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Decrease in 3/6 Nicotinic Receptors but Not Nicotine-Evoked Dopamine Release in Monkey Brain after Nigrostriatal Damage

来源:分子药理学杂志 作者:Sarah E. McCallum, Neeraja Parameswaran, Tanuja Bo 2007-5-15
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摘要: ) Abstract 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 ......


    The Parkinson's Institute, Sunnyvale, California (S.E.M., N.P., T.B., M.Q.)
    Institute for Behavioral Genetics, University of Colorado, Denver, Colorado (S.R.G.)
    Department of Biology and Psychiatry, University of Utah, Salt Lake City, Utah (J.M.M.)

    Abstract

    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 (70%) of release was inhibited by the 3/6 antagonist -conotoxinMII (-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 3/6 nAChRs in caudate and putamen. In contrast, 3/6 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 3/6 nAChR sites with no decrease in 3/6 receptor-evoked dopamine release. No declines in -CtxMII-resistant nAChR (4) binding or nicotine-evoked release were observed in any region. These results show a selective preservation of 3/6 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.

    Parkinson's disease (PD) is characterized by motor and cognitive deficits and is manifested by reductions in dopaminergic neurons in substantia nigra and dopamine levels in striatum (Olanow, 2004). PD is also associated with decreases in striatal nicotinic acetylcholine receptors (nAChRs), including those containing 4, 3, and/or 6 but not 7 subunits ( denotes nicotinic receptors containing the indicated  and/or  subunit and also additional undefined subunits; Quik, 2004). Similar declines in nAChRs in Parkinsonian animal models support these findings (Quik et al., 2001, 2002, 2003; Champtiaux et al., 2002; Kulak et al., 2002; Zoli et al., 2002).

    Receptor binding and immunoprecipitation studies have revealed multiple nAChR subtypes on striatal dopaminergic terminals, including both 4 and 6 in rodents (Klink et al., 2001; Champtiaux et al., 2002, 2003; Zoli et al., 2002; Cui et al., 2003) and 3, 4, and 6 in monkeys (Kulak et al., 2002; Quik et al., 2001, 2002, 2005). In monkey, moderate nigrostriatal damage induced by the dopaminergic toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) preferentially affects receptors sensitive to -conotoxinMII (-CtxMII) or 3/6 nAChRs (refers to receptors containing 3 and/or 6 subtypes; Kulak et al., 2002; Quik et al., 2002). The function of nAChRs is of particular interest to PD because of their presence on dopamine nerve terminals. In mammalian brain, nicotine stimulates dopamine release by acting on nAChRs on striatal dopamine terminals and somatodendritic receptors in the substantia nigra (Rapier et al., 1990; Pidoplichko et al., 1997; Wonnacott, 1997; Wonnacott et al., 2000; Grady et al., 2001).

    Recent studies using nAChR-null mutant mice and subtype-selective -conotoxins suggest that both 6 (-CtxMII-sensitive) and 4 (-CtxMII-resistant) nAChRs contribute to nicotine-evoked dopamine release in striatum (Champtiaux et al., 2003; Cui et al., 2003; Salminen et al., 2004; Azam and McIntosh, 2005). Whereas the specific nAChR populations reduced by nigrostriatal damage have been identified, the effect of these receptor losses on nAChR function is much less clear with only one report in rodent to date. This work showed that MPTP lesioning reduced dopamine release in mouse striatum with the 6 and 42 populations equally affected (Quik et al., 2003).

    The aim of the present study was to determine the functional relevance of nAChR declines after nigrostriatal damage in a nonhuman primate model of PD. At a molecular level, the nAChR composition in human striatum may exhibit a closer resemblance to that in nonhuman primates because striatal -CtxMII-sensitive 3 nAChRs have been identified in monkey and human but not rodent striatum (Champtiaux et al., 2002; Whiteaker et al., 2002; Quik, 2004). We first characterized nicotine-evoked [3H]dopamine release from monkey striatal synaptosomes. Thereafter, changes in dopamine release, nAChR binding, and dopamine transporter levels were measured after administration of MPTP. Because a loss of dopamine receptors in nucleus accumbens may be observed in PD (Hornykiewicz, 1998) and is associated with other features of the disease, such as depression or anhedonia, we also measured changes in nAChR binding and function in this region. Our findings reveal a selective preservation of -CtxMII-sensitive nAChR function after nigrostriatal damage.

    Materials and Methods

    Materials. The radioligands [3H]dopamine (3,4-[ring-2,5,6-3H]; 30-60 Ci/mmol), [125I]RTI-121 (2200 Ci/mmol), and 125I-epibatidine (2200 Ci/mmol) were obtained from PerkinElmer Life and Analytical Sciences (Boston, MA). -CtxMII was synthesized as described by Cartier et al. (1996) and labeled with 125I (Whiteaker et al., 2000). HEPES was purchased from Roche Applied Science (Indianapolis, IN), and Econo-Safe and BudgetSolve scintillation cocktail were purchased from Research Products International (Mount Prospect, IL). The following compounds were obtained from Sigma Chemical (St. Louis, MO): -bungarotoxin, ascorbic acid, bovine serum albumin, MPTP, mecamylamine HCl, (-)-nicotine tartrate, nomifensine, pargyline, and sucrose.

    Animals. Female squirrel monkeys (Saimiri sciureus) were purchased from Osage Research Primates (Osage Beach, MO) or the Primate Research Laboratory at the University of South Alabama (Mobile, AL). Animals weighing between 0.5 and 0.7 kg were housed in a room with a 13-h/11-h light/dark cycle. Immediately after arrival, the monkeys were quarantined and tested according to standard veterinary practice. The monkeys had free access to water and were given food once daily. Procedures conformed to National Institutes of Health Guide for the Care and Use of Laboratory Animals guidelines and were approved by the Institutional Animal Care and Use Committee.

    Drug Treatment and Behavioral Evaluation. After an acclimation period, monkeys were randomly assigned to treatment with a single saline or MPTP injection (1.75-2 mg/kg s.c.). Two to four weeks after saline or MPTP injection, animals were rated for parkinsonism using a modified Parkinson rating scale for the squirrel monkey (Quik et al., 2001). The disability scores ranged from 0 to a maximum of 20 for a severely parkinsonian animal. Animals were rated for parkinsonism by assessment of 1) spatial hypokinesia (reduction in use of the available cage space), 2) body bradykinesia (increased slowness in body movement), 3) manual dexterity, 4) balance, and 5) freezing. If the animals were not parkinsonian (rating score <3), they were administered a second MPTP injection. Our previous experience indicated that some animals were more resistant to the effects of MPTP, possibly because of differences in pharmacokinetics or metabolism of MPTP. The second dose of MPTP (1.75 mg/kg) was a somewhat lower dose than that originally administered, because previous studies showed that the animals were more susceptible to the toxic effects of MPTP upon its readministration. A second injection to these animals led to declines in dopaminergic measures similar to that in the more sensitive animals. Parkinsonism was again assessed. The monkeys were euthanized 2 to 4 weeks after the final MPTP administration, at which time the effects of the lesion were maximal with little regeneration (Stanic et al., 2003; Lai et al., 2004).

    Tissue Preparation. Animals were euthanized in accordance with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association and conforming to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Ketamine hydrochloride (15-20 mg/kg i.m.) was administered for sedation approximately 5 min before a lethal i.v. injection of 0.22 ml/kg euthanasia solution (390 mg/ml sodium pentobarbital and 50 mg/ml phenytoin sodium). The brains were then immediately removed and divided along the midline. One half of each brain was placed in a mold and cut into 6-mm thick blocks that were frozen in isopentane on dry ice and stored at -80°C for the autoradiographic studies. The other half was sliced into 2-mm sections. Brain regions, including medial caudate, ventral putamen, and nucleus accumbens, were dissected from sections between 15 and 13.5 mm anterior to bregma, according to a squirrel monkey atlas (Fig. 1; Emmers and Akert, 1963). These subregions of the caudate and putamen were selected because they have a greater proportion of -CtxMII-sensitive nAChR-binding sites (Quik et al., 2002). For the autoradiographic studies, 20-e sections were cut from the 6-mm blocks using a cryostat (Leica Microsystems, Inc., Deerfield, IL) cooled to -15°C. After thaw-mounting onto Superfrost Plus slides (Fisher Scientific Co., Pittsburgh, PA), sections were air-dried and stored at -80°C.

    [3H]Dopamine Release from Synaptosomes. The procedure for measuring nicotine-evoked [3H]dopamine release from synaptosomal preparations of control and lesioned tissue was adapted from the method of Grady et al. (2001). Portions of medial caudate (20-40 mg wet weight), ventral putamen (20-40 mg), and nucleus accumbens (12-25 mg) were each homogenized (20 strokes by hand) using a glass Teflon homogenizer in a 2-ml volume of ice-cold sucrose (0.32 M) buffered with 5 mM HEPES, pH 7.5. To stay within a linear range of release, a 0.5- to 2-mg tissue aliquot was used for each sample (filter). The homogenized tissue was centrifuged at 12,000g for 20 min to obtain a P1 pellet. Pellets were then resuspended in 0.8 ml of uptake buffer containing 128 mM NaCl, 2.4 mM KCl, 3.2 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, 25 mM HEPES, pH 7.5, 10 mM glucose, 1 mM ascorbic acid, and 0.01 mM pargyline.

    Synaptosomes were incubated in uptake buffer for 10 min at 37°C, and then 100 nM [3H]dopamine was added (4 e藽i for 0.8-ml synaptosomes), and incubation continued for an additional 5 min. An 80-e蘬 aliquot of synaptosomes (approximately 0.5-2 mg of tissue) was then pipetted onto 5-mm-diameter A/E glass fiber filters (Gelman Instrument Co., Ann Arbor, MI) mounted on polypropylene platforms. Filters were perfused with uptake buffer [plus 0.1% bovine serum albumin (BSA) and 10 e nomifensine] for 10 min at a rate of 1 ml/min before fractions of release were collected. Release was stimulated by an 18-s exposure to 20 mM K+ or 0.001 to 100 e nicotine. Some filters were perfused with 50 nM -CtxMII or 100 e mecamylamine for 3 min just before the nicotine exposure. For experiments with -bungarotoxin, synaptosomes were incubated with or without 1 e toxin for 30 min at 37°C before the addition of [3H]dopamine. For each filter, 15 fractions (18 s) were collected, which included fractions of basal release before and after the stimulated release. Econosafe cocktail was added to the vials, and radioactivity was determined by scintillation counting on a Beckman LS6500 counter (Fullerton, CA).

    [125I]RTI-121 Autoradiography. Dopamine transporter binding was measured using [125I]RTI-121 (2200 Ci/mmol; PerkinElmer Life and Analytical Sciences) as described previously (Quik et al., 2001). Frozen sections were thawed and then incubated twice for 15 min each at room temperature in 50 mM Tris-HCl, pH 7.4, 120 mM NaCl, and 5 mM KCl. Sections were incubated next for 2 h in buffer with 0.025% BSA, 1 e fluoxetine, and 50 pM [125I]RTI-121. Nonspecific binding was determined using 100 e nomifensine. Sections were washed four times at 0°C for 15 min each in buffer and once in ice-cold water, air-dried, and exposed for 2 days to Kodak MR film (PerkinElmer Life and Analytical Sciences) with 125I microscale standards (Amersham Biosciences, Piscataway, NJ).

    125I--CtxMII Autoradiography. -CtxMII was iodinated (Whiteaker et al., 2000), and receptor autoradiography was performed as described previously (Quik et al., 2001). Thawed sections were first incubated at room temperature for 15 min in 20 mM HEPES buffer, pH 7.5, containing 144 mM NaCl, 1.5 mM KCl, 2 mM CaCl2, 1 mM MgSO4, 0.1% BSA, and 1 mM phenylmethylsulfonyl fluoride. This was followed by 1-h incubation with 125I--CtxMII (0.5 nM) at room temperature in buffer with 0.5% BSA, 5 mM EDTA, 5 mM EGTA, and 10 e蘥/ml each of aprotinin, leupeptin, and pepstatin A. Slides were washed for 30 s in 1x HEPES buffer at room temperature, 30 s in ice-cold 1x buffer, twice for 5 s each in 0.1x buffer (0°C), and twice for 5 s each at 0°C in deionized water. Nonspecific 125I--CtxMII binding was determined using 0.1 e epibatidine. The sections were then air-dried and exposed to Kodak MR film with 125I standards for 2 to 5 days.

    [125I]Epibatidine Autoradiography. Binding was performed as described previously (Perry and Kellar, 1995). Sections were thawed and incubated at room temperature for 40 min in buffer containing 50 mM Tris, pH 7.0, 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl2, and 1.0 mM MgCl2 plus 0.015 nM 125I-epibatidine (2200 Ci/mmol; PerkinElmer Life and Analytical Sciences). Inhibition by -CtxMII was performed with 0.1 e added to the incubation buffer, and incubation continued for an additional 40 min. Nonspecific binding was defined using 0.1 mM nicotine. After incubation, sections were washed twice for 5 min each in incubation buffer at 4°C and once for 10 s in ice-cold water. After drying, sections were exposed to Kodak MR film for 1 to 3 days with 125I standards.

    Data Analysis. For dopamine release experiments, a curve-fitting algorithm of SigmaPlot 5.0 for DOS (SPSS Inc., Chicago, IL) was used. Release was plotted as counts per minute versus fraction number. Basal release was then calculated by selecting the fractions collected just before and after the peak and plotting them as a single exponential decay function. The first-order equation, Rt = R0 (e-kt), was used to determine basal release, where Rt = release at time t, R0 = initial release, and k = rate of decline of basal release. Fractions of release that were at least 10% higher than baseline were added to achieve total cpm released. The calculated baseline release was subtracted from each fraction, and the corrected cpm were then normalized to the wet weight of the tissue sample on each filter to obtain units of counts per minute per milligram of tissue. To obtain Rmax (a determination of maximal response) and EC50 values for dose-response curves, data were fit using a nonlinear regression equation in SigmaPlot 2001 for Windows (SPSS Inc.). Statistical comparisons were performed using either Student's unpaired t test or two-way repeated measures ANOVA followed by Bonferroni post hoc tests where appropriate (Prism; GraphPad Software Inc., San Diego, CA). A level of p < 0.05 was considered significant.

    For analysis of autoradiographic data, quantitation of optical densities associated with medial caudate, ventral putamen, and nucleus accumbens was done using the ImageQuant system from Amersham Biosciences Inc. The optical density values were converted to nanocuries per milligram of tissue using standard curves generated from 125I standards simultaneously exposed to the films. The activity levels of the 125I standards ranged from 0.5 to 200 fmol/mg of tissue for 20-e tissue samples per the manufacturer's specifications (Amersham Biosciences Inc.). Specific binding was determined by subtracting background from total values. Sample optical density readings were within the linear range of the film. The receptor binding value for a brain area for any one animal was obtained from two independent experiments with one or two consecutive sections per experiment. All values are expressed as the mean ± S.E.M. of the indicated number of animals. Statistical analyses were done using Student's unpaired t test where p < 0.05 was considered significant (GraphPad Prism).

    Results

    Characterization of Nicotine-Evoked [3H]Dopamine Release from Control Monkey Brain Synaptosomes. Although stimulation-evoked release has been measured from monkey striatum in vitro using voltammetry (Cragg et al., 2000) and, more recently, nicotine-stimulated release has been measured in vivo with positron emission tomography (Marenco et al., 2004), nicotine-evoked [3H]dopamine release from synaptosomes has not previously been studied using monkey brain, although this approach is widely used with rodent preparations (Wonnacott, 1997; Grady et al., 2001; Champtiaux et al., 2003; Quik et al., 2003). Because this technique offers the advantage that it allows for the quantitative determination of evoked-release under well defined experimental conditions, we conducted experiments to establish its feasibility using monkey synaptosomes. Because the striatum in primates is anatomically heterogeneous (Kemel et al., 1989; Haber et al., 2000), we measured the release separately in synaptosomes prepared from the caudate and putamen, as well as nucleus accumbens. We focused on portions closer to the midline (medial caudate and ventral putamen) where a denser distribution of -CtxMII-sensitive sites exists, especially in the medial caudate (Quik et al., 2001, 2002).

    Tissue concentration curves using caudate, putamen, and nucleus accumbens from control animals revealed a linear relationship for [3H]dopamine release between 0.5 and 5 mg (wet weight) tissue/sample (data not shown). With larger amounts of tissue, the release evoked by either nicotine or 20 mM K+ showed a tendency to plateau. Therefore, tissue was kept within a range of 0.5 to 2 mg for all release experiments.

    Nicotine stimulated the release of [3H]dopamine from monkey synaptosomes prepared from caudate, putamen, and nucleus accumbens in a concentration-dependent manner. A sample trace from a single stimulation in caudate from one monkey is shown in Fig. 2. Release evoked by 20 mM K+ was similar to that stimulated by a maximal concentration of nicotine (10 e) (Fig. 2).

    Experiments were done next in control monkey caudate to ascertain whether nicotine-evoked [3H]dopamine release was calcium-dependent and nAChR-receptor-mediated. The removal of Ca2+ from the release and uptake buffers resulted in no measurable dopamine release (3.2 mM Ca2+ = 4384 ± 1008 cpm versus 0 mM Ca2+ = 119 ± 72 cpm; p < 0.05; n = 3). This clearly demonstrated that nicotine-evoked [3H]dopamine release from monkey brain is a calcium-dependent process. A 3-min pretreatment of synaptosomes with 100 e mecamylamine, a noncompetitive nAChR antagonist, blocked nicotine-evoked [3H]dopamine release in caudate (p < 0.01; Table 1), whereas release stimulated by 20 mM K+ was unaffected by mecamylamine pretreatment (data not shown). Pretreatment of synaptosomes for 3 min with 50 nM -CtxMII, an 3/6 nAChR antagonist, blocked 70% [3H]dopamine release (p < 0.01; Table 1), suggesting that dopamine release in monkey striatum is largely mediated by 3/6 nAChRs. A 30-min incubation of synaptosomes with 1 e -bungarotoxin had no effect on release stimulated by either 20 mM K+ (data not shown) or nicotine (Table 1), suggesting that striatal nicotine-evoked [3H]dopamine release is not mediated by 7 nAChRs, as in the rodent.

    Mean counts per minute (± S.E.M.) are from experiments performed in monkey caudate (n = 3-8 monkeys) and represent a single stimulation with 10 or 30 e nicotine.

    Nicotinic Receptors Containing 3 and/or 6 Subunits Mediate a Large Proportion of Nicotine-Evoked [3H]Dopamine Release in Monkey Brain. To identify the nAChR population(s) mediating nicotine-stimulated [3H]dopamine release in monkey striatum, release was performed in the presence and absence of -CtxMII (Kulak et al., 1997; Kaiser et al., 1998; Quik et al., 2003; Salminen et al., 2004). -CtxMII inhibition curves for nicotine-evoked [3H]dopamine release in monkey caudate yielded an IC50 value of 4.0 nM (95% confidence interval, 0.64 to 21.2 nM; n = 2) with maximal inhibition at 30 nM -CtxMII, similar to the mouse (Salminen et al., 2004). Therefore, we used 50 nM -CtxMII in the present experiments to provide a maximal blockade of release in monkey brain. The component of dopamine release abolished by 50 nM -CtxMII was labeled "-CtxMII-sensitive", whereas that remaining in the presence of -CtxMII was considered "-CtxMII-resistant". In control monkey caudate, -CtxMII inhibited 70% of nicotine-evoked [3H]dopamine release (Table 2); thus, 70% of dopamine release was -CtxMII-sensitive and mediated by 3/6 nAChRs, whereas 30% was resistant to inhibition by -CtxMII. The proportion of -CtxMII-sensitive dopamine release was similar in putamen (70%) and nucleus accumbens (80%) (Table 2).

    Note that -CtxMII-sensitive nicotine-evoked dopamine release represents 70% of the total dopamine release in control monke y striatum compared with 30% in the rodent. In nucleus accumbens, 80% dopamine release was mediated via 3/6 nAChR. Rmax values (± S.E.M.) expressed as counts/minute were obtained from nonlinear curve fits of dose-response curves (n = 6-8 monkeys/group)

    Declines in Dopamine Transporter and Nicotinic Receptor Binding Accompany Behavioral Deficits after Damage to the Nigrostriatal System. We measured striatal dopamine transporter levels, behavioral ratings of parkinsonism, and striatal 125I-epibatidine binding after MPTP exposure to evaluate the behavioral and biochemical changes after nigrostriatal damage.

    As a marker of dopamine terminal integrity, the dopamine transporter was measured in caudate, putamen, and nucleus accumbens using [125I]RTI-121 binding. The MPTP regimen used in this study resulted in a 50% decline in the dopamine transporter in both caudate (p < 0.01; Fig. 3) and putamen (p < 0.01; Fig. 3) measured using [125I]RTI-121. The mean [125I]RTI-121 binding in caudate was 28.5 ± 1.9 fmol/mg of tissue (n = 7) in control animals and 12.1 ± 2.5 fmol/mg of tissue (n = 10) in MPTP-lesioned animals. In putamen, these values were 23.0 ± 2.1 fmol/mg of tissue in control (n = 7) and 10.1 ± 2.3 fmol/mg of tissue (n = 10) in lesioned animals. These values are for medial caudate and ventral putamen, consistent with the striatal areas used for the release studies. Dopamine transporter levels in the nucleus accumbens were not decreased by MPTP treatment (p = 0.24; Fig. 3). The mean [125I]RTI-121 binding in this region was 12.03 ± 1.27 fmol/mg of tissue for control animals (n = 4) and 17.88 ± 3.20 fmol/mg of tissue for lesioned animals (n = 7).

    After MPTP or saline treatment, we also determined the behavioral effects of nigrostriatal damage in these same animals. Parkinsonism averaged 6.3 ± 1.6 of a maximum of 20 (n = 10) after MPTP treatment, similar to that previously obtained (Quik et al., 2001). Control monkeys showed no signs of parkinsonism.

    MPTP exposure decreased total 125I-epibatidine binding in the medial caudate (44%; p < 0.01) and ventral putamen (44%; p < 0.01; Fig. 3). Total 125I-epibatidine binding in caudate was 0.50 ± 0.05 fmol/mg of tissue (n = 7) for control animals and 0.29 ± 0.02 fmol/mg of tissue (n = 9) for MPTP-treated animals. In putamen, these values were 0.49 ± 0.05 fmol/mg of tissue in control animals (n = 7) and 0.28 ± 0.04 fmol/mg of tissue in MPTP-treated animals (n = 9). These declines were somewhat greater than previously observed (Kulak et al., 2002; Quik et al., 2002), probably because of interanimal variability. Total 125I-epibatidine binding in nucleus accumbens was unaffected after MPTP treatment (1.47 ± 0.06 versus 1.39 ± 0.05 fmol/mg; n = 4-5) (Fig. 3).

    Reduction in Total Nicotine-Evoked [3H]Dopamine Release Is Smaller than Declines in Striatal Dopamine Transporter Levels or [125I]Epibatidine Binding after MPTP Exposure. In marked contrast to the consistent decreases in striatal [125I]RTI-121 and 125I-epibatidine binding, nicotine-evoked [3H]dopamine release was less affected or unaffected by MPTP administration. Representative nicotine and K+-evoked [3H]dopamine release traces in control and MPTP lesioned monkey striatum are shown in Fig. 4. In caudate (Fig. 4, left), a decrease in nicotine-evoked [3H]dopamine release was observed. A determination of maximal response (Rmax) was generated from nicotine dose-response curves (see Materials and Methods). In caudate, nicotine-evoked [3H]dopamine release was significantly reduced (p < 0.01; Fig. 3) but by a lesser extent (30%) than the dopamine transporter (44%) in this region. The mean Rmax value for nicotine-evoked release in caudate from control animals was 4800 ± 302 cpm/mg of tissue, and MPTP lesioning decreased this value to 3626 ± 136 cpm/mg of tissue.

    However, in putamen (Fig. 4, right), no reduction of nicotine-evoked [3H]dopamine release occurred after MPTP treatment, despite a nearly 50% decrease in dopamine transporter levels (as measured by [125I]RTI-121 binding) and 125I-epibatidine binding (Fig. 3). Rmax values for dopamine release in putamen were 3620 ± 236 cpm/mg of tissue for control animals and 4050 ± 97.6 cpm/mg of tissue for MPTP-treated animals (Fig. 3). Nicotine-evoked [3H]dopamine release was also not decreased in the nucleus accumbens after MPTP treatment and, in fact, showed a tendency for an increase, a finding not unexpected because of the lack of reduction of dopamine transporter binding in this region.

    [3H]Dopamine release evoked by 20 mM K+ was examined for control and MPTP-treated monkeys (Fig. 4, middle). Although a 17% decrease in K+-evoked release was observed after lesioning in caudate, this was not significant (p = 0.20; Table 3). In fact, no significant decreases in K+-evoked release were found after MPTP treatment in any region measured.

    Mean (± S.E.M.) values for K+-evoked release are shown for control and MPTP-treated monkeys. No differences in K+-evoked release were observed between treatment groups in any region measured.

    We investigated whether there were differences in basal [3H]dopamine release in caudate, putamen, and nucleus accumbens with lesioning that may account for the differential declines in nicotine-evoked release in caudate compared with putamen. Declines in basal release correlated with the dopamine transporter in a similar manner in the caudate (r = 0.78; p < 0.01) and putamen (r = 0.80; p < 0.01) as shown in Fig. 4. No decreases in basal release were observed with MPTP treatment in nucleus accumbens (data not shown).

    Reduced Nicotine-Evoked [3H]Dopamine Release in Caudate of Lesioned Monkey Is the Result of Selective Reduction of -CtxMII-Sensitive Sites. To evaluate the effect of MPTP treatment on 3/6 nAChRs, 125I-epibatidine binding and nicotine-evoked [3H]dopamine release were performed in the presence of -CtxMII. There were no significant declines in -CtxMII-resistant 125I-epibatidine-binding sites after MPTP treatment in any region examined (Table 4). Likewise, nigrostriatal damage resulted in no measurable reduction in -CtxMII-resistant [3H]dopamine release in striatum or nucleus accumbens (Table 4). In fact, a 20% increase in -CtxMII-resistant release from nucleus accumbens was observed in lesioned animals compared with controls (p < 0.01; n = 6-7).

    In contrast to the lack of effect of MPTP treatment on -CtxMII-resistant binding, there was a decrease in 3/6 nAChRs in both caudate and putamen, determined using either 125I--CtxMII binding or -CtxMII inhibition of 125I-epibatidine binding. 125I--CtxMII binding was significantly decreased in caudate (72%; p < 0.01) and putamen (56%; p < 0.01) (Fig. 5). Likewise, -CtxMII-sensitive 125I-epibatidine binding was significantly reduced by 50% in both caudate (p < 0.05; Fig. 5) and putamen (p < 0.05; Fig. 5) after nigrostriatal damage.

    It is interesting that a significant decrease in 3/6 nAChR binding was also found in the nucleus accumbens of MPTP-treated monkeys. The -CtxMII-sensitive component of 125I-epibatidine binding was reduced by 62% (Fig. 5). Mean binding levels were 0.31 ± 0.03 fmol/mg of tissue in control animals (n = 11) and 0.13 ± 0.04 fmol/mg of tissue in lesioned animals (n = 5; p < 0.01). 125I--CtxMII binding was also reduced by 60% after lesioning (0.52 ± 0.04 versus 0.21 ± 0.03 fmol/mg of tissue, p < 0.01; n = 3-4) (Fig. 5).

    Decreases in 3/6 nAChR binding were accompanied by a reduction in -CtxMII-sensitive nicotine-evoked [3H]dopamine release only in caudate where the Rmax for nicotine-stimulated release was reduced by 43% (p < 0.01; Fig. 5). The Rmax for striatal dopamine release in untreated animals was 3261 ± 241 cpm, and the Rmax for release in lesioned animals was 1862 ± 96 cpm. Despite reductions in 3/6 nAChR binding in both putamen and nucleus accumbens, no decreases in -CtxMII-sensitive nAChR function were observed in these regions (Fig. 5).

    Nicotine Stimulates -CtxMII-Sensitive Dopamine Release Significantly More Potently than -CtxMII-Resistant Release in Monkey Brain. EC50 values for -CtxMII-sensitive and -resistant release were generated from the dose-response curves from control and MPTP-lesioned monkeys (Fig. 6; Table 5). Regardless of treatment group or region, the -CtxMII-sensitive component of dopamine release consistently exhibited a 2- to 5-fold higher affinity for nicotine than the -CtxMII-resistant component. MPTP treatment had no significant effects on EC50 values for either the -CtxMII-resistant or the -CtxMII-sensitive component of dopamine release (Table 5). The EC50 values for nicotine-evoked release in control monkey brain were approximately 0.5 e in each brain region measured (Table 5). These values are similar to those for nicotine-evoked dopamine release from mouse striatal synaptosomes (Salminen et al., 2004).

    Note that -CtxMII-sensitive sites exhibit a 2- to 5-fold higher affinity for nicotine-evoked dopamine release. EC50 values (mean ± S.E.M.) are from five to eight animals/group.

    The analyses of the dose-response curves by two-way ANOVA were in agreement with previous comparisons of the Rmax values with reductions in dopamine release after MPTP treatment only in caudate. In this region, both total release [two-way ANOVA by treatment: F(1,130) = 6.5; p < 0.05; Fig. 6] and -CtxMII-sensitive release [F(1,127) = 6.7; p < 0.01] were significantly affected by nigrostriatal damage but dopamine release in putamen [F(1,132) = 0.60; p = 0.44] and nucleus accumbens [F(10,103) = 1.29; p = 0.26] was not reduced by MPTP treatment.

    Discussion

    This investigation is the first report of nicotine-evoked [3H]dopamine release using striatal synaptosomes from non-human primates, a species selected for our studies because of its usefulness as a model for PD. Our data demonstrate robust dopamine release mediated by both -CtxMII-sensitive (3/6) and -resistant (4) nAChR subtypes, similar to previous measurements from rodent striatum. However, distinct from the rodent, a substantially greater proportion (70%) of release was mediated through the 3/6 nAChR population. Furthermore, after nigrostriatal damage, we show that there is a decline in 3/6 nAChR-evoked [3H]dopamine release in caudate but not putamen, although lesioning decreased 3/6 nAChRs in both caudate and putamen. A dissociation between the number of 3/6 nAChR sites and the amount of nicotine-evoked dopamine release was also seen in the nucleus accumbens with a decline in 3/6 receptor binding but no decrease in 3/6 receptor-evoked release. These results demonstrate that 3/6 nAChR-mediated dopamine release can function at control levels in the presence of a partial receptor loss in selected dopaminergic pathways.

    Stimulation-evoked release has previously been measured from control monkey striatum in vitro using voltammetry (Cragg et al., 2000); nicotine-stimulated release has been measured in vivo with positron emission tomography (Marenco et al., 2004). However, nicotine-evoked [3H]dopamine release has not yet been investigated in control monkey brain or after nigrostriatal damage. The control studies reported here show that nicotine elicited a robust calcium-dependent increase in dopamine release in both striatum and nucleus accumbens with EC50 values of 0.5 e, similar to that in mouse (Salminen et al., 2004). The pharmacology of dopamine release was comparable with that in the rodent with a complete block with the nonselective nAChR antagonist mecamylamine, a partial block with the 3/6 antagonist -CtxMII, and no effect of the 7 receptor antagonist -bungarotoxin. These combined data suggest the involvement of non-7 nAChRs in release, including 3, 4, and/or 6 subtypes.

    Although there are many similarities in release characteristics in monkey and mouse brain, one important distinction is the ratio of -CtxMII-sensitive versus -resistant nAChR-mediated dopamine release. In rodents, 30% striatal dopamine release is sensitive to inhibition by -CtxMII (Kulak et al., 1997; Quik et al., 2003; Salminen et al., 2004). In contrast, in both monkey striatum and nucleus accumbens, 70% nicotine-evoked [3H]dopamine release was blocked by the toxin. This species difference in release may be related to the greater proportion of -CtxMII-sensitive nAChRs measured by binding assays in monkey compared with rodent brain (Whiteaker et al., 2000; Quik et al., 2001, 2003). Although the molecular basis for this interspecies heterogeneity in nAChR subtype proportions remains unknown, it probably reflects species differences in nAChR composition. -CtxMII interacts with both 3 and 6 nAChRs (McIntosh et al., 2004). However, studies using 3- and 6-null mutant mice have shown that primarily 6 but not 3 nAChR subtypes are found in mouse striatum (Champtiaux et al., 2002; Whiteaker et al., 2002; Zoli et al., 2002). In contrast, recent immunoprecipitation and radioligand binding studies demonstrate that both 3 and 6 subtypes are located in monkey striatum (Quik et al., 2005). Thus, the presence of 3 nAChR may contribute, at least in part, to the greater proportion of -CtxMII-sensitive receptors in monkey striatum.

    Our previous work has shown that striatal nAChR-binding sites in monkeys were differentially affected by moderate nigrostriatal damage with a selective decline in 3/6 nAChRs and no change in subtypes containing other subunits such as 4 (Quik et al., 2001). The present results (summarized in Table 6) show that, in caudate, only the -CtxMII-sensitive component (3/6 receptors) of nicotine-stimulated [3H]dopamine release was decreased after nigrostriatal damage. This was consistent with the receptor decline and with the dopamine transporter decline (Quik et al., 2001). In addition, the present data show that there was no significant decrease in -CtxMII-resistant function (4 nAChRs), in agreement with the lack of effect of moderate MPTP treatment on -CtxMII-resistant-binding sites (Quik et al., 2001). The small but nonsignificant decrease in K+-evoked [3H]dopamine release in caudate may suggest that function in only a subset of striatal dopaminergic terminals is affected by moderate nigrostriatal damage.

    All values are expressed as a percentage of the unlesioned control samples. Note the selective functional preservation of 3/6 receptor-evoked dopamine release in monkey putamen and nucleus accumbens after nigrostriatal damage (boldface). Values for the 3/6 subtype represent the average of the -CtxMII-sensitive and 125I--CtxMII sites, because these two measures were very similar (see Fig. 5).

    Similar to the caudate, neither -CtxMII-resistant binding sites nor evoked-dopamine release mediated by these sites was significantly decreased in the putamen after nigrostriatal damage. As in the caudate, 3/6 nAChRs, as well as the dopamine transporter, were decreased 50% in the putamen with moderate nigrostriatal damage. We were surprised to find, however, that 3/6 receptor-mediated dopamine release in putamen was not reduced with nigrostriatal damage. Thus, the functional consequences of lesioning are quite distinct in the caudate and putamen, despite similar receptor changes in the two regions after MPTP treatment,

    The regulation of nAChR expression in the mesolimbic system seems distinct from that in the nigrostriatal system, as evidenced by the differential pattern of changes obtained in nucleus accumbens in 3/6 nAChR sites and function after MPTP treatment (summarized in Table 6). The nucleus accumbens receives dopaminergic projections from the ventral tegmental area, a region more resistant to MPTP treatment than the substantia nigra, with no decline in the dopamine transporter (Hung and Lee, 1996). Consistent with such a finding, no decrease was observed in -CtxMII-resistant nAChR number or function; in fact, release was increased. However, we observed an unexpected decline in 3/6 nAChR binding, measured using either 125I--CtxMII- or -CtxMII-sensitive 125I-epibatidine binding. One possible interpretation of these findings is that dopamine 3/6 nAChRs in the nucleus accumbens are present on a very small proportion of the dopamine terminals that are selectively vulnerable to MPTP treatment. Another possibility is that nucleus accumbens 3/6 nAChRs are down-regulated by striatal inputs that are lost with nigrostriatal damage. Despite the decline in 3/6 nAChRs, -CtxMII-sensitive dopamine release remained intact in nucleus accumbens, providing another example of functional sparing of 3/6 nAChRs similar to that seen in putamen.

    Lesion-induced changes previously measured in mouse brain (Quik et al., 2003) had a pattern different from that seen in any region measured in monkey brain. In mouse striatum, both -CtxMII-sensitive (6) and -resistant (4) components of dopamine release were reduced to the same extent with a moderate lesion (Quik et al., 2003). In contrast, the present data show that only -CtxMII-sensitive and not -CtxMII-resistant nAChR function is affected by moderate nigrostriatal damage in nonhuman primates. These variations most probably relate to species differences in nicotinic receptor distribution, number, or regulation, and/or the mechanisms that mediate nigrostriatal damage in the two species. These data suggest that species heterogeneity in striatal dopamine function must be considered when extrapolating responses from rodent to primate brain.

    An extensive literature indicates that the basal ganglia are anatomically heterogeneous and composed of distinct nigral neuronal populations that project to divergent striatal areas (Parent et al., 1983; Fearnley and Lees, 1991; Haber et al., 2000). Moreover, various neurotransmitter receptors, transporters, and enzymes are organized in distinct ventromedial-to-dorsolateral gradients in the striatum (Kemel et al., 1989; Haber et al., 2000). Thus, the differential effects in nAChR-evoked dopamine release in the caudate and putamen may be possible. Although the dopamine transporter and 3/6 nAChRs were decreased in both caudate and putamen by nigrostriatal damage and a similar percentage decline in 3/6-mediated release was found in caudate, the function in the putamen was unaffected. A different pattern was seen in nucleus accumbens where there was no change in dopamine transporter, a decrease in 3/6 nAChRs, and, as in putamen, no change in 3/6 nAChR function. In fact, a slight increase in function was observed in nucleus accumbens. These data indicate that function can be maintained in the presence of a receptor decline in both putamen and nucleus accumbens. Although the mechanism(s) by which this enhanced evoked-release in selected regions may occur is not presently known, the possibilities include: 1) spare receptors or a larger intracellular pool of receptors in some regions that can be mobilized to the nerve terminal surface under conditions of damage; 2) enhanced coupling of nicotinic receptors to the dopamine exocytotic process; 3) a change in release properties through decreased autoinhibition by the D2 receptor in the presence of lower synaptic dopamine levels; and/or 4) compensatory increases in the vesicular monoamine transporter, such that quantal release from vesicles is enhanced. There may also be selective recovery of dopaminergic nerve terminals in the putamen, although compensatory sprouting and other regenerative changes generally tend to occur over a longer time frame (Stanic et al., 2003; Lai et al., 2004).

    It is interesting that PD is associated with differential declines in the dopamine transporter and dopamine levels by region in striatum. There is an uneven pattern of dopaminergic nerve terminal damage with a more severe decline in dopamine levels and the dopamine transporter in putamen (80-90%) than caudate (50%) (Kish et al., 1988; Brooks et al., 1990). The present data suggest that mechanisms develop with nigrostriatal damage to maintain nicotine-evoked dopamine release in the form of enhanced nicotinic receptor-evoked function, possibly because of the critical role of dopamine in movement and posture control.

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