Introduction

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Parkinson's disease (PD) is a neurodegenerative disorder characterized by a progressive loss of dopamine neurons in the substantia nigra (Lang and Lozano, 1998). The ensuing dopaminergic deficit results in motor symptoms that are relieved with administration of the dopamine precursor L-dopa. However, motor and other complications develop with long-term use of L-dopa, and furthermore, this drug does not halt disease progression. These observations raise the need for alternative therapeutic approaches. Accumulating evidence suggests that activation of nicotinic acetylcholine receptors (nAChRs) may have therapeutic potential for PD. This is based on findings showing 1) an apparent protective effect of tobacco use on PD (Morens et al., 1995; Quik and Jeyarasasingam, 2000), 2) positive effects of nicotine administration on parkinsonian symptomatology in humans (Kelton et al., 2000) and in monkeys (Schneider et al., 1998), and 3) the ability of nicotine to stimulate dopamine release in the caudate-putamen (MacDermott et al., 1999).

Multiple nAChR subunits have been identified in the basal ganglia of rodents and nonhuman primates, including 2 to 7 and 2 to 4 (Jones et al., 1999; Quik et al., 2000a; Han et al., 2000). Although the composition of basal ganglia nAChRs is still uncertain, the presence of these transcripts would allow for numerous pentameric subunit combinations. For example, the majority of nAChRs expressed in rodent brain that bind [³H]nicotine and [³H]cytisine seem to be composed of 4 and 2 subunits (Flores et al., 1991; Davila-Garcia et al., 1997); however, receptor studies using [³H]epibatidine, ¹²⁵I--bungarotoxin, and ¹²⁵I--conotoxin MII indicate that 7- or 3/6-containing nAChRs are also present (Marks et al., 1986; Whiteaker et al., 2000c). In human basal ganglia, [³H]nicotine, [³H]cytisine, and [³H]epibatidine binding sites have been identified (Gotti et al., 1997; Court et al., 2000; Perry et al., 2000), but the nAChR subtypes contributing to the binding sites have not been extensively characterized. The subtypes of nAChRs expressed in control and PD brains is an important issue when considering the therapeutic potential of nicotinic ligands. Although nAChR expression declines in the caudate-putamen and substantia nigra of PD brains (Gotti et al., 1997; Court et al., 2000; Perry et al., 2000), a large portion (30 to 70%) of the receptors remains as potential therapeutic targets.

As an approach to determine the nAChRs that may be altered with nigrostriatal degeneration in primates, we initiated a series of experiments using squirrel monkeys treated with the nigrostriatal toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). This treatment results in loss of dopaminergic terminals in the caudate-putamen, decreased dopamine levels, and symptoms similar to idiopathic PD (Przedborski et al., 2001). For this study, we used ¹²⁵I-epibatidine, a ligand that binds with high affinity to nAChRs containing 2 to 6 subunits (Davila-Garcia et al., 1997). The work indicates that in MPTP-induced parkinsonism, there is a preferential loss of a specific subset of nAChRs recognized by ¹²⁵I-epibatidine.

	Experimental Procedures

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Materials. ¹²⁵I-Epibatidine (2200 Ci/mmol) and [¹²⁵I]RTI-121 (2200 Ci/mmol) were purchased from PerkinElmer Life Sciences (Boston, MA). Nicotine hydrogen tartrate and cytisine were obtained from Sigma (St. Louis, MO), and A85380 was obtained from Fisher Scientific (Pittsburg, PA). -Conotoxin MII was synthesized as described previously (Cartier et al., 1996).

Animals. Twenty adult, drug-free squirrel monkeys (Saimiri sciureus) were used for these studies (Osage Research Primates, Osage Beach, MO). They were housed individually on a 13-h light/11-h dark cycle and fed once daily with free access to water. MPTP treatment and behavioral testing was done as described previously (Quik et al., 2001). Briefly, baseline locomotor activity was evaluated daily for a 1-h period for 8 to 11 consecutive days using a computerized movement monitor cage (Quik et al., 2001). Animals were then treated with saline or 2 mg/kg MPTP s.c. Starting 2.5 weeks after treatment, locomotor activity was again measured for a 10-day period. The severity of the parkinsonian syndrome after MPTP treatment was rated using a modified monkey parkinsonian rating scale (Langston et al., 2000; Quik et al., 2000b). Five clinical parameters were evaluated including spatial hypokinesia, body bradykinesia, manual dexterity, balance, and freezing; each of which has a 5-point range with 0 being normal and 4 being severely affected, allowing for a composite score ranging between 0 (normal) to 20 (severely parkinsonian). If the parkinsonian scored was less than 3, monkeys were given a second injection of MPTP at a lower dose (1.75 mg/kg) because the animals were usually more susceptible to MPTP with the second injection. Monkeys were killed 4 weeks after the final MPTP injection in accordance with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association. Ketamine hydrochloride (15-20 mg/kg i.m.) was administered to sedate the animals, followed by an injection of 0.22 ml/kg i.v. euthanasia solution (390 mg of sodium pentobarbital and 50 mg of phenytoin sodium/ml).

Tissue Preparation. The brains were removed, chilled, cut into 6-mm-thick blocks, quick frozen in isopentane on dry ice, and kept at 80°C until use. Twenty-micrometer-thick brain sections were prepared at 20°C in a Leica cryostat, thaw mounted onto poly-L-lysine coated slides, dried, and stored at 80°C. A squirrel monkey atlas (Emmers and Akert, 1963) was used to identify different brain regions in Nissl-stained tissue sections from each monkey. Level assignments indicate the distance anterior (in millimeters) to the interaural line; for example, level A15.0 is 15 mm anterior to the interaural line.

¹²⁵I-Epibatidine Binding. Binding was conducted as described previously (Quik et al., 2000b). Briefly, 20-µm-thick monkey tissue sections were thawed and incubated with or without competing ligand at room temperature for 40 min in buffer (50 mM Tris, pH 7.0, 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl₂, and 1.0 mM MgCl₂) plus ¹²⁵I-epibatidine (2200 Ci/mmol; PerkinElmer Life Sciences). The concentration of radiolabeled ligand ranged from 0.01 to 0.08 nM, which is well below the K_d of 0.10 nM (P. Whiteaker, personal communication). Nonspecific binding was defined in the presence of 0.1 mM nicotine. Sections were washed twice for 5 min in buffer at 4°C and once for 10 s in ice-cold doubly distilled H₂O. After drying at room temperature, slides were exposed for 3 to 5 days to Hyperfilm -Max film (Amersham Biosciences, Piscataway, NJ).

[¹²⁵I]RTI-121 Binding. Dopamine transporter (DAT) density in the caudate-putamen was assessed using [¹²⁵I]RTI-121 binding as described previously (Quik et al., 2001). Monkey sections were preincubated twice for 15 min in preincubation buffer (50 mM Tris-HCl, pH 7.4, 120 mM NaCl, and 5 mM KCl). Sections were then incubated for 2 h with 50 pM [¹²⁵I]RTI-121 in preincubation buffer plus 0.025% bovine serum albumin and 1 µM fluoxetine. Nonspecific binding was determined in the presence of 100 µM nomifensine, a dopamine uptake inhibitor. Sections were washed four times for 15 min in ice-cold preincubation buffer, once for 10 s in ice-cold doubly distilled H₂O, dried, and exposed for 2 days to Hyperfilm max film.

Data Analysis and Quantitation. Quantitative differences in radioligand binding were determined by computer-assisted densitometry (ImageQuant, Molecular Dynamics, Sunnyvale, CA). Absorbances of autoradiographic film images were corrected for background and converted to femtomole per milligram of tissue by comparison with curves generated from known ¹²⁵I standards exposed to film with the sections. Absorbances for tissue sections and standards were within the linear range of the film. For the caudate and putamen, density was measured at level A14.5, A13.5, and A12.5, and values were averaged to obtain the amount of ¹²⁵I-epibatidine binding in each region per monkey.

	Results

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¹²⁵I-Epibatidine Binding in Control Monkey Brain. The regional distribution of nAChRs was evaluated in control monkey brain using ¹²⁵I-epibatidine (0.03 nM). ¹²⁵I-Epibatidine binding sites are distributed throughout the monkey brain, with the highest density in the medial habenula, interpeduncular nucleus, and thalamus (Fig. 1; Table 1). In the basal ganglia, binding was greatest in the substantia nigra, with moderate levels in the caudate-putamen and nucleus accumbens and low levels in the globus pallidus. Nonspecific binding in the presence of 100 µM nicotine was indistinguishable from film background.

View larger version (57K): [in this window] [in a new window]   Fig. 1.   Autoradiograms depicting the distribution of 125I-epibatidine binding sites at different anatomical levels throughout control monkey brain (A15.0 to A3.5). Note the presence of 125I-epibatidine sites in the caudate, putamen, and substantia nigra. Blank represents nonspecific binding in the presence of 100 µM nicotine. Scale bar is 5 mm. AC, anterior commissure; Amg, amygdala; CC, corpus callosum; Cd, caudate; Cxf, frontal cortex; Cxpa, parietal cortex; Cxpi, piriformis cortex; Cxt, temporal cortex; Hip, hippocampus; Hyp, hypothalamus; IP, interpeduncular nucleus; LG, lateral geniculate nucleus; MH, medial habenula; NAc, nucleus accumbens; OT, olfactory tubercle; Put, putamen; RN, reticular nucleus; SN, substantia nigra; Th, thalamus.

nAChR Ligands Differentially Inhibit ¹²⁵I-Epibatidine Binding. As an approach to identify nAChR subtypes present in monkey brain, ¹²⁵I-epibatidine inhibition studies were performed using nicotine, cytisine, A85380, and -conotoxin MII (Fig. 2). Nicotine, cytisine, and A85380 completely inhibited ¹²⁵I-epibatidine binding in all brain regions investigated and were not different from film background at the highest concentrations tested. In contrast, -conotoxin MII (3 µM) maximally inhibited binding by 50% in the caudate and putamen and did not affect binding in the frontal cortex or thalamus. The results suggest that monkey caudate-putamen express both -conotoxin MII-sensitive and -insensitive nAChR populations; each comprising ~50% of the total amount of ¹²⁵I-epibatidine sites.

View larger version (37K): [in this window] [in a new window]   Fig. 2.   125I-Epibatidine binding in control brain is differentially inhibited by nAChR ligands. Sections were incubated with 125I-epibatidine in the absence or presence of 0.1 pM to 10 µM nicotine, cytisine, A85380, or -conotoxin MII (-Ctx MII). Note that nicotine, cytisine, and A85380 completely inhibited binding at the highest concentrations tested, whereas -Ctx MII only blocked 50% of 125I-epibatidine binding sites in the caudate-putamen and had no affect on binding in the frontal cortex or thalamus. All curves fit best to a one-site model (see Table 2 for Ki values). Points are mean ± S.E.M. of three to four experiments. If no error bars are depicted, S.E.M. was within the size of the symbol.

MPTP Treatment Resulted in Nigrostriatal Damage and Motor Deficits. The effect of MPTP treatment was evaluated biochemically by measuring DAT density in the caudate and putamen and behaviorally by monitoring baseline locomotor activity and parkinsonism (Figs. 3 and 5). MPTP-treated animals were separated into two treatment groups as follows. Monkeys with striatal dopamine transporter levels reduced by about 70% were considered moderately lesioned; they exhibited 60% declines in locomotor activity compared with pretreatment values and had only mild parkinsonism (1.57 ± 0.32). In contrast, the group designated severely lesioned had 95% declines in the dopamine transporter in both caudate and putamen, exhibited a 90% decrease in baseline locomotor activity, and were decidedly parkinsonian (7.75 ± 0.89).

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Parkinson ratings and free activity in monkeys after MPTP treatment. Monkeys were administered 1.75 to 2.0 mg/kg MPTP or saline s.c., and behavioral testing was reevaluated 3 to 4 weeks after treatment, as described under Experimental Procedures. Monkeys were divided into control (Con), moderate (Mod), and severe (Sev) groups based upon the extent of nigrostriatal damage (see Results). Each value represents the mean ± S.E.M. of six to seven animals. *, p < 0.05 and **, p  0.01 to control; #, p < 0.05 to moderates.

Nigrostriatal Lesioning Decreases nAChR Expression in the Caudate-Putamen. Autoradiographs demonstrating the effect of MPTP treatment on nAChR and dopamine transporter expression are shown in Fig. 4. Monkeys with moderate (n = 7) and severe (n = 6) nigrostriatal damage had similar (50 and 57%, respectively) decreases in ¹²⁵I-epibatidine binding in the caudate and putamen (Fig. 5), whereas the frontal cortex and thalamus were unaffected (data not shown). In contrast, there were significant differences between moderate and severe treatment groups in the amount of decrease of dopamine transporter expression (70 versus 95%, p < 0.05).

View larger version (95K): [in this window] [in a new window]   Fig. 4.   MPTP treatment decreases nAChR expression in the caudate-putamen. Film autoradiograms of representative sections from a control and severely lesioned (MPTP) monkey. Note the selective decrease in 125I-epibatidine binding (EPI, 0.03 nM) in the caudate (Cd) and putamen (Put). Decreases in [125I]RTI-121 binding (0.05 nM) to DAT were used to evaluate the extent of nigrostriatal damage. Scale bar is 5 mm.

View larger version (42K): [in this window] [in a new window]   Fig. 5.   Quantitative analysis of declines in nAChR and dopamine transporter expression in the caudate and putamen of parkinsonian monkeys. Monkeys were administered 1.75 to 2.0 mg/kg MPTP or saline s.c., as described under Experimental Procedures. Alterations in nAChR expression were investigated using 125I-epibatidine binding (0.03 nM) and decreases in the dopamine (DA) transporter were studied using [125I]RTI-121 (0.05 nM). Note that nAChR expression is decreased ~50% in monkeys, with either moderate (Mod, n = 7) or severe (Sev, n = 6) nigrostriatal lesions compared with controls (Con, n = 7), whereas dopamine transporter expression is differentially affected. Each bar represents the mean ± S.E.M. of the indicated number of animals. *, p < 0.001 compared with control; #, p < 0.05 to moderate.

-Conotoxin MII-Sensitive nAChRs Are Selectively Affected by MPTP Lesioning. We investigated the possibility that -conotoxin MII-sensitive nAChRs are selectively decreased after nigrostriatal damage using -conotoxin MII inhibition of ¹²⁵I-epibatidine binding in the caudate and putamen of control, moderate, and severely lesioned monkeys (Fig. 6). The four moderate monkeys used for these experiments averaged slightly higher DAT levels (~37% of control) and ¹²⁵I-epibatidine binding (~75% of control) compared with the total moderate treatment group (n = 7), whereas the DAT levels (~5% of control) and ¹²⁵I-epibatidine binding (~40% of control) of the five severely lesioned monkeys were similar to the entire severe treatment group (n = 7). Interestingly, the inhibition curves of moderately lesioned monkeys overlap those of controls at high concentrations of -conotoxin MII, implying that decreases in ¹²⁵I-epibatidine binding in these animals are due to a selective loss of -conotoxin MII-sensitive nAChRs. -Conotoxin MII does not inhibit ¹²⁵I-epibatidine binding in severely lesioned monkeys, which correlates with our previous results using ¹²⁵I--conotoxin MII (Quik et al., 2001). At 1 µM -conotoxin MII, the amount of ¹²⁵I-epibatidine binding is the same for control and moderately lesioned monkeys (~50%), whereas severely lesioned monkeys show an additional ~15% decrease in ¹²⁵I-epibatidine binding (p < 0.05). This implies that -conotoxin MII-sensitive nAChRs are selectively decreased with nigrostriatal lesioning and that severe dopaminergic deficits are necessary to affect expression of other receptor subtypes.

View larger version (29K): [in this window] [in a new window]   Fig. 6.   -Conotoxin MII-sensitive nAChRs are selectively decreased in the caudate-putamen after nigrostriatal damage. 125I-Epibatidine inhibition studies using -conotoxin MII were performed in the caudate and putamen of control (Con), moderate (Mod), and severely (Sev) lesioned monkeys. The putative nAChR subtypes present in the caudate-putamen, and their relative proportions in the treatment groups are indicated to the right (see Discussion). Points are mean ± S.E.M. of four monkeys for Mod and five monkeys for Con and Sev.

The ¹²⁵I-Epibatidine Sites That Remain in Severely Lesioned Monkeys Are Sensitive to Other nAChR Ligands. The results of nicotine, cytisine, and A85380 inhibition of ¹²⁵I-epibatidine binding in severely lesioned monkeys are presented in Fig. 7. As was the case with control animals, all inhibition curves in severely lesioned monkeys were monophasic. Furthermore, in all areas, the K_i values for cytisine and A85380 inhibition remained unchanged with MPTP treatment (Table 2), whereas there were statistically significant decreases in the K_i values in the caudate and putamen for nicotine (p < 0.05 to control, reflecting a change in the IC₅₀ from 10 to 5 nM). These results provide further evidence that cytisine and A85380 do not distinguish between the -conotoxin MII-sensitive and -insensitive nAChR populations.

View larger version (31K): [in this window] [in a new window]   Fig. 7.   -Conotoxin MII-insensitive nAChRs in the caudate of severely lesioned monkeys are sensitive to other nAChR ligands. 125I-Epibatidine inhibition studies were conducted using the nAChR ligands nicotine, cytisine, A85380, and -conotoxin MII in control and severely lesioned monkeys (MPTP). All curves fit best to a one-site model (see Table 2 for Ki values). Inhibition in putamen was similar. Points are mean ± S.E.M. of three to four experiments. If no error bars are shown, the S.E.M. was within the size of the symbol.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The results from the present study are the first to show that nicotinic receptors containing 6 and/or 3 are the predominant receptor population affected after moderate nigrostriatal damage in the monkey. Our data demonstrate that striatal ¹²⁵I-epibatidine sites can be subdivided into two populations of approximately equal magnitude based on sensitivity to the cone snail toxin -conotoxin MII and that, in these latter sites, nAChRs are selectively decreased after MPTP treatment. Previous work suggested that -conotoxin MII-sensitive receptors are present on dopaminergic neurons (Quik et al., 2001). Our present results show that in monkey striatum the nAChR populations have similar affinities for nicotine, cytisine, and A85380, that -conotoxin MII can distinguish between nAChR populations, and that -conotoxin MII-sensitive nAChRs comprise a large proportion (50%) of total ¹²⁵I-epibatidine sites. These combined data may indicate that the predominant presynaptic nAChR population on dopaminergic neurons is sensitive to -conotoxin MII and possibly contains 3 and/or 6 subunits.

The present results, combined with our previous work (Kulak and Quik, 2000; Quik et al., 2001), suggest that at least three populations of nAChRs are expressed in monkey caudate-putamen, distinguished by their sensitivity to the nAChR ligands -conotoxin MII and -bungarotoxin (Table 3). -Conotoxin MII-sensitive nAChRs contribute to 50% of ¹²⁵I-epibatidine binding sites, are selectively decreased with a moderate nigrostriatal lesion, and completely eliminated with severe nigrostriatal damage. -Conotoxin MII-insensitive nAChRs also contribute to 50% of ¹²⁵I-epibatidine binding sites, although they are only partially decreased (20 to 25%) with severe nigrostriatal damage. The third population of nAChRs expressed in the caudate-putamen do not bind ¹²⁵I-epibatidine but do bind ¹²⁵I--bungarotoxin, a ligand selective for 7-containing nAChRs (Kulak and Quik, 2000). 7-Containing nAChRs do not seem to be localized to dopaminergic neurons because 95% dopaminergic depletion in the caudate-putamen causes ¹²⁵I--bungarotoxin binding to increase 100 to 150% (Kulak and Quik, 2000). The nAChR populations that remain in the caudate-putamen after MPTP treatment are likely to be present on nondopaminergic neurons. They may be expressed at the presynaptic terminals of glutamatergic, GABAergic, serotonergic, or other striatal afferents, or postsynaptically on striatal GABAergic or cholinergic neurons (Gotti et al., 1997; Jones et al., 1999; MacDermott et al., 1999).

View this table: [in this window] [in a new window]   TABLE 3 nAChR populations expressed in monkey caudate-putamen Subunits are inferred from mRNA expression and sensitivity to nAChR ligands (Quik et al., 2000a,b)

The subtypes of nAChR that bind -conotoxin MII are currently under intense scrutiny. Early reports using Xenopus laevis oocytes or cell lines expressing simple / subunit combinations (such as 32 or 42) indicated that -conotoxin MII preferentially binds to nAChRs with an 32-interface (Cartier et al., 1996; McIntosh et al., 1999). Recent work using oocytes that expressed complex nAChRs composed of multiple  and  subunits, including 6, indicates that -conotoxin MII may also bind to nAChRs that contain an 6 subunit (Vailati et al., 1999; Kuryatov et al., 2000). Because 6 mRNA is prominently present in monkey substantia nigra (Quik et al., 2000a; Quik et al., 2000b; Han et al., 2000) and 3 mRNA is present in much lower abundance, if at all (Cimino et al., 1992; Han et al., 2000), these results may suggest that the primary presynaptic nicotinic receptor population is one that contains the 6 nicotinic receptor subunit. The  subunit present in combination with the 6 subunit in -conotoxin MII-sensitive nAChRs is also under investigation. Studies with knockout mice show that 2 and 3 subunits are necessary for the majority of -conotoxin MII binding in rodent striatum (Cordero-Erausquin et al., 2000; Grady et al., 2001). Oocyte expression with combinations of 3, 2, and 3 or 6, 2, and 4 subunits indicates that -conotoxin MII can bind to nAChRs containing combinations of 3/2/3 or 6/2/4 subunits (Kuryatov et al., 2000; McIntosh et al., 2000). Therefore, the -conotoxin MII-sensitive ¹²⁵I-epibatidine sites (~50%) in the primate caudate-putamen may contain 6, possibly in combination with 2, 3, or 4 nAChR subunits.

The population of ¹²⁵I-epibatidine binding sites insensitive to -conotoxin MII may contain 2, 4, or 5 acetylcholine recognition subunits expressed with 2, 3, and/or 4 nAChR subunits (Quik et al., 2000a; Han et al., 2000). In rodents, the majority of high-affinity [³H]nicotine and [³H]cytisine binding sites contain 4 and 2 subunits (Flores et al., 1991; Davila-Garcia et al., 1997). Nicotine, cytisine, and A85380 have high affinity for 42 nAChRs expressed in man, whereas other combinations, such as 22, 24, and 44, with or without 5, have marked decreases in affinities for these ligands (Ramirez-Latorre et al., 1996). Based on these findings, it is possible that the -conotoxin MII-insensitive component of ¹²⁵I-epibatidine binding consists of nAChRs containing at least an 42 interface, with or without 2, 5, 3, or 4.

The relative proportions and ligand affinities of the -conotoxin MII-sensitive and -insensitive nAChR populations in primate caudate-putamen seem to be different from those previously reported in rodent striatum. In monkey caudate-putamen, receptor binding studies show that ~50% of the ¹²⁵I-epibatidine sites are -conotoxin MII-sensitive, whereas in rodent striatum, the -conotoxin MII-sensitive nAChRs comprise only ~15% of epibatidine binding sites (Whiteaker et al., 2000c). In addition, throughout monkey brain, the nAChR populations sensitive and insensitive to -conotoxin MII have affinities for cytisine and nicotine in the low nanomolar range. In contrast, the nAChR populations expressed in rodent brain seem to have very different affinities for these ligands, with IC₅₀ values of 18 and 481 nM for cytisine and nicotine, respectively (Marks et al., 1998; Whiteaker et al., 2000a,b). It has previously been reported in rodents that [³H]nicotine and [³H]cytisine bind predominantly, if not exclusively, to 42 nAChRs (Flores et al., 1991; Davila-Garcia et al., 1997). In monkeys, the similar affinities that cytisine and nicotine have for the -conotoxin MII-sensitive and -insensitive nAChRs imply that [³H]nicotine and [³H]cytisine binding in the caudate-putamen of humans (Houghtling et al., 1995; Court et al., 2000) may not exclusively label 42 nAChRs and that the decreases in nAChR binding in PD brains could be due to loss of other nAChR subtypes, such as 3/6-containing nAChRs sensitive to -conotoxin MII.

The relationship of the nAChR populations expressed in the caudate-putamen to basal ganglia function in primates remains to be investigated. In rodents, activation of -conotoxin MII-sensitive nAChRs accounts for 40% of nicotine-evoked dopamine release from striatum (Kulak et al., 1997; Kaiser et al., 1998; Grady et al., 2001), although -conotoxin MII maximally inhibits ~15% of epibatidine binding (Whiteaker et al., 2000c). The results from this work show that in the striatum of monkeys, ~50% of the nAChRs that bind epibatidine are -conotoxin MII-sensitive. If the -conotoxin MII-sensitive nAChRs in the caudate-putamen of monkeys are involved in nicotine-evoked dopamine release in a manner similar to rodents, these nAChRs may contribute to the majority of presynaptic nicotine-evoked dopamine release in the caudate-putamen. The results presented here imply that drugs that activate -conotoxin MII-sensitive nAChRs may represent a novel nAChR population for increasing dopamine release in the caudate-putamen of primates.

Our data in the monkey suggest that the -conotoxin MII-sensitive nAChR population may represent a target for the treatment of PD symptoms because these receptors are selectively decreased after nigrostriatal degeneration. However, a question that arises is the therapeutic potential of nicotinic drugs in the presence of substantially reduced receptor expression. Although the receptor sites are greatly reduced in MPTP-treated monkeys, these animals also exhibited 95 to 99% declines in the striatal dopamine transporter. In contrast, striatal dopamine levels are less severely decreased (70 to 80%) in the early stages of PD (Lang and Lozano, 1998), with a potentially greater number of residual -conotoxin MII-sensitive nAChR. Postmortem studies with brains from individuals with PD are essential to clarify these issues.

Neuronal nAChRs may also represent a target for neuroprotective strategies to halt disease progression. Considerable epidemiological evidence demonstrates that cigarette smokers have a decreased risk for PD (Morens et al., 1995). The mechanism whereby smoking protects against nigrostriatal degeneration is not yet known, although animal studies indicated that nicotine is a potential candidate (Quik and Jeyarasasingam, 2000; Balfour and Fagerstrom, 1996). Considering the fact that PD symptoms develop in humans when 20 to 40% of dopamine levels remain, in contrast to monkeys, agonists directed to nAChRs containing 3/6 or 42 subunits may have the potential for neuroprotective benefits.

In summary, these studies indicate that monkey striatum expresses multiple nAChR populations discriminated by -conotoxin MII but not nicotine, cytisine, or A85380 and that the decrease in ¹²⁵I-epibatidine binding in the caudate-putamen of parkinsonian monkeys is due to a loss of -conotoxin MII-sensitive nAChRs. It has previously been shown that in Parkinson's disease there is a decrease in nAChR binding in the striatum but no decline in 3, 4, 7, or 2 immunoreactivity (Martin-Ruiz et al., 2000; Perry et al., 2000). 6-Containing nAChRs are primarily affected by nigrostriatal lesioning and severe damage is necessary to see an effect upon 42-containing nAChRs. Thus, in the early stages of PD, ligands directed toward 6-containing nAChRs (-conotoxin MII-sensitive) may be important for therapeutics, whereas nAChR ligands with a wider spectrum of activities may be more relevant for advanced PD.