α6ß2* and α4ß2* nicotinic receptors both regulate dopamine signaling with increased nigrostriatal damage: relevance to Parkinson's disease

Abstract

Nicotinic receptors (nAChRs) are important modulators of dopaminergic transmission in striatum, a region critical to Parkinson's disease. The nAChRs mainly involved are the α6β2* and α4β2* subtypes. Lesion studies show that the α6β2* receptor is decreased to a much greater extent with nigrostriatal damage than the α4β2* subtype raising the question whether this latter nAChR population is more important with increased nigrostriatal damage. To address this, we investigated the effect of varying nigrostriatal damage on α6β2* and α4β2* receptor-modulated dopamine signaling using cyclic voltammetry. This approach offers the advantage that changes in dopamine release can be observed under different neuronal firing conditions. Total single-pulse-evoked dopamine release decreased in direct proportion to declines in the dopamine transporter and dopamine uptake. We next used α-conotoxinMII and mecamylamine to understand the role of the α4β2* and α6β2* subtypes in release. Single-pulse-stimulated α6β2* and α4β2* receptor dopamine release decreased to a similar extent with increasing nigrostriatal damage, indicating that both subtypes contribute to the control of dopaminergic transmission with lesioning. Total burst-stimulated dopamine release also decreased proportionately with nigrostriatal damage. However, the role of the α4β2* and α6β2* nAChRs varied with different degrees of lesioning, suggesting that the two subtypes play a unique function with burst firing, with a somewhat more prominent and possibly more selective role for the α6β2* subtype. These data have important therapeutic implications because they suggest that drugs directed to both α4β2* and α6β2* nAChRs may be useful in the treatment of neurological disorders such as Parkinson's disease.

Fig. 1.

Striatal dopamine transporter declines with 6-OHDA lesioning. Rats received unilateral 6-OHDA injections at two different sites in the medial forebrain bundle as described under Materials and Methods. Various doses of the toxin were injected to achieve different degrees of nigrostriatal damage. Alterations in striatal dopamine transporter expression were assessed using [¹²⁵I]RTI-121 autoradiography. After quantitative analyses, rats were grouped as shown. The numerical values represent the mean ± S.E.M. of the indicated number of rats. ***, p < 0.001 indicates significance of difference from control using a Newman-Keuls multiple comparisons post hoc test.

Fig. 2.

Motor deficits with progressive nigrostriatal damage. Parkinsonism was assessed using the limb asymmetry or cylinder test. The percentage use of the affected limb was determined during a 5-min rating period for each animal. A statistically significant decrease in the use of the contralateral paw was observed only in rats with moderately severe and severe lesions. The values represent the mean ± S.E.M. of four to nine rats. **, p < 0.01; ***, p < 0.001 indicate significance of difference from control using a Newman-Keuls multiple comparisons post hoc test.

Fig. 3.

Decreases in both single and burst-evoked dopamine release correlate with nigrostriatal damage. Evoked endogenous dopamine release across the different range of lesions was measured after one pulse, four pulses at 30 Hz, and four pulses at 100 Hz electrical stimulation. Peak dopamine release decreased in proportion to the extent of lesion regardless of stimulation frequency. Sample traces of dopamine signals measured from a representative animal from each lesion group are shown to the left of the average group data. The scale bar represents 100 nM and 0.5 s. Values represent the mean ± S.E.M. of four to nine rats. *, p < 0.05; **, p < 0.01; ***, p < 0.001 indicate significance of difference from control using a Newman-Keuls multiple-comparisons post hoc test.

Fig. 4.

Uptake rate constants decrease in proportion to the extent of nigrostriatal damage. A, representative traces of dopamine release from each group. Uptake rate constants were calculated by fitting the clearance portion of the curve to one-phase exponential decay (r > 0.9) B, uptake rate constant values were significantly decreased in proportion to lesion size. Values represent mean ± S.E.M. of four to nine rats per group. **, p < 0.01; ***, p < 0.001 indicate significance of difference from control using a Newman-Keuls multiple comparisons post hoc test.

Fig. 5.

nAChR blockade decreases dopamine release with single but not burst stimulation in control rat striatum. Dopamine release was measured in the absence (total) and presence of the α6β2* nAChR antagonist α-CtxMII (100 nM) or the general nAChR blocker mecamylamine (100 μM). Sample dopamine signals after a 1 pulse (A), 4 pulses at 30 Hz (B), and 4 pulses at 100 Hz (C) electrical stimulation are shown. The scale bar represents 100 nM and 2.5 s. Quantitative analyses of peak dopamine release show that with a one-pulse stimulus (D), ∼45% of endogenous dopamine release is mediated through α6β2* nAChRs whereas ∼25% is modulated by α4β2* nAChRs, as evidenced by significant decreases in release in the presence of α-CtxMII or mecamylamine. In contrast, dopamine release-stimulated at higher frequencies was affected by perfusion of neither α-CtxMII nor mecamylamine (E and F). Values represent the mean ± S.E.M. of four to nine rats. ***, p < 0.001 indicates significance of difference from total; +, p < 0.05 indicates significance of difference from α-CtxMII using a Newman-Keuls multiple comparisons post hoc test.

Fig. 6.

Effect of α6β2* and/or α4β2* nAChR blockade on single-pulse stimulated dopamine release with varying nigrostriatal damage. Dopamine release was measured in the absence (total) and presence of the α6β2* nAChR antagonist α-CtxMII (100 nM) or the general nAChR blocker mecamylamine (100 μM). Release was normalized to total release for each lesioned group. NAChR inhibition with either α-CtxMII or mecamylamine significantly decreased dopamine release in rats with mild (B) and moderate (C) lesions, although to a lesser extent than in control rats (A). No significant changes were observed in the rats with moderately severe (D) and severe (E) lesions. Values represent the mean ± S.E.M. of four to nine rats. *, p < 0.05; ***, p < 0.001 indicate significance of difference from total; +, p < 0.05 indicates significance of difference from α-CtxMII using a Newman-Keuls multiple comparisons post hoc test.

Fig. 7.

Both α6β2* and α4β2* nAChR-modulated release decline with nigrostriatal damage. NAChR-mediated release was determined by subtracting release in the presence of mecamylamine from total release. α6β2* mediated release was determined by subtracting release in the presence of α-CtxMII from total release. α4β2* mediated release was determined by subtracting release in the presence of mecamylamine from that in the presence of α-CtxMII. Quantitative analyses showed a significant decrease in nAChR-mediated release in proportion to the extent of lesioning. This was accompanied by a significant decrease in α6β2* and α4β2* mediated release. Values represent the mean ± S.E.M. of four to nine rats. *, p < 0.05; **, p < 0.01; ***, p < 0.001 indicate significance of difference from control using a Newman-Keuls multiple comparisons post hoc test.

Fig. 8.

NAChR antagonism results in similar effects on burst-stimulated dopamine release in control and dopamine-depleted striatum. Left, representative traces of dopamine release in the absence or presence of α-CtxMII or mecamylamine after a single pulse or at 4 pulses at either 30 or 100 Hz. Middle, quantitative analyses of the data for control (n = 9 rats) and lesioned rats (n = 4 rats per lesion group) at varying frequency, as indicated. The frequency dependence of release in the absence and presence of the antagonists was similar in striatum of control and lesioned rats. Thus, the relief of short-term depression with nAChR blockade during burst stimulation is observed in both control striatum and with nigrostriatal damage. *, p < 0.05; ***, p < 0.001 indicate significance of difference from total release using a Bonferroni post hoc test. Right, normalization of the data to one pulse at the same condition for each lesion paradigm. nAChR antagonism effectively relieves short-term depression at the higher stimulation frequencies, although there was less of an increase with greater nigrostriatal damage. Thus both α4β2* and α6β2* nAChRs influence burst-evoked release throughout the neurodegenerative process. Values represent the mean ± S.E.M. of nine control rats and four rats per lesioned group. *, p < 0.05; **, p < 0.01; ***, p < 0.001 indicate significance of difference from total release; +, p < 0.05; ++, p < 0.01 indicates significance of difference from release in the presence of α-CtxMII using a Bonferroni post hoc test.