in vivo responses to acute or chronic nicotine exposure. OBJECTIVES: This review capitalizes on the authors' collective decades of in vivo nicotine experimentation to clarify the issues and to identify the variables to be considered in choosing a dosaging regimen. Nicotine dose ranges tolerated by humans and their animal models provide guidelines for experiments intended to extrapolate to human tobacco exposure through cigarette smoking or nicotine replacement therapies. Just as important are the nicotine dosaging regimens used to provide a mechanistic framework for acquisition of drug-taking behavior, dependence, tolerance, or withdrawal in animal models. RESULTS: Seven species are addressed: humans, nonhuman primates, rats, mice, Drosophila, Caenorhabditis elegans, and zebrafish. After an overview on nicotine metabolism, each section focuses on an individual species, addressing issues related to genetic background, age, acute vs chronic exposure, route of administration, and behavioral responses. CONCLUSIONS: The selected examples of successful dosaging ranges are provided, while emphasizing the necessity of empirically determined dose-response relationships based on the precise parameters and conditions inherent to a specific hypothesis. This review provides a new, experimentally based compilation of species-specific dose selection for studies on the in vivo effects of nicotine.

Chronic oral nicotine treatment protects against striatal degeneration in MPTP-treated primates.

The present studies were done to investigate the effect of long-term nicotine treatment against nigrostriatal damage in non-human primates. Monkeys were administered nicotine in drinking water for 6 months to provide chronic but intermittent delivery as with smoking. Plasma nicotine levels ranged from 10 to 15 ng/mL, which were within the range in cigarette smokers. Animals were then lesioned with low doses of the dopaminergic neurotoxin MPTP for several months while nicotine was continued. The results showed that levels of striatal tyrosine hydroxylase, dopamine transporter, vesicular monoamine transporter, dopamine and nicotinic receptors were greater in nicotine-treated MPTP-lesioned primates than in lesioned animals not receiving nicotine. Nicotine had no effect in unlesioned animals. Monoamine oxidase activity was similar in unlesioned and lesioned animals treated with or without nicotine, suggesting that nicotine did not exert its effects through changes in MPTP or dopamine metabolism. MPTP-induced cell loss in the substantia nigra was unaffected by nicotine treatment, indicating that nicotine acts at the striatal level to restore/maintain dopaminergic function. These data further support the possibility that nicotine contributes to the lower incidence of Parkinson's disease in smokers.

Partial recovery of striatal nicotinic receptors in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys with chronic oral nicotine.

Recent studies in nonhuman primates show that chronic nicotine treatment protects against nigrostriatal degeneration, with a partial restoration of neurochemical and functional measures in the striatum. The present studies were done to determine whether long-term nicotine treatment also protected against striatal nicotinic receptor (nAChR) losses after nigrostriatal damage. Monkeys were administered nicotine in the drinking water for 6 months and subsequently lesioned with the dopaminergic neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) over several months while nicotine was continued. (125)I-Epibatidine, [(125)I]5-[(125)I]iodo-3(2(S)-azetidinylmethoxy)-pyridine (A85380), and (125)I-alpha-conotoxinMII autoradiography was performed to evaluate changes in alpha4beta2* and alpha3/alpha6beta2* nAChRs, the major striatal subtypes. Nicotine treatment increased alpha4beta2* nAChRs by > or =50% in striatum of both unlesioned and lesioned animals. This increase in alpha4beta2* nAChRs was significantly greater in lesioned compared with unlesioned monkey striatum. Chronic nicotine treatment led to a small decrease in alpha3/alpha6beta2* nAChR subtypes. The decline in alpha3/alpha6beta2* subtypes, defined using alpha-conotoxinMII-sensitive (125)I-epibatidine or [(125)I]A85380 binding, was significantly smaller in striatum of nicotine-treated lesioned monkeys compared with unlesioned monkeys. This difference was not observed for alpha3/alpha6beta2* nAChRs identified using (125)I-alpha-conotoxinMII. These data suggest that there are at least two striatal alpha3/alpha6beta2* subtypes that are differentially affected by chronic nicotine treatment in lesioned animals. In addition, the results showing an improvement in striatal alpha4beta2* and select alpha3/alpha6beta2* nAChR subtypes, combined with previous work, demonstrate that chronic nicotine treatment restores and/or protects against the loss of multiple molecular markers after nigrostriatal damage. Such findings suggest that nicotine or nicotinic agonists may be of therapeutic value in Parkinson's disease.

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.

Chronic oral nicotine normalizes dopaminergic function and synaptic plasticity in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-lesioned primates.

Our recent studies show that chronic oral nicotine partially protects against striatal damage in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated nonhuman primates. To identify the cellular changes associated with this protective action, we investigated the effects of nicotine treatment on stimulus-evoked dopamine release, dopamine turnover, and synaptic plasticity in striatum from lesioned and unlesioned animals. Monkeys were chronically (6 months) treated with nicotine in the drinking water and subsequently lesioned with the dopaminergic neurotoxin MPTP (6 months) while nicotine was continued. Nigrostriatal damage increased nicotinic acetylcholine receptor (nAChR)-mediated fractional dopamine release from residual terminals, primarily through changes in alpha3*/alpha6* nAChRs. In contrast, fractional receptor-evoked dopamine release was similar to control in unlesioned and lesioned animals with chronic oral nicotine. Long-term nicotine administration also attenuated the enhanced K(+)-evoked fractional dopamine release from synaptosomes of MPTP-lesioned animals, suggesting that nicotine treatment had a generalized effect on dopaminergic function. This premise was further supported by experiments showing that nicotine dosing decreased the elevated dopamine turnover that occurs after nigrostriatal damage. We next investigated changes in synaptic plasticity with lesioning and nicotine treatment. Nicotine treatment alone enhanced synaptic plasticity by lowering the threshold for long-term depression (LTD) in the corticostriatal pathway. MPTP lesioning led to a loss of LTD, a measure of short-term synaptic plasticity. In contrast, LTD was preserved in nicotine-treated lesioned animals. Thus, the present data show that the disruptions in striatal dopaminergic function after nigrostriatal damage were attenuated with chronic nicotine administration. These cellular alterations may underlie the ability of nicotine to maintain/restore normal function with nigrostriatal damage.

Increases in alpha4* but not alpha3*/alpha6* nicotinic receptor sites and function in the primate striatum following chronic oral nicotine treatment.

Knowledge of the effects of chronic nicotine is critical considering its widespread use in tobacco products and smoking cessation therapies. Although nicotine is well known to up-regulate alpha4* nAChR sites and function in the cortex, its actions in the striatum are uncertain because of the presence of multiple subtypes with potentially opposing effects. We therefore investigated the effect of long-term nicotine treatment on nAChR sites and function in the primate striatum, which offers the advantage of similar proportions of alpha3*/alpha6* and alpha4* nAChRs. Nicotine was given in drinking water, which resembles smoking in its intermittent but chronic delivery. Plasma nicotine and cotinine levels were similar to smokers. Chronic nicotine treatment (> 6 months) enhanced alpha4* nAChR-evoked [(3)H]dopamine release in striatal subregions, with an overall pattern of increase throughout the striatum when normalized to uptake. This increase correlated with elevated striatal alpha4* nAChRs. Under the same conditions, striatal alpha3*/alpha6* nAChR sites and function were decreased or unchanged. These divergent actions of chronic nicotine treatment on alpha4* versus alpha6* nAChRs, as well as effects on dopamine uptake, allow for a complex control of striatal activity to maintain dopaminergic function. Such knowledge is important for understanding nicotine dependence and the consequences of nicotine administration for the treatment of neurological disorders.

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.

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.