Long-term nicotine treatment differentially regulates striatal alpha6alpha4beta2* and alpha6(nonalpha4)beta2* nAChR expression and function

Abstract

Nicotine treatment has long been associated with alterations in alpha4beta2(*) nicotinic acetylcholine receptor (nAChR) expression that modify dopaminergic function. However, the influence of long-term nicotine treatment on the alpha6beta2(*) nAChR, a subtype specifically localized on dopaminergic neurons, is less clear. Here we used voltammetry, as well as receptor binding studies, to identify the effects of nicotine on striatal alpha6beta2(*) nAChR function and expression. Long-term nicotine treatment via drinking water enhanced nonburst and burst endogenous dopamine release from rat striatal slices. In control animals, alpha6beta2(*) nAChR blockade with alpha-conotoxin MII (alpha-CtxMII) decreased release with nonburst stimulation but not with burst firing. These data in control animals suggest that varying stimulus frequencies differentially regulate alpha6beta2(*) nAChR-evoked dopamine release. In contrast, in nicotine-treated rats, alpha6beta2(*) nAChR blockade elicited a similar pattern of dopamine release with nonburst and burst firing. To elucidate the alpha6beta2(*) nAChR subtypes altered with long-term nicotine treatment, we used the novel alpha-CtxMII analog E11A in combination with alpha4 nAChR knockout mice. (125)I-alpha-CtxMII competition studies in striatum of knockout mice showed that nicotine treatment decreased the alpha6alpha4beta2(*) subtype but increased the alpha6(nonalpha4)beta2(*) nAChR population. These data indicate that alpha6beta2(*) nAChR-evoked dopamine release in nicotine-treated rats is mediated by the alpha6(nonalpha4)beta2(*) nAChR subtype and suggest that the alpha6alpha4beta2(*) nAChR and/or alpha4beta2(*) nAChR contribute to the differential effect of higher frequency stimulation on dopamine release under control conditions. Thus, alpha6beta2(*) nAChR subtypes may represent important targets for smoking cessation therapies and neurological disorders involving these receptors such as Parkinson's disease.

Fig. 1

Chronic nicotine treatment differentially regulates α4β2* and α6β2* nAChRs in rat striatum. Rats were given nicotine in the drinking water for 14 days, after which receptor autoradiography was done using ¹²⁵I-epibatidine and ¹²⁵I-α-CtxMII. Changes in the α4β2* and α6β2* nAChRs were determined by measurement of ¹²⁵I-epibatidine binding in the absence and presence of α-CtxMII (300 nM), with an increase in α4β2* nAChRs or α-CtxMII-resistant ¹²⁵I-epibatidine binding sites (A) and a decrease in α6β2* nAChRs or α-CtxMII-sensitive ¹²⁵I-epibatidine binding sites (B). α6β2* nAChRs were also measured using ¹²⁵I-α-CtxMII, with a significant decline with nicotine treatment (C). Data represent mean ± SEM of 6-8 rats. Significance of difference from control using a t-test, *p < 0.05; ***p < 0.001.

Fig. 2

Chronic nicotine treatment increases electrically-evoked dopamine release in rat striatum. Top and middle panels show representative traces of dopamine release in striatum of control (solid line) and nicotine-treated (dashed line) rats after 1 pulse and 4 pulse stimulation, respectively. Insets: typical voltammograms for dopamine with an oxidation peak at 500-600 mV and a reduction peak around -200 mV. Quantitative analyses of peak dopamine release (bottom panel) for each treatment group normalized to release by 1 pulse in controls. The values represent the mean ± SEM of 4-6 rats (15-25 observations per animal). Significance of difference from control using a Bonferroni post hoc test after a 1 pulse stimulus, **p < 0.01; after a 4 pulse stimulus, *p < 0.05.

Fig. 3

Chronic nicotine treatment prevents the enhancement of burst-stimulated dopamine release after α6β2* nAChR blockade. Representative traces for dopamine release after 1 pulse (top panel) and 4 pulse (middle panel) stimulation in the absence and presence of α-CtxMII for both control (left) and nicotine-treated rats (right). Insets: typical voltammograms for dopamine with oxidation peaks at 500-600 mV and reduction peaks around -200 mV. Quantitative analyses (bottom panel) of peak dopamine release normalized to control total release by 1 pulse in control (left) and nicotine-treated (right) rats induced by non-burst and burst stimulation before and after application of α-CtxMII. The values represent the mean ± SEM of 6 rats (15-20 observations per animal). Significance of difference from total release using a Bonferroni post hoc test, *p < 0.05; ***p < 0.001. Significance of difference from release with 1 pulse stimulation in the presence of α-CtxMII using a Bonferroni post hoc test, ⁺⁺⁺p < 0.001.

Fig. 4

Chronic nicotine treatment preferentially downregulates a subpopulation of α6β2* nAChRs in rat striatum. To further characterize the effect of nicotine treatment on α6β2* nAChRs, ¹²⁵I-α-CtxMII competition assays were done using varying concentrations (10^-15 to 10^-7 M) of the α-CtxMII analog E11A. A biphasic inhibition curve (data fit best to two site model) was obtained in control striatum indicating that E11A discriminates between at least two α6β2* nAChRs, previously shown to represent the α6α4β2* and α6(nonα4)β2* subtypes. Competition analysis of E11A inhibition of ¹²⁵I-α-conotoxin MII binding in striatum of rats receiving nicotine showed a preferential decline in the very high affinity α6β2* nAChR, that is, the α6α4β2* subtype. Symbols represent mean ± SEM of 6-8 rats. Where the SEM is not depicted, it fell within the symbol.

Fig. 5

Nicotine treatment differentially alters α6β2* nAChRs in wildtype and α4 (-/-) nAChR mice. Mice were maintained at the final dose of nicotine in the drinking water for 14 days, after which ¹²⁵I-epibatidine binding was done in the absence and presence of 300 nM unlabeled α-CtxMII to identify α-CtxMII-resistant (α4β2*) and α-CtxMII-sensitive (α6β2*) binding sites. A, Striatal α4β2* nAChRs in wildtype mice were significantly increased with nicotine treatment, with no binding in striatum of α4 (-/-) mice, as expected. B, α6β2* nAChR binding was significantly decreased in α4 (-/-) mice and nicotine-treated wildtype mice. Unexpectedly, there was a significant increase in α6β2* nAChR binding in striatum of α4 (-/-) mice receiving nicotine. Since α4 (-/-) mice do not express α6α4β2* nAChRs, these data suggest there is a selective upregulation of α6(nonα4)β2* nAChRs with nicotine treatment. C, For a direct measure of changes in α6β2* nAChRs with nicotine treatment, ¹²⁵I-α-CtxMII binding was done using striatal sections from wildtype and α4 nAChR-null mutant mice. Again, nicotine treatment decreased α6β2* nAChRs in the wildtype mice. There was also a decrease in the α4 (-/-) mice due to a loss of the α6α4β2* subtype. Again, however, there was a significant increase in α6β2* binding in nicotine-treated α4 (-/-) mice, further supporting the idea that there is a selective upregulation of α6(nonα4)β2* nAChRs with nicotine treatment. Data represent mean ± SEM of 3 mice. Significance of difference from control using a Bonferroni post hoc test, *p < 0.05; **p < 0.01; ***p < 0.001. NB, indicates no binding.

Fig. 6

Decrease in striatal α6α4β2* nAChRs but increase in α6(nonα4)β2* nAChRs with nicotine treatment. Wildtype and α4 nAChR-null mutant (-/-) mice were maintained at the final dose of nicotine in the drinking water for 14 days. Striatal ¹²⁵I-α-CtxMII binding was subsequently determined in the presence and absence of varying concentrations of E11A (10^-15 to 10^-7 M). Two site binding curves were obtained in striatum from wildtype mice suggesting the presence of at least two α6β2* nAChR populations. In striatum of α4 (-/-) mice, E11A inhibited ¹²⁵I-α-Ctx MII binding in a monophasic manner, as expected since α4 (-/-) mice do not express α6α4β2* nAChRs. Competition analysis of E11A inhibition of ¹²⁵I-α-CtxMII binding in nicotine-treated wildtype and α4 (-/-) mice yielded similar monophasic curves suggesting the presence of only α6(nonα4)β2* nAChRs. This site was preferentially increased with nicotine treatment in α4(-/-) mice, as depicted in the summary graph (lower panel). Values represent mean ± SEM of 3 mice. Where the SEM is not depicted, it fell within the symbol. Significance of difference from respective non-treated control using a Bonferroni post hoc test, ** p < 0.01; ***p < 0.001. NB, indicates no binding.