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Subunit Composition of Nicotinic Receptors in Monkey Striatum: Effect of Treatments with 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine or L-DOPA

The Parkinson's Institute, Sunnyvale, California (M.Q., T.B., J.M.K.); Consiglio Nazionale delle Ricerche, Institute of Neuroscience, Cellular and Molecular Pharmacology, Department of Medical Pharmacology, University of Milan, Milan, Italy (S.V., F.C., C.G.); Department of Radiology, The Johns Hopkins University School of Medicine, Baltimore, Maryland (H.F.); and Department of Biology and Psychiatry, University of Utah, Salt Lake City, Utah (J.M.M.).

Received August 9, 2004; accepted October 5, 2004

	Abstract

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Nicotinic acetylcholine receptors (nAChRs) represent an important modulator of striatal function both under normal conditions and in pathological states such as Parkinson's disease. Because different nAChR subtypes may have unique functions, immunoprecipitation and ligand binding studies were done to identify their subunit composition. As in the rodent, 2, 4, 6, 2, and 3 nAChR subunit immunoreactivity was identified in monkey striatum. However, distinct from the rodent, the present results also revealed the novel presence of 3 nAChR subunit-immunoreactivity in this same region, but not that for 5 and 4. Relatively high levels of 2 and 3 subunits were also identified in monkey cortex, in addition to 4 and 2. Experiments were next done to determine whether striatal subunit expression was changed with nigrostriatal damage. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment decreased 6 and 3 subunit immunoreactivity by 80% in parallel with the dopamine transporter, suggesting that they are predominantly expressed on nigrostriatal dopaminergic projections. In contrast, 3, 4, and 2 subunit immunoreactivity was decreased 50%, whereas 2 was not changed. These data, together with those from dual immunoprecipitation and radioligand binding studies ([³H]cytisine, ¹²⁵I--bungarotoxin, and ¹²⁵I--conotoxin MII) suggest the following: that 623, 6423, and 32^* nAChR subtypes are present on dopaminergic terminals and that the 42 subtype is localized on both dopaminergic and nondopaminergic neurons, whereas 22^* and 7 receptors are localized on nondopaminergic cells in monkey striatum. Overall, these results suggest that drugs targeting non-7 nicotinic receptors may be useful in the treatment of disorders characterized by nigrostriatal dopaminergic damage, such as Parkinson's disease.

Because animal models represent an excellent first step, studies have been done in both rodents and monkeys to address this question. In rodents, numerous nAChR subunit mRNAs (2–7 and 2–4) have been localized to the substantia nigra (Marks et al., 1992; Le Novere et al., 1996; Whiteaker et al., 2000, 2002; Champtiaux et al., 2002). Moreover, receptor binding and antibody immunoprecipitation studies indicate that these transcripts are expressed with multiple nAChR subtypes present in the striatum, including those expressing 42, 425, 6223, and 623 (Whiteaker et al., 2000, 2002; Klink et al., 2001; Zoli et al., 2002; Champtiaux et al., 2003, 2002; Salminen et al., 2004). Nigrostriatal damage, produced by administration of the selective dopaminergic neurotoxins 6-hydroxydopamine or 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) results in losses of both 4^* and 6^* nAChR populations in rodents (Zoli et al., 2002; Champtiaux et al., 2003; Quik et al., 2003b). Moreover, these receptor losses are associated with functional deficits at both the cellular (Quik et al., 2003b) and behavioral (Le Novere et al., 1999) levels.

Studies to identify the nicotinic receptor subtypes and the effects of nigrostriatal damage and dopamine precursor treatment have also been done in nonhuman primates, which bear a close resemblance to humans at the genetic, molecular, and behavioral level. In addition, monkeys with nigrostriatal damage exhibit symptoms that resemble those in Parkinson's disease, with the motor deficits reversed by the same drug used to treat this disorder. Studies have shown that the 2–7 and 2–4 nAChR transcripts are present in monkey substantia nigra (Han et al., 2000; Quik et al., 2000a,b) and that binding sites for ¹²⁵I-epibatidine, [³H]cytisine, ¹²⁵I-A85380, ¹²⁵I--conotoxin MII, and ¹²⁵I--bungarotoxin are expressed in the striatum and substantia nigra (Quik et al., 2001; Kulak et al., 2002a,b; Han et al., 2003). Furthermore, there are differential changes in nAChRs after MPTP treatment, with a complete loss of ¹²⁵I--conotoxin MII sites and also declines in -conotoxin MII-resistant ¹²⁵I-epibatidine sites. Thus, radioligand binding studies suggest that 62^* and/or 32^*, as well as 42^*, nAChRs are present in monkey striatum, with preferential declines in 62^* and/or 32^* sites, and smaller losses in 42^*-expressing receptors with nigrostriatal damage. Treatment with L-DOPA, the most frequently used therapy for Parkinson's disease, also resulted in changes in nAChRs with a selective loss of a low-affinity -conotoxin MII-sensitive site (Quik et al., 2003a).

The objective of the present study was to further identify the nAChR subunit composition in monkey striatum and the effect of nigrostriatal damage and L-DOPA treatment on the different receptor populations. To approach this, receptor binding studies using nAChR-directed radioligands and immunoprecipitation experiments using subunit-selective antibodies were done in striata from control and treated monkeys.

	Materials and Methods

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Animals and Treatment
Adult squirrel monkeys (Saimiri sciureus) weighing 0.5 to 0.8 kg were purchased from Osage Research Primates (Osage Beach, MO), and quarantined upon arrival. They were housed in a 13-h/11-h light/dark cycle. They had free access to water and were given food pellets and fruit once daily. All procedures used conform to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. MPTP (2 mg/kg, s.c.) treatment was as described previously (Quik et al., 2000b). To evaluate the behavioral effects of the lesion, animals were rated for parkinsonism using a modified Parkinson rating scale for the squirrel monkey, in which the disability scores ranged from 0 to 20. The composite score was evaluated based on 1) spatial hypokinesia, 2) body bradykinesia, 3) manual dexterity, 4) balance, and 5) freezing. A group of unlesioned animals was administered L-DOPA (15 mg/kg) in combination with carbidopa by oral gavage twice daily, 4 h apart, on a 5-day/2-day on/off schedule for 8 weeks.

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, followed by injection of 0.22 ml/kg i.v. euthanasia solution (390 mg/ml of sodium pentobarbital and 50 mg/ml phenytoin sodium). When the heart had stopped, the brains were rapidly removed, placed in a mold, and cut into 6-mm blocks. These were frozen in isopentane on dry ice and stored at -80°C. Striatal and cortical tissue was dissected from half the brain and used for the antibody immunoprecipitation studies. The other half of the brain was used for the autoradiographic studies. Sections (20 µm) were cut using a cryostat, thaw-mounted onto poly-L-lysine–coated slides, air-dried, and stored at -80°C. For the receptor binding studies, MPTP-treated monkeys were separated into two groups as reported previously (Quik et al., 2001; Kulak et al., 2002a). Monkeys with striatal dopamine transporter levels 30% of control were defined as moderately lesioned, whereas those with transporter levels 5% of control were defined as severely lesioned. Only tissue from severely lesioned animals was used for the immunoprecipitation studies.

Antibody Production and Characterization
The polyclonal antibodies against the human 2, 3, 4, 5, 6, 2, 3, or 4 monkey nAChR peptide subunits (Table 1) were produced in rabbit as described previously (Zoli et al., 2002; Champtiaux et al., 2003) and affinity-purified. The peptides obtained from monkey or human sequences were located in the putative cytoplasmic loop between M3 and M4. The affinity-purified antisera were bound to CNBr-activated Sepharose at a concentration of 1 mg/ml, and the columns used for subtype immunopurification.

Because no cloned nAChR monkey subunits are available, the specificity of the antibodies produced against the human peptides was tested by quantitative immunoprecipitation experiments using extracts obtained from human embryonic kidney cells transfected with different combinations of the human 2, 3, 4, 5, 6, 2, and 4 subunits (a generous gift from Dr. E. Sher of Eli Lilly and Co Ltd, Bristol, UK) or from tissues obtained from wild-type and nAChR-null mutant mice. Triton X-100 (2%) extracts, labeled with 2 nM [³H]epibatidine, prepared from the transfected cells or from tissues obtained from wild-type or knockout animals, were incubated with the saturating concentrations of the antibodies directed against all the subunits. In these tissues, the antibodies recognized only the receptors containing the corresponding subunits. The immunoprecipitation capacity of these antibodies versus the human and rodent subtypes was very high (>80%) (Zoli et al., 2002; Champtiaux et al., 2003; Moretti et al., 2004). The anti-4, -6, -2, and -3 antibodies were also tested on monkey-purified subtypes (see Results), where they also had a very high immunoprecipitation capacity. Binding values 6% were at the detection level of the assay, so this value was used as our cut-off for subunit expression.

Preparation of Membranes and 2% Triton X-100 Extracts from Monkey Brain
Monkey striatum and cortex, obtained as described above, was separately homogenized in an excess of 50 mM sodium phosphate pH 7.4, 1 M NaCl, 2 mM EDTA, 2 mM EGTA, and 2 mM phenylmethylsulfonyl fluoride for 2 min using an UltraTurrax homogenizer. The homogenates were then diluted and centrifuged for 1.5 h at 60,000g. The homogenization, dilution, and centrifugation of the indicated tissue was performed twice, after which the pellets were collected, rapidly rinsed with 50 mM Tris HCl, pH 7, 120 mM NaCl, 5 mM KCl, 1 mM MgCl₂, 2.5 mM CaCl₂, and 2 mM phenylmethylsulfonyl fluoride and then resuspended in the same buffer containing a mixture of 20 µg/ml of each of the following protease inhibitors: leupeptin, bestatin, pepstatin A, and aprotinin. Triton X-100 at a final concentration of 2% was added to the washed membranes, which were extracted for 2 h at 4°C. The extracts from tissues were then centrifuged for 1.5 h at 60,000g and recovered. An aliquot of the resultant supernatants was collected for protein measurement using the bicinchoninic acid protein assay (Pierce, Rockford, IL) with bovine serum albumin as the standard.

[³H]Epibatidine Binding Assays for Immunoprecipitation Studies
Membrane binding experiments were performed by incubating membrane homogenates overnight with 2 nM [³H]epibatidine (56 Ci/mmol; Amersham Biosciences, Piscataway, NJ) at 4°C. To prevent binding of [³H]epibatidine to -bungarotoxin-binding receptors, membranes were preincubated with 2 µM -bungarotoxin and then with [³H]epibatidine. Specific radioligand binding was defined as total binding minus nonspecific binding determined in the presence of 100 nM unlabeled epibatidine. The 2% Triton X-100 extracts of tissues were preincubated with 2 µM -bungarotoxin for 3 h and then labeled with 2 nM [³H]epibatidine. Tissue extract binding was performed using DE52 ion-exchange resin (Whatman, Maidstone, UK) as described previously (Vailati et al., 1999).

Immunoprecipitation of [³H]Epibatidine-Labeled Receptors by Anti-Subunit–Specific Antibodies
Striatal and cortical extracts or purified receptors were preincubated with 2 µM -bungarotoxin, labeled with 2 nM [³H]epibatidine, and incubated overnight with a saturating concentration of affinity-purified IgG (20–30 µg; Sigma Chemical, St. Louis). The immunoprecipitation was recovered by incubating the samples with beads containing bound anti-rabbit goat IgG (Technogenetics, Milan, Italy). The level of antibody immunoprecipitation was expressed as the percentage of [³H]epibatidine-labeled receptors immunoprecipitated by the antibodies (taking the amount present in the 2% Triton X-100 extract solution before immunoprecipitation as 100%) or as femtomoles od immunoprecipitated receptors per milligram of protein.

For each purification experiment, the 2% Triton X-100 extract obtained from striatal membranes, prepared as described above, was incubated three times with 5 ml of Sepharose-4B bound anti-6 antibody to remove the 6^* receptors. The flow-through of the 6 column was analyzed for the subunit content of the remaining receptors and then incubated two times with 5 ml of anti-2 antibody bound to Sepharose-4B. The bound 2^* nAChRs were then eluted with the 2 peptide and analyzed for their subunit composition by quantitative immunoprecipitation.

The 2% Triton X-100 cortical extract was incubated with 5 ml of Sepharose-4B bound anti-4 antibodies to remove the 4 receptors. The bound receptors were eluted by competition with 100 µM concentrations of the corresponding 6 or 4 peptides used for antiserum production.

Receptor Autoradiography
[¹²⁵I]RTI-121 Autoradiography. [¹²⁵I]RTI-121 (2200 Ci/mmol; PerkinElmer Life and Analytical Sciences, Boston, MA) was used to measure binding to the dopamine transporter (Quik et al., 2001). Sections were preincubated twice for 15 min each in 50 mM Tris-HCl buffer, pH 7.4, containing 120 mM NaCl and 5 mM KCl. Incubation (2 h) was done in the same buffer plus 0.025% BSA, 1 µM fluoxetine, and 50 pM [¹²⁵I]RTI-121. The sections were washed four times for 15 min each at 4°C in preincubation buffer, dipped in ice-cold water, air-dried, and placed against Kodak MR film (PerkinElmer Life and Analytical Sciences) for 1 to 3 days with ¹²⁵I microscale standards (Amersham Biosciences). Nomifensine (100 µM) was used to define nonspecific binding.

¹²⁵I-Epibatidine Autoradiography. ¹²⁵I-Epibatidine binding to striatal sections was done as described previously (Perry and Kellar, 1995; Kulak et al., 2002a). In brief, sections were preincubated for 30 min, and then incubated for 40 min at room temperature in 50 mM Tris buffer, pH 7, 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl₂,and1mM MgCl₂, containing 0.015 nM ¹²⁵I-epibatidine (2200 Ci/mmol; PerkinElmer Life and Analytical Sciences). For competition studies, a concentration range of 10 pM to 10 µM -conotoxin MII was used. Sections were subsequently washed (4°C) for 5 min with buffer (2x) and for 10 s in ice-cold H₂O and then air-dried. They were exposed for 2 to 5 days to Kodak MR film (PerkinElmer Life and Analytical Sciences), together with ¹²⁵I standards (Amersham Biosciences). Nicotine (10 µM) was used to determine nonspecific binding, which was the same as film blank.

¹²⁵I-A-85380 Autoradiography. Preparation of ¹²⁵I-A85380 (specific activity, 1500 Ci/mmol) and binding to brain membranes was perfromed as described previously (Mukhin et al., 2000). Preincubation was for 20 min in the same buffer used for ¹²⁵I-epibatidine binding assays, followed by a 40-min incubation in fresh buffer containing ¹²⁵I-A-85380 (80 pM). Sections were washed in buffer at 4°C twice for 5 min each, followed by a 10-s wash in deionized H₂O (4°C). Air-dried slides were exposed to Kodak MR film (PerkinElmer Life and Analytical Sciences) for 1 to 2 days with ¹²⁵I standards (Amersham Biosciences). Nicotine (10 µM) was used to determine nonspecific binding, which was the same as film blank.

[³H]Cytisine Autoradiography. [³H]Cytisine (specific activity, 37.5 Ci/mmol; PerkinElmer Life and Analytical Sciences) binding was performed as described previously (Perry and Kellar, 1995; Sihver et al., 1998). Sections were incubated at room temperature for 60 min in buffer (50 mM Tris, pH 7, 120 mM NaCl, 5 mM KCl, 2.5 mM CaCl₂, and 1 mM MgCl₂) plus 2 nM [³H]cytisine. After incubation, sections were washed twice for 5 min each in buffer at 4°C and 1 x 10 s in ice-cold H₂O. After drying at room temperature, slides were exposed for 8 to 12 weeks to ³H-sensitive Hyperfilm (Amersham), along with ³H standards (American Radiolabeled Chemicals, Inc., St. Louis, MO). Nicotine (10 µM) was used to determine nonspecific binding.

¹²⁵I--Conotoxin MII Autoradiography. ¹²⁵I--conotoxin MII (specific activity, 2200 Ci/mmol) was synthesized and radiolabeled as described previously (Whiteaker et al., 2000). For assay (Whiteaker et al., 2000; Quik et al., 2001), sections were preincubated at room temperature for 15 min in binding buffer (144 mM NaCl, 1.5 mM KCl, 2 mM CaCl₂, 1 mM MgSO₄, 20 mM HEPES, and 0.1% BSA, pH 7.5) plus 1 mM phenylmethylsulfonyl fluoride. This was followed by a 1-h incubation at room temperature in binding buffer plus 0.5% BSA, also containing 5 mM EDTA, 5 mM EGTA, and 10 µg/ml each of aprotinin, leupeptin, and pepstatin A, and 0.5 nM ¹²⁵I--conotoxin MII. To terminate the assay, slides were rinsed for 30 s in binding buffer at room temperature followed by 30 s in ice-cold Buffer, two 5-s rinses in 0.1x binding buffer (0°C) and two washes in water (0°C). The sections were air-dried and exposed to Kodak MR film (PerkinElmer Life and Analytical Sciences) for 2 to 5 days together with ¹²⁵I-standards (Amersham Biosciences). Epibatidine (0.1 µM) was used to determine nonspecific binding.

¹²⁵I--Bungarotoxin Autoradiography. Sections were preincubated at room temperature in 50 mM Tris HCl, pH 7, for 30 min (Clarke and Pert, 1985). They were next incubated for 1 h in the same buffer containing 3 nM ¹²⁵I--bungarotoxin (specific activity 128 Ci/mmol, PerkinElmer Life and Analytical Sciences). The sections were then rinsed four times for 15 min each in ice-cold buffer and once in ice-cold water, air-dried, and placed against Kodak MR film for 1 to 2 weeks (PerkinElmer Life and Analytical Sciences). Nicotine (100 µM) was used to define nonspecific binding.

Analyses of Autoradiographic Data. A squirrel monkey (Saimiri sciureus) brain atlas was used to identify brain regions, as described previously (Quik et al., 2000a). The optical density values, determined using an ImageQuant system (Amersham Biosciences), were assessed by subtracting background from tissue values. This was followed by conversion to femtomoles per milligram of tissue using standard curves generated from radioactive standards simultaneously exposed to the films. Sample optical density readings were within the linear range of the film. Receptor binding data for any one animal represents the mean from one to two sections each from two or more independent experiments.

Competition curves were compared and best-fit to one- and two-site models using Prism (GraphPad Software, San Diego, CA). Statistical analyses were done using one-way analysis of variance followed by Newman-Keuls multiple comparison test where p 0.05 was considered significant. All values are expressed as the mean ± S.E.M. of the indicated number of animals.

	Results

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Characterization of nAChR Subunit Antibodies. The identification of nAChR subtypes in monkey brain relied on the use of a series of antisera raised against unique amino acid sequences of the different human or monkey subunits. All of the antibodies (except for the anti-3 antibody, which was not tested) selectively interacted with receptors expressing the appropriate human nAChR subunit in transfected HEK cells. Because of the sequence identity between 3 and 6 subunits, we also tested whether identification of 3^* nAChRs (14%) in striatum might be caused by cross-reactivity of the anti-3 antibodies with 6^* receptors; however, the 3 antibody recognized only 3% of purified 6^* receptors. In addition, the immunoprecipitation capacity and specificity of the antibodies was investigated on purified 6^* receptors obtained from striatum and on 4^* receptors purified from the cortex. We found that the 4, 6, 2, and 3 antibodies had an immunoprecipitation capacity of more than 60%. We did not consider the contribution of subunits to receptor composition that were immunodetected in amounts of 6% or less, and therefore minor nAChR subtypes may have been excluded from the analyses.

NAChR Subunit Expression in Control Monkey Striatum and Cortex. Experiments were first done to quantify the relative contribution of each nicotinic subunit to [³H]epibatidine binding present in the striatum. To approach this, we performed quantitative immunoprecipitation experiments using subunit-specific antibodies and [³H]epibatidine-labeled receptors. Receptor levels in control monkey striatum were 55.5 ± 4.1 and 69.6 ± 5.5 fmol/mg of protein in the membrane preparation and 2% Triton extract, respectively. The receptors immunoprecipitated by specific nAChR subunit antibodies (calculated as the percentage of the total number of [³H]epibatidine receptors) were: 2 (91%), 4 (55%), 6 (25%), 3 (18%), 3 (14%), and 2 (12%) (Fig. 1A). The 5 and 4 subunit containing receptors fell below the detection limit of the assay (6%). Values represent the mean ± S.E.M. of six immunoprecipitation experiments performed in duplicate for each antibody.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Immunoprecipitation analyses of the subunit composition of [3H]epibatidine receptors expressed in monkey striatum (A) and cortex (B). Triton X-100 (2%) membrane extracts from control monkey striatum or cortex were labeled with 2 nM [3H]epibatidine. Immunoprecipitation was done as described using saturating concentrations (20–30 µg) of anti-subunit antibodies. The amount immunoprecipitated by each antibody was subtracted from the value obtained in control samples containing an identical concentration of normal rabbit IgG. Note the presence of 2, 3, 4, 6, 2, and 3 nAChR subunit immunoreactivity in monkey striatum, and 2, 3, 4, and 2 nAChR subunit immunoreactivity in cortex. The remaining subunits were below the detection limit of the assay (< 6%). In A and B, values represent the mean ± S.E.M. of six (striatum) and three (cortex) separate immunoprecipitation experiments. In each immunoprecipitation experiment, each antibody was tested in duplicate. C, dual immunoprecipitation analyses of the subunit composition of striatal 6* nAChRs. Control striatal extracts were loaded onto an anti-6 affinity column to bind the 6* nAChR population. The receptors were eluted from the resin using 6 peptide, labeled with 2 nM [3H]epibatidine and then immunoprecipitated with the indicated subunit-specific antibodies. Note that all 6* nAChRs have 2 subunits and that the 6 and 3 subunits do not coassemble. D, dual immunoprecipitation of 4* nAChRs in monkey cortex. Experiments were performed using an anti-4 affinity column, followed by elution with 4 peptide. Results are expressed as femtomoles of [3H]epibatidine binding per milligram of protein. Each data point in C and D represents the mean ± S.E.M. of two experiments performed in triplicate.

A similar approach using [³H]epibatidine-labeled sites was used to identify the major nAChR subtypes in monkey cortex. Receptor levels in control monkey cortex were 41.6 ± 3.6 and 49.9 ± 2.6 fmol/mg of protein in the membrane preparation and 2% Triton extract, respectively. Immunoprecipitation studies using crude membrane extracts showed that receptors contained the 2 (96%), 4 (77%), 2 (21%), and 3 (10%) subunits, whereas the 5, 6, 3, and 4 subunits were below the level of detection of the assay. Results represent the mean ± S.E.M. of three immunoprecipitation experiments performed in duplicate for each antibody (Fig. 1B).

Thus, similar to the rodent, the major nicotinic receptor subtypes in monkey cortex contain the 4 and 2 subunits, whereas in the striatum, they contain 4, 6, 2, and 3 subunits. On the other hand, the 2 and 3, but not 5 and 4, subunits are present in monkey striatum and cortex, distinct from rodent brain.

Subunit Composition of 6^* nAChRs in Monkey Striatum. Our immunoprecipitation experiments, as well as previous receptor studies, indicate that there is a selective expression of 6^* nAChRs in monkey striatum. To identify the subunits that coassemble with 6, we immunodepleted striatal extract of 6^* receptors using an affinity column with a bound anti-6 antibody. Selective 6^* nAChR immunodepletion was confirmed by the fact that immunoprecipitated 6^* [³H]epibatidine-labeled receptors decreased from 25% in the total striatal extract to 1% in the flow-through of the 6 column. In addition, 4^* receptors were increased (from 58.5 to 71.6%), suggesting that an appreciable portion of the 4 subunit pool is not assembled with the 6 subunit. 2^* nAChRs were also substantially increased in the flow through (from 12 to 30%), suggesting they may primarily be associated with non-6^* nAChRs. On the other hand, 3^* receptors markedly decreased suggesting a colocalization with 6. 2^* nAChRs remained unchanged indicating they are present in the majority of receptor subtypes.

To identify their subunit composition, 6^* receptors were eluted from the affinity column with 6 peptide and labeled with [³H]epibatidine; the eluate was immunoprecipitated with nAChR subunit-specific antisera. As shown in Fig. 1C, the anti-4, -2, and -3 sera immunoprecipitated 47, 100, and 61% of the purified [³H]epibatidine-labeled receptors, respectively. In contrast, the anti-2, -3, -5, and -4 sera immunoprecipitated 6% (detection limit of the assay) of [³H]epibatidine binding, suggesting they do not coassemble with 6.

The dual immunoprecipitation data suggest that 6^* nAChRs may be composed of 623 and/or 6423 subunits. In addition, analyses of the 6-affinity column flow-through indicate that 42^* nAChRs also form major striatal subtypes.

Subunit Composition of non-6^* nAChRs in Monkey Striatum. To identify striatal nAChRs not containing the 6 subunit, we also immunopurified the flow-through of the 6 affinity column using an anti-2 column. We then eluted the bound receptors with 2 peptide and performed immunoprecipitation studies using subunit specific antisera. The anti 2, 3, and 4 antibodies immunoprecipitated 22.9 ± 3.9, 20.4 ± 5.6, and 73.4 ± 2.4% (mean ± S.E.M., n = 2) of the [³H]epibatidine-labeled purified 2^* receptors, respectively. The other antibodies yielded no detectable immunoreactive material.

These studies clearly show that, in addition to 6^* nAChRs, 42^* receptors are also present in monkey striatum together with a minor population of 22^* and 32^* nAChRs. Because of the low recovery of the 2^* and 3^* subtypes in the 2 purified receptor preparation, it was not feasible to further investigate their subunit composition.

Subunit Composition of 4^* nAChRs in Monkey Cortex. Because 4 is the major acetylcholine binding subunit in cortex, experiments were done to determine with which subunits 4 is coexpressed (Fig. 1D). Cortical extracts were incubated with anti-4 antibody linked to Sepharose beads. Bound 4^* receptors were then eluted with 4 peptide. Immunoprecipitation experiments showed that 95% of these receptors contained the 2 subunit, 17% the 2 subunit, and 8% the 3 subunit. Therefore, all 4^* receptors most likely couple with 2, whereas a subpopulation of 42^* subtypes also contain the 2 and 3 subunits.

Nigrostriatal Damage Decreases Select nAChR Subunits in Monkey Striatum. Studies were next done to determine the effect of nigrostriatal damage on nAChR subunit expression in monkey striatum (Table 2). Animals were lesioned with the selective dopaminergic neurotoxin MPTP and euthanized 1 month later when the effects of the lesion were maximal. [³H]epibatidine binding in monkey striatum was significantly (p < 0.005) reduced from 55.5 ± 4.1 to 30.0 ± 3.5 fmol/mg of protein (n = 6 experiments) in the membrane preparation and from 69.6 ± 5.5 to 35.2 ± 1 fmol/mg of protein in the 2% Triton extract (n = 6 experiments), similar to previous results (Kulak et al., 2002a). Immunoprecipitation of solubilized [³H]epibatidine binding sites using subunit-specific antibodies (Table 2) showed that MPTP-lesioning produced the greatest decline (expressed as percentage decrease) in 6^* (83%) and 3^* (86%) subtypes, as well as significant reductions in receptors containing 3 (50%), 4 (32%), and 2 (48%) subunits but not 2 subunits. Because 6^* and 3^* nAChRs were decreased in parallel with the dopamine transporter, these subtypes are most likely coexpressed on dopamine terminals. In contrast, receptors expressing the 4 and 2 subunits seem to be present on both dopaminergic and nondopaminergic neurons, whereas 2^* subtypes are on nondopaminergic cells. Putative receptor subtypes in striatum thus include 623, 6a42b3, 42, and 22^*.

View this table: [in this window] [in a new window]   TABLE 2 Selective declines in nAChR subunit-immunoreactivity in monkey striatum after MPTP treatment NAChR subunit immunoprecipitation was done as described in the Fig. 1 legend using striatal and cortical extracts prepared from control and MPTP-lesioned monkey brain. Note the similar declines in 6 and 3-subunit-immunoreactivity after MPTP treatment, suggesting that these two subunits are decreased in parallel with the dopamine transporter. Declines were also observed in 3, 4, and 2 subunit immunoreactivity. Binding in extracts from control and MPTP-lesioned striatum was 69.6 ± 5.55 and 35.2 ± 1.0 fmol/mg of protein, respectively, and in cortex was 49.9 ± 2.6 and 47.3 0 ± 0.8 fmol/mg of protein, respectively. Values represent the mean ± S.E.M. of six (striatum control and MPTP-treated) and three (cortex control and MPTP-treated) immunoprecipitation experiments. In each immunoprecipitation experiment, each antibody was tested in duplicate.

Consistent with previous results, cortical [³H]epibatidine receptors were unaffected by MPTP-treatment with 41.6 ± 3.6 and 41.4 ± 5.8 fmol/mg of protein in membranes from control animals and MPTP-treated animals, respectively. The 2% Triton extracts were also similar in controls and MPTP treated animals with values of 49.9 ± 2.7 and 47.3 ± 0.8 fmol/mg of protein, respectively. Immunoprecipitation studies performed on cortical tissues confirmed that there was no change in the expressed subtypes after MPTP lesioning.

Radioligand Binding Studies—Effect of Nigrostriatal Damage. Earlier work had shown that receptors labeled with ¹²⁵I-epibatidine, a ligand that identifies multiple receptor subtypes (2^* through 6^*) were reduced with nigrostriatal damage (Kulak et al., 2002a), consistent with the present immunoprecipitation data. Other studies using the more selective radioligand ¹²⁵I--conotoxin MII further demonstrated specific declines with lesioning in 3^* and/or 6^* nAChRs (Quik et al., 2001). In the present experiments, we investigated binding of ¹²⁵I--bungarotoxin to 7 receptors and [³H]cytisine, which interacts with 42^* and 22^* subtypes (Luetje and Patrick, 1991). Autoradiographic studies showed there was a decrease in [³H]cytisine binding in caudate and putamen (Fig. 2A) but no change in ¹²⁵I--bungarotoxin binding (Fig. 2B).

View larger version (39K): [in this window] [in a new window]   Fig. 2. Computer-generated autoradiograms of nAChR binding in striatum of control and MPTP-treated monkeys. Note the decline in binding of [3H]cytisine to 4* nAChRs (A) in monkey striatum with nigrostriatal damage, but not in 125I--bungarotoxin (-BGT) binding (B) to 7 receptors.

Previous work in rodents had indicated that [³H]cytisine binds to an 4^* nAChR (Flores et al., 1992). The present results (Fig. 3A) show that -conotoxin MII does not compete with [³H]cytisine in striatal slices from either control or MPTP-lesioned animals. This observation suggests that [³H]cytisine binds at a similar receptor interface (that is, 42) in monkey striatum. Nicotine completely blocked [³H]cytisine binding in striatum from both control and MPTP-lesioned monkeys (Fig. 3B), demonstrating that the radioligand binds to a receptor with nicotinic characteristics. Previous studies (Quik et al., 2001) had shown that the nAChRs decreased with nigrostriatal damage were -conotoxin MII-sensitive (that is, 3^* and/or 6^*). This work, combined with the present experiments showing that [³H]cytisine binding (4^*) receptors are decreased after MPTP treatment (Fig. 4), suggests that these nAChRs may have both an 42 and an 62 interface (that is, 6242^*).

View larger version (20K): [in this window] [in a new window]   Fig. 3. Competition analyses of [3H]cytisine binding to striatum from control and MPTP-treated animals. A, [3H]cytisine binding in the presence of increasing concentration of -conotoxin MII. The lack of inhibition by -conotoxin MII suggests that [3H]cytisine binds exclusively to a receptor with an 42 interface in monkey striatum. B, [3H]cytisine binding in the presence of increasing concentrations of nicotine. Each value represents the mean ± S.E.M. from three monkeys.

View larger version (25K): [in this window] [in a new window]   Fig. 4. Nicotinic receptor subunit composition in monkey striatum based on receptor binding and immunoprecipitation data from control and MPTP-lesioned animals. Left column, quantitative analyses of binding using radioligands that label different nAChR subtypes. A, 125I--bungarotoxin (BGT) labeling of 7 nAChRs was similar in striatum from control and lesioned animals. B, [3H]cytisine binding was partially reduced with MPTP treatment, indicating that subtypes containing at least one 42 interface are decreased with nigrostriatal damage. C, 125I--conotoxin MII (CtxMII), a radioligand that binds to nAChRs with an 32 and/or 62 interface, is completely abolished with nigrostriatal damage (Quik et al., 2001) suggesting that these receptors are primarily localized to nigrostriatal dopaminergic terminals. D, 125I-epibatidine binds multiple (2* through 6*) nAChR subtypes and is partially reduced with lesioning (Kulak et al., 2002a). These data, coupled with the immunoprecipitation results (see Fig. 1) showing the presence of the 2, 3, 4, 6, 2, and 3 subunits in control and lesioned striatum, indicate that multiple subtypes are present in monkey striatum, including 7, 42, 6423, 623, 22*, 32*, and possibly others. These are similar to the subtypes identified in rodent brain with some differences, including 1) the presence of nAChRs expressing 3, but not 5 and 4 subunits in monkey striatum and cortex, and 2) a larger proportion 6* and/or 3* nAChRs in monkey (50%) compared with rodent (15%) striatum. For A and B, each value represents the mean ± S.E.M of three to seven animals. Significance of difference from control, ***, p < 0.001. -CtxMII, -conotoxin MII.

L-DOPA Treatment Decreases nAChRs in Monkey Striatum. Previous studies had shown that 2 weeks of L-DOPA treatment (15 mg/kg twice daily, every 4 h) reduced striatal ¹²⁵I-epibatidine sites (Quik et al., 2003a). To determine whether a longer course of treatment might result in a differential decline, we investigated the effect of 8 weeks of administration. Results (Fig. 5A) show that there was a somewhat greater decline in ¹²⁵I-epibatidine binding (25%), with similar results obtained using ¹²⁵I-A85380. No change was observed in ¹²⁵I--conotoxin MII binding sites or [¹²⁵I]RTI-121 binding to the dopamine transporter.

View larger version (28K): [in this window] [in a new window]   Fig. 5. Effect of L-DOPA treatment on striatal nAChRs. 125I-Epibatidine and 125I-A-85380 binding were significantly decreased in monkey caudate after 8 weeks of L-DOPA treatment, with no decline in the 125I--conotoxin MII and [125I]RTI-121 sites. Values represent the mean ± S.E.M. of three to twelve animals. B, -conotoxin MII competition of 125I-epibatidine binding in control and L-DOPA–treated monkeys. Competition analyses demonstrate the presence of both a high- and a low-affinity -conotoxin MII sensitive in control striatum (fit best to a two-site model) but only a high-affinity component after L-DOPA treatment (fit best to a one-site model), consistent with the lack of effect of L-DOPA on 125I--conotoxin MII binding sites. Values represent the mean ± S.E.M. of three to four animals. C, immunoprecipitation analyses suggest that nAChR subunit immunoreactivity is similar before and after L-DOPA treatment. The results are expressed as percentage of control and are the mean ± S.E.M. value of three experiments done in duplicate. Significance of difference from control, *, p < 0.05; **, p < 0.01.

Competition studies of ¹²⁵I-epibatidine binding by -conotoxin MII were then done to determine whether L-DOPA treatment had selective effects on different nAChR populations after 8 weeks of treatment. Analyses of the inhibition curves demonstrated a biphasic -conotoxin MII inhibition of striatal ¹²⁵I-epibatidine binding in control animals but not in L-DOPA–treated animals. The control data best fit to a two-site competition model with IC₅₀ values of 1.78 nM (CI 0.7 to 4.0 nM) and 1.14 µM (CI 0.10 to 9.0 µM), whereas the data from the treated animals fit best to a one-site competition model with an IC₅₀ value of 8.37 nM (CI 2.4 to 28 nM). Thus, 8 weeks of L-DOPA treatment led to a selective decrease in low-affinity but not high-affinity -conotoxin MII–sensitive sites consistent with the lack of change in ¹²⁵I--conotoxin MII (Fig. 5B).

As an approach to understand the subunit composition of the striatal nAChR sites affected by L-DOPA treatment, immunoprecipitation studies were done (Fig. 5C). No significant declines were observed in nAChR subunit-immunoreactivity with L-DOPA treatment compared with control animals.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Using a combined molecular and pharmacological approach, we investigated nAChR subunit composition in striatum using control and MPTP-lesioned monkeys. The results show that several major populations are present in striatum including 7, 42^*, 62^*, 32^*, and 22^* nAChRs. Detailed analyses of the present data, combined with previous receptor binding and recent functional studies suggest the following: 1) 62^* nAChRs contain 3 and also, in part, 4 to form 623 and 4623 subtypes, 2) the presence of striatal 42 and 22^* nAChRs, and 3) the existence of a novel 32^* nAChR population. A detailed rationale for the existence of these subtypes and their localization (Fig. 6) in monkey striatum is discussed below.

View larger version (26K): [in this window] [in a new window]   Fig. 6. Schematic localization of nAChR subtypes on dopaminergic and nondopaminergic neurons in the primate nigrostriatal pathway. ACh, acetylcholine; DA, dopamine; Glu, glutamate; SN, substantia nigra.

Receptor Subtypes Present in Monkey Striatum. Our postulated composition of striatal nAChR subtypes is based on the current hypothesis that heteromeric nAChRs have at least two subunits bearing the principal amino acid loops for acetylcholine binding interfaces (2, 3, 4, or 6 subunits) and two subunits bearing the complementary amino acid loops (2 or 4 subunits), whereas the fifth subunit can be either a complementary or a purely structural subunit (5 or 3 subunits).

Receptor Subtypes Present on Striatal Dopaminergic Terminals—6423, 623, 32^*. Our previous data had shown that nigrostriatal damage leads to a selective decline in striatal nAChRs that bind ¹²⁵I--conotoxin MII, a ligand that interacts at an 32^* and/or 62^* interface, with no change in other receptor subtypes (McIntosh et al., 1999; Quik et al., 2001; Kulak et al., 2002a; Nicke et al., 2004). These findings suggested that receptors expressing 62 and/or 32 subunits are localized to dopaminergic terminals in monkey striatum. The present results show that [³H]cytisine, a ligand that interacts at an 42 receptor interface (Flores et al., 1992), binds to monkey striatum and, in addition, that [³H]cytisine binding is reduced with moderate nigrostriatal damage. Previous data using ¹²⁵I-epibatidine had shown that a moderate lesion decreased only -conotoxin MII-sensitive nAChRs (Quik et al., 2001; Kulak et al., 2002a). These combined data can most readily be explained by postulating the existence of a receptor subtype with both an 62 and also an 42 interface (that is, an 6242^* subtype).

The current antibody experiments support and extend the results from the receptor studies. The dual immunoprecipitation shows that all striatal 6-subunit-immunoreactivity is precipitated by the anti-2 antibody, suggesting an absolute requirement for an 62 interface, in agreement with the ¹²⁵I--conotoxin MII binding data. In addition, the lesion studies show that the 6 and 3 subunit are decreased in parallel after nigrostriatal damage, suggesting they are coexpressed, thus forming an 623^* receptor. The anti-4 but not the anti-2 and anti-3 antibodies also immunoprecipitated 6^* nAChRs, whereas the 5 and 4 subunits were not detectable in striatum. Together, these observations reduce the potential subunit combinations to 623 and 6423. These subtypes may both be present in striatum, because the anti-4 antibody only precipitated a portion of the 6^* sites.

The immunoprecipitation data also show that 3 subunit-immunoreactivity is present in striatal extracts. Furthermore, our studies using a purified 2^* receptor preparation clearly show that the 3 and 2 subunit coprecipitate. These results provide direct evidence that -conotoxin MII binds at an 32 interface in monkey striatum, as suggested previously (McIntosh et al., 1999; Kulak et al., 2002b; Nicke et al., 2004). Lesion studies show that all -conotoxin MII-sensitive receptors are lost with nigrostriatal damage, suggesting that they are present on striatal dopaminergic terminals. These combined data suggest that 32^* nicotinic receptors are located on nigrostriatal terminals in monkey brain, together with the 6423 and 623 subtypes.

Receptor Subtypes Present on Dopaminergic and Nondopaminergic Striatal Neurons—42 and 22^*. As discussed earlier, results show that 30% of the [³H]cytisine sites (containing an 42 interface) are decreased with moderate nigrostriatal damage, suggesting they form an 62423 subtype. The remaining [³H]cytisine binding sites would represent non-6 42^* nAChRs, which may be both pre- and postsynaptic. The presence of this latter population is also confirmed from the results of the dual label 2 immunoprecipitation experiments using the non-6^* receptor preparation. Evidence for a presynaptic localization for a portion of the 42^* receptors stems from the results of our functional studies showing that 30% of nicotine-evoked [³H]dopamine release from striatal synaptosomes is resistant to inhibition by -conotoxin MII (McCallum et al., 2004).

The immunoprecipitation data are consistent with these findings and allow us to speculate as to the remaining composition of the 42^* sites. They do not seem to contain 3 or 6 because they are -conotoxin MII-resistant. They are also most probably not expressed with the 3 subunit because the lesion studies indicate that 3 is coexpressed with 6. The absence of the 5 and 4 subunits in monkey striatum rules out their presence in the 42^* pentamer. Thus, the only remaining subunit that can form a receptor with 42^* receptors is 2, yielding 42 and 422 nAChRs. This finding is supported by our studies using total striatal extracts and a non-6 containing 2 purified receptor preparation, which showed that a large proportion of 2^* receptors contain the 4 subunit, and a minority contained the 2 subunit.

The 4 and 2 subunits may be present within the same or on distinct nAChR subtypes, allowing for the presence of 42 and 22^* nAChRs. Because 2 is not affected by nigrostriatal damage, the 22^* receptors are most likely to be found on nondopaminergic neurons, as in the rodent (Zoli et al., 2002). In summary, dopaminergic terminals exclusively express 42 receptors, whereas 42 and 22^* receptors may be expressed on nondopaminergic neurons.

Receptors Present Exclusively on Nondopaminergic Striatal Elements. The ¹²⁵I--bungarotoxin binding studies show that striatal 7 receptor expression is relatively low and unaffected by nigrostriatal damage. These data suggest that these sites are localized on striatal GABA-ergic and cholinergic neurons, glutamatergic inputs, and/or nonneuronal cells (Kaiser and Wonnacott, 2000; Rogers et al., 2001). With respect to number of binding sites per receptor, homomeric 7 nAChRs are likely to have five acetylcholine sites, whereas heteromeric receptors with several different subunits contain at least two binding sites and possibly more depending on the nature of the other subunits.

L-DOPA Treatment Differentially Affects Striatal Nicotinic Receptors. Previous studies had shown that a 2-week treatment with L-DOPA, a commonly used therapy for Parkinson's disease, resulted in a 20% decline in striatal -conotoxin MII–sensitive ¹²⁵I-epibatidine sites with no change in ¹²⁵I--conotoxin MII binding (Quik et al., 2003a). Because patients are treated with L-DOPA for extended times, we next investigated the effect of an 8-week treatment course. The results show that the decline in striatal -conotoxin MII–sensitive ¹²⁵I-epibatidine sites persists with continued L-DOPA treatment. The finding that there is no change in binding of ¹²⁵I--conotoxin MII (0.5 nM) to high-affinity sites, suggests a preferential loss in low- but not high-affinity -conotoxin MII–sensitive receptors. This is supported by the competition data, which best fit to a one-site model after L-DOPA treatment but to a two-site model in the control condition.

These data are in apparent contradiction with the immunoprecipitation results, which show no significant difference in nAChR subunit-immunoreactivity in animals treated with L-DOPA compared with control. These results may suggest that L-DOPA treatment induces a change/redistribution in composition of 3^* and/or 6^* nAChR subtypes (as detected in the radioligand binding assays) without affecting the total amount of these two subunit (as measured by the immunoprecipitation assay). On the other hand, or as well, the varying results between the two assays may reflect a greater sensitivity of the autoradiographic binding technique compared with immunoprecipitation.

Receptor Subtypes Present in Monkey Cortex. The major nAChR receptor populations in cortex seem to contain 42 subunits, in agreement with previous studies in rodents (Flores et al., 1992; Zoli et al., 2002; Champtiaux et al., 2003). In contrast, 2^* nAChRs were also identified in monkey cortex, an observation consistent with the identification of 2 mRNA in monkey brain (Han et al., 2003). Both 42 and 422 nAChRs seem to be present with this latter subtype representing 16% of the 42^* cortical receptor population. We also identified 3^* receptors in monkey cortex (8%), an observation consistent with recent findings demonstrating the presence of 32^* and/or 62^* nAChRs in human cortex (Amtage et al., 2004; Quik et al., 2004). Neither MPTP-lesioning nor L-DOPA treatments affected cortical nAChRs, as previously shown (Kulak et al., 2002a; Quik et al., 2003a).

Summary. The present results show that several major nAChR populations are present in monkey brain. In cortex, we identified 7 and