Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

Parkinson's disease is characterized by loss of dopaminergic neurons innervating the striatum. The most effective therapy is replacement of dopamine with L-dopa or dopamine receptor agonists, but virtually all patients treated for extended periods develop motor complications: "wearing off," the abrupt loss of effectiveness of the medication at the end of the dosing interval, and "dyskinesias," abnormal involuntary choreiform movements. These symptoms are often disabling (Standaert and Stern, 1993). An animal model for Parkinson's disease is the unilateral, 6-hydroxydopamine (6-OHDA)-lesioned rat (Ungerstedt and Arburthnott, 1970). These animals respond to dopamine agonists by circling, and with chronic treatment they exhibit responses of increasing amplitude and decreasing duration, analogous to the motor effects observed in human patients (Papa et al., 1995).

N-Methyl-D-aspartate (NMDA) glutamate receptors play a key role in the regulation of movement by the striatum, and are thought to be promising therapeutic targets for treatment of Parkinson's disease. NMDA-binding sites are very abundant in the striatum (Albin et al., 1992). In rodents, injection of NMDA agonists into the striatum causes contralateral rotation, whereas bilateral injection causes parkinsonism (Thanos et al., 1992; Klockgether and Turski, 1993). NMDA antagonists potentiate the immediate effects of dopamine on striatal function (Klockgether and Turski, 1990; Morelli et al., 1992; Papa et al., 1993; Kaur and Starr, 1997) and are highly effective in attenuating the motor effects of chronic dopaminergic therapy (Papa et al., 1995; Marin et al., 1996; Blanchet et al., 1997). Recent evidence suggests that the basis for this effect is that chronic dopaminergic treatment modifies the properties of striatal NMDA receptors (Chase et al., 1998).

NMDA receptors are heteromeric assemblies of NMDA receptor subunits (NRs) NR1 and NR2; seven isoforms of NR1 are produced by alternative splicing of a single gene, whereas four distinct genes encode NR2A, NR2B, NR2C, and NR2D (Dingledine et al., 1999). In the rat striatum, the mRNAs for NR1, NR2A, and NR2B are abundant, whereas the relative levels of NR2C and NR2D mRNA are low (Standaert et al., 1994). Both the abundance as well as the subunit composition of receptor complexes probably have an important influence on NMDA receptor function. Protein phosphorylation is an additional means of modulating the properties of NRs. The NR1 subunit has at least three distinct serine phosphorylation sites in the carboxy tail region, which affect the interaction with calmodulin as well as subcellular distribution (Hisatsune et al., 1997; Tingley et al., 1997). The NR2s are tyrosine phosphorylated, and modulation of NR2 phosphorylation has been observed in several models of synaptic plasticity, including taste learning (Lau and Huganir, 1995; Rostas et al., 1996; Rosenblum et al., 1997; Dunah et al., 1998b).

Only limited direct studies of striatal NMDA receptors in Parkinson's disease models have been reported. Ligand-binding studies with [³H]glutamate have produced inconclusive results, suggesting relatively small changes in binding to NMDA receptor sites (Wüllner et al., 1993; O'Dell and Marshall, 1996; Ulas and Cotman, 1996), whereas with [³H]MK801 a bilateral reduction in binding, greater on the lesioned side, was observed (Porter et al., 1994). An in situ hybridization study demonstrated a small-magnitude up-regulation of the NR2A (Ulas and Cotman, 1996). Recently, two reports (Menegoz et al., 1996; Oh et al., 1998) described increased tyrosine phosphorylation of NMDA receptors after nigrostriatal denervation and chronic L-dopa treatment.

In the current study, biochemical methods were used to study the subunit abundance, composition, and the serine and tyrosine phosphorylation of NRs present in the normal rat striatum, and to determine how these properties are altered in the rat unilateral 6-OHDA model of parkinsonism and by chronic treatment with L-dopa. NR proteins are known to be present in the cytoplasm as well as in association with the cell membrane of neurons (Petralia et al., 1994) and trafficking of receptor proteins from the cytoplasm to the cell surface is a potentially important means of functional regulation (Shi et al., 1999). Therefore, we analyzed separately the receptors present in total striatal homogenate and those associated with neuronal membranes.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials

Protein A-Sepharose, dimethylpimelimidate, and benserazide were purchased from Sigma Chemical Co. (St. Louis, MO). Sodium deoxycholate (S285-100, lot 951636), L-glutamate, L-glycine, sodium fluoride, and Triton X-100 were obtained from Fisher Scientific (Pittsburgh, PA). L-Dopa and 6-OHDA were from Research Biochemicals (Natick, MA). The chemiluminescence detection system (Super Signal) was from Pierce (Rockford, IL). The subunit-specific monoclonal NR1 (Luo et al., 1997), polyclonal NR2A (Wang et al., 1995), and monoclonal NR2B (Wang et al., 1995) antibodies were developed in the laboratory of Dr. Barry B. Wolfe, Georgetown University. The phosphoserine specific polyclonal NR1 antibodies (Tingley et al., 1997) were produced in the laboratory of Dr. Richard L. Huganir, Johns Hopkins University. The polyclonal -Actinin-2 antibody (Wyszynski et al., 1997) was a generous gift from Dr. Morgan Sheng, Harvard Medical School. The following antibodies were obtained from commercial sources: polyclonal anti-GluR2/3 (Chemicon International, Temecula, CA ); monoclonal PSD-95 (K28/43) (Upstate Biotechnology, Lake Placid, NY); monoclonal antiphosphotyrosine (PY20) and recombinant antiphosphotyrosine monoclonal (RC20) (Transduction Laboratories, Lexington, KY); monoclonal tyrosine hydroxylase antibody and nonimmune purified rabbit immunoglobulins (Sigma Chemical Co.); horseradish peroxidase-linked goat anti-rabbit and horseradish peroxidase-linked goat anti-mouse (Jackson ImmunoResearch, West Grove, PA ).

Methods

Experimental protocols involving the use of vertebrate animals were approved by the Massachusetts General Hospital Subcommittee on Research Animal Care and conformed to the National Institutes of Health guidelines.

Unilateral 6-OHDA Lesions. Adult male Sprague-Dawley rats weighing 200 to 250 g were treated with imipramine (25 mg/kg i.p.) and anesthetized with sodium pentobarbital (50 mg/kg i.p.). The rats were positioned in a stereotaxic frame and nigrostriatal lesions were produced by injecting 16 µg of 6-OHDA dissolved in 4 µl of water containing 0.02% ascorbate into the left medial forebrain bundle with a 10-µl Hamilton syringe. The stereotaxic coordinates were anterior 1.6 mm from the interaural line, 2.2 mm lateral, and 7.7 mm below the surface of dura with the atlas of Paxinos and Watson (1986). Sham lesions were performed with the same procedure, with injection of 4 µl of saline. The rats were maintained in cages with free access to food and water and housed on a 12-h light/dark cycle for 14 days before rotational testing.

Behavioral Screening. To determine the success of nigrostriatal denervation, rats were injected with apomorphine (0.25 mg/kg i.p.) in saline containing 0.1% sodium meta-bisulfite 14 days after the lesion and placed in an automated rotameter (Omnitech Electronics, Columbus, OH). Complete rotations ipsilateral and contralateral to the side of lesion were recorded for 60 min. Rats that exhibited >300 rotations contralateral to the side of lesion were used in this study. To study the effects of the lesion, rats were sacrificed by rapid decapitation 48 h after behavior screening. The brains were removed, the left striatum and right striatum dissected separately, and frozen on dry ice. Tissue samples were stored at 80°C.

Chronic L-dopa Treatment. Additional groups of rats that met the above-mentioned behavioral criteria were treated with repeated injections of L-dopa, as previously described (Papa et al., 1995). Treatment was started 24 h after testing with apomorphine, and consisted of L-dopa methyl ester (25 mg/kg) and benserazide (6.25 mg/kg) in saline, or saline injected i.p. twice daily for 21 consecutive days. These rats were sacrificed by rapid decapitation 12 h after the final injection, and the left and right striata were dissected and frozen at 80°C.

Nondenaturing Conditions of Protein Solubilization for Subunit Composition Studies. The immunoprecipitation and immunoblot methods used in this investigation had been characterized and used in previous studies (Wang et al., 1995; Luo et al., 1997; Dunah et al., 1998a). Two different methods of protein extraction were used in this study. Rat brain tissues were homogenized with a polytron (Brinkmann, Dallas, TX) in ice-cold Tris-EDTA buffer (10 mM Tris-HCl, 5 mM EDTA, pH 7.4) and centrifuged at 700g at 4°C. The pellet containing mainly nuclei and large debris was discarded. The resulting supernatant was centrifuged at 10,000g at 4°C and the pellet (P2 fraction) was used as total striatal protein homogenate. Membrane enriched proteins were prepared by homogenizing the P2 fraction in ice-cold Tris-EDTA buffer containing 320 mM sucrose. The sample was centrifuged at 25,000g at 4°C to pellet the synaptosomal membrane fraction. Extracts prepared by either method were solubilized by the addition of one-tenth volume of 10% sodium deoxycholate in 500 mM Tris-HCl, pH 9.0, and incubated at 36°C for 30 min. A one-tenth volume of a buffer containing 1% Triton X-100, and 500 mM Tris-HCl, pH 9.0, was added and the samples were dialyzed against binding buffer (50 mM Tris-HCl, pH 7.4, 0.1% Triton X-100) overnight in cold room. Samples were centrifuged at 37,000g at 4°C. The supernatants were used for immunoblot and immunoprecipitation.

Denaturing Conditions of Protein Solubilization for Phosphorylation Studies. The total striatal homogenate and synaptosomal striatal membrane fractions were prepared as described above with TEVP buffer (10 mM Tris-HCl, pH 7.4, 5 mM NaF, 1 mM Na₃VO₄, 1 mM EDTA, 1 mM EGTA). The resultant pellets were solubilized with 2% SDS in TEVP buffer and centrifuged at 15,000g. The supernatants were used for immunoblot and immunoprecipitation studies.

Precoupling Antibodies to Protein A-Sepharose. The monoclonal NR1 and the monoclonal antiphosphotyrosine (PY20) antibodies were incubated with protein A-Sepharose beads at a concentration of 20 µg of antibody per 50 µl of hydrated protein A-Sepharose beads for 2 h at room temperature (RT) in 100 mM sodium borate, pH 8.0, with gentle rotation. To maximize immunoprecipitation efficiency, the NR1 antibody was chemically coupled to the protein A-Sepharose with dimethylpimelimidate in 200 mM sodium borate, pH 9, for 30 min at RT. Nonspecific sites on the beads were blocked with 200 mM ethanolamine, pH 8.0, for 2 h at RT. The beads were washed with 100 mM sodium borate, pH 8.0, and used for immunoprecipitation. This covalent coupling procedure was not necessary for the antiphosphotyrosine (PY20) antibody.

Immunoprecipitation. Solubilized protein samples were diluted 20-fold with immunoprecipitation buffer (150 mM NaCl, 50 mM Na₃SO_4, pH 7.2, 1% sodium deoxycholate, 2 mM EDTA, 1% Triton X-100). The diluted samples were incubated with 50 µl of protein A-Sepharose/antibody-coupled beads for each 200 µg of soluble protein for 3 h in cold room with gentle rotation. The immunoprecipitation pellets were washed three times with ice-cold immunoprecipitation buffer after brief centrifugation. For the determination of NR composition, the supernatants were transferred to separate tubes and aliquots were taken and diluted with loading buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 50 mM DTT, 7.5% glycerol). The pellets were resuspended in a suitable volume of loading buffer so that their fractional loads are directly comparable with diluted samples from the supernatants. The serine phosphorylation of NR1 subunit was studied with previously characterized antibodies that specifically recognize the NR1 protein only when it is phosphorylated at serine residues 890, 896, and 897 (Tingley et al., 1997).

Gel Electrophoresis, Quantitative Immunoblotting, and Statistical Analysis. SDS-polyacrylamide gel electrophoresis (PAGE) and transfer of separated proteins to polyvinylidene difluoride (PVDF) were performed as previously described (Wang et al., 1995; Dunah et al., 1996; Luo et al., 1996). In all experiments, 7.5% polyacrylamide gels were used for protein separation, and the concentration of antibodies used for immunoblotting was 1 to 2 µg/ml. Protein concentration was determined with the BioRad protein assay kit. Bands were visualized on film by enhanced chemiluminescence (SuperSignal) and their net intensities were quantified with computer-assisted densitometry (Kodak 1-D System; Kodak, Rochester, NY). For the analyses of NMDA receptor subunit abundance, samples from sham and 6-OHDA-lesioned rats were loaded onto a single gel, and the intensities of the bands were expressed as a percentage of the lesioned striatum of the sham-operated rat. For the phosphorylation and subunit composition studies, the primary analyses were conducted by running samples from the lesioned and unlesioned sides of each rat on the same gel, and expressing the lesioned intensity as a percentage of that found on the unlesioned side. These values were then used to calculate group means, and reported as means ± S.E. Differences between groups were analyzed with ANOVA with post hoc tests (Scheffe's). For all analyses, statistical significance was taken to be P < .05.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Characterization of 6-OHDA Nigrostriatal Lesions. The extent of denervation of the nigrostriatal system induced by stereotactic unilateral 6-OHDA injection was determined by behavioral and biochemical methods. All rats were selected for study with a threshold criterion of 300 ipsilateral rotations per hour after administration of 0.25 mg of apomorphine s.c. The rats studied averaged 398.4 ± 24 contralateral rotations in an hour with negligible ipsilateral rotations, whereas apomorphine did not elicit rotational behavior in the sham-operated animals. Striatal protein extracts from a subset of sham- and 6-OHDA-lesioned rats (n = 5) were examined for tyrosine hydroxylase immunoreactivity, a marker for dopaminergic neurons (Fig. 1). In sham-operated rats, tyrosine hydroxylase immunoreactivity in the lesioned striatum was 92.3 ± 1.6% of that on the unlesioned side (Fig. 1A). In contrast, tyrosine hydroxylase immunoreactivity in the lesioned striatum was reduced to 3.6 ±1.8% of that on the unlesioned side in 6-OHDA-lesioned rats (Fig. 1B). The extent of tyrosine hydroxylase depletion was not altered by chronic treatment with saline (Fig. 1C) or L-dopa (Fig. 1D).

View larger version (35K): [in this window] [in a new window]   Fig. 1.   Striatal tyrosine hydroxylase immunoreactivity is decreased in rats with unilateral nigrostriatal dopaminergic lesions. Solubilized proteins from either sham-operated rats (A), or 6-OHDA (B)-lesioned rats, and 6-OHDA-lesioned rats that were treated with saline (C) and L-dopa (D) were resolved on SDS-PAGE. The blots were probed with anti-tyrosine hydroxylase antibody. Lanes 1 and 2 contain samples from the lesioned striatum (L) and unlesioned striatum (U), respectively. Each lane contains 5 µg of protein. The positions and sizes of molecular weight markers are indicated in kilodaltons. Immunoreactivity was reduced in each of the lesioned striata (*).

Unilateral Nigrostriatal Denervation: Effect on Abundance, Subunit Composition, and Phosphorylation of NMDA Receptors in Total Striatal Protein Homogenate. We first examined the NMDA receptor subunit proteins in total striatal homogenates (membrane and cytoplasmic compartments) extracted as described in Methods. For each rat, the lesioned and unlesioned striatum were studied in parallel on the same gel, and the results are expressed as ratio of the lesioned to the unlesioned side. There were no significant differences in the abundance of NR1 (102 ± 2.9%), NR2A (106 ± 2.7%), and NR2B (100 ± 3.2%) in the striatum of 6-OHDA-lesioned rats (Fig. 2, A and B). Similarly, sham-operated rats showed no difference in the amounts of NR1, NR2A, and NR2B between the lesioned and unlesioned sides (Fig. 2, A and B). Immunoblots that were probed with antibodies to the -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid glutamate receptor subunits GluR2/3 and the postsynaptic proteins -Actinin-2 and PSD-95 revealed that these proteins also were not altered in total striatal homogenates (Fig. 2, A and B).

View larger version (72K): [in this window] [in a new window]   Fig. 2.   The subunit abundance, composition, and phosphorylation of NMDA receptor subunits in total striatal protein homogenates are not altered in rats with unilateral nigrostriatal ablation. Abundance: A, total protein homogenates from sham-operated rats (lanes 1 and 2) and 6-OHDA-lesioned rats (lanes 3 and 4) were solubilized and separated on SDS-PAGE. The blots were probed with antibodies specific for NR1, NR2A, NR2B, GluR2/3, -Actinin-2, and PSD-95. The lesioned striatum (L) and unlesioned striatum (U) are indicated on top of the figure. Each lane contains equal amounts (5 µg) of total protein. B, densitometric quantification of the relative abundance of NR1, NR2A, NR2B, GluR2/3, -Actinin-2, and PSD-95 in the lesioned striatum () and unlesioned striatum () of sham-operated and 6-OHDA-lesioned rats. None of the proteins studied are significantly altered in the total striatal extracts. Values on the ordinate represent intensity of the signal relative to the unlesioned side of the sham-operated rats. Data are means ± S.E. obtained from four rats. Composition: C and D, total protein homogenates from the lesioned striatum (lanes 1-5) and unlesioned striatum (lanes 6-10) of sham-operated rats (C) and 6-OHDA-lesioned rats (D) were solubilized with nondenaturing conditions. The soluble proteins were immunoprecipitated with anti-NR1 antibody. Samples from input (I; lanes 1 and 6; 5 µg), immunoprecipitation pellet (P and p; 5 µg and 2.5 µg of protein, respectively), and supernatant (S and s; 5 µg and 2.5 µg of protein, respectively) were resolved on SDS-PAGE. The blots were probed with anti-NR1, anti-NR2A, and anti-NR2B antibodies. Phosphorylation: E and F, total protein homogenates from the lesioned striatum (lanes 1-3) and unlesioned (lanes 4-6) of sham-operated (E) and 6-OHDA-lesioned rats (F) were solubilized and immunoprecipitated with anti-phosphotyrosine (PY) antibody. The pellets were separated on SDS-PAGE and the blots were probed with anti-NR2A, anti-NR2B, and anti-phosphotyrosine (PY) antibodies. Lanes 1 and 6 contain 2.5 µg of protein from the input. Lanes 2 to 5 contain samples from immunoprecipitation pellets (P and p; 20 µg and 10 µg of protein, respectively).

View larger version (69K): [in this window] [in a new window]   Fig. 3.   Specificity of immunoprecipitation of tyrosine-phosphorylated proteins by antiphosphotyrosine antibody. Rat striatal membrane proteins were solubilized and immunoprecipitated with anti-phosphotyrosine antibody in the presence of 10 mM phosphotyrosine (+PY; lanes 3 and 4), absence of 10 mM phosphotyrosine (PY; lanes 5 and 6), presence of 10 mM phosphoserine (+PS; lanes 7 and 8); and with protein A-Sepharose-coupled rabbit immunoglobulins (IgG; lanes 9 and 10) and protein A-Sepharose beads (PAS; lanes 11 and 12). The pellets were subjected to SDS-PAGE, and the blots were probed with anti-NR2A, anti-NR2B, and anti-phosphotyrosine (PY). Lanes 1 and 2 contain 2.5 and 1.25 µg, respectively, of input. Lanes 3 to 12 contain 2-fold (40 and 20 µg) load of protein from the immunoprecipitation pellet.

Unilateral Nigrostriatal Denervation: Effect on Abundance, Subunit Composition, and Phosphorylation of NRs in Striatal Membranes. In contrast to the results obtained studying total striatal protein extracts, analysis of NRs present in striatal membrane fractions revealed striking alterations in subunit abundance, composition, and phosphorylation. In 6-OHDA-lesioned rats, the abundance of NR1 and NR2B, but not NR2A was reduced in the lesioned striatum (NR1, 68± 3.2%; NR2B, 62 ± 4.4%) relative to the unlesioned striatum (Fig. 4, A and B). There were no significant changes in the relative abundance of GluR2/3, -Actinin-2 and PSD-95 (Fig. 4, A and B). The abundance of NR1, NR2A NR2B, GluR2/3, -2-Actinin, and PSD-95 were similar on the lesioned and unlesioned sides of the sham-operated rats (Fig. 4, A and B). The abundance of NR1, NR2A, and NR2B in the cortex, thalamus, and midbrain of the 6-OHDA-lesioned rats also was examined and no difference between the lesioned and unlesioned sides in these regions was found (data not shown).

View larger version (70K): [in this window] [in a new window]   Fig. 4.   NMDA receptor subunits NR1 and NR2B, but not NR2A are decreased in the striatal membrane protein extracts of rats with unilateral nigrostriatal depletion. Abundance: A, striatal membrane proteins from sham-operated rats (lanes 1 and 2) and 6-OHDA-lesioned rats (lanes 3 and 4) were separated on SDS-PAGE. The blots were probed with anti-NR1, anti-NR2A, anti-NR2B, anti-GluR2/3, anti--2-Actinin, and anti-PSD-95 antibodies. Each lane contains 5 µg of protein. B, relative abundance of NR1, NR2A, NR2B, GluR2/3, -2-Actinin, and PSD-95 in the lesioned striatum () and unlesioned striatum () of sham and 6-OHDA-lesioned rats. Values on the ordinate represent the amount of each protein as a percentage of the unlesioned side of the sham-operated animals. The abundance of NR1 and NR2B are reduced (P < .05), whereas the other proteins measured were unchanged. Data are means ± S.E. values obtained from five rats. Values on the ordinate represent intensity of the signal on lesioned striatum relative to the unlesioned striatum obtained from five rats. Composition: C and D, membrane proteins from the lesioned striatum (lanes 1-5) and unlesioned striatum (lanes 6-10) of sham-operated rats (A) and 6-OHDA-lesioned rats (B) were extracted with nondenaturing conditions and immunoprecipitated with NR1 antibody. Lanes 1 and 6 contain 5 µg of protein from the input (I). Lanes 2, 3, 7, and 8 contain samples from the immunoprecipitation pellet (P and p; 5 and 2.5 µg of protein, respectively). Lanes 4, 5, 9, and 10 contain samples from the supernatant (S and s; 5 and 2.5 µg of protein, respectively). The blots were probed with anti-NR1, anti-NR2A, and anti-NR2B antibodies. Phosphorylation: E and F, membrane proteins from the lesioned striatum (lanes 1-3) and unlesioned striatum (lanes 4-6) were immunoprecipitated with antiphosphotyrosine antibody. The blots were probed with antibodies against NR2A, NR2B, and phosphotyrosine (PY). Lanes 1 and 6 contain 2.5 µg of the input. Lanes 2 to 5 contain samples from immunoprecipitation pellet (P and p; 20 and 10 µg of protein, respectively). Asterisks indicate significant differences between the lanes marked with bars.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Densitometric quantification of serine phosphorylated-NR1, and tyrosine phosphorylated NR2A and NR2B in sham-operated and 6-OHDA-lesioned rats, and chronic L-dopa-treated nigrostriatal lesioned rats. Values on the abscissa represent the relative amount of phosphorylated NR1 at serine 890, serine 896, and serine 897, and tyrosine-phosphorylated NR2A and NR2B given as percentage of the unlesioned striatum. Data are means ± S.E. obtained from five rats. Asterisks indicate significant differences between 6-OHDA lesion and sham-operated (B) or L-dopa and saline treatment (D).

View larger version (51K): [in this window] [in a new window]   Fig. 6.   The serine phosphorylation of NMDA receptor NR1 is decreased in the striatal membranes of rats with unilateral nigrostriatal lesions, but increased after chronic treatment with L-dopa. Soluble membrane protein extracts from the lesioned striatum (lanes 1-3) and unlesioned striatum (lanes 4-6) of sham-operated rats (A) and 6-OHDA-lesioned rats (B), and 6-OHDA-lesioned rats treated with saline (C) and L-dopa (D) were immunoprecipitated with anti-NR1 antibody. The pellets were resolved on SDS-PAGE. The blots were probed with phosphoserine specific NR1 antibodies, NR1-S890, NR1-S896, and NR1-S897 as indicated across the figure. Lanes 1 and 6 contain 5-µg load of the input. Lanes 2 to 5 contain samples from immunoprecipitation pellet (P and p; 20- and 10-µg protein load, respectively). The arrows indicate the expected position of serine phosphorylated NR1 protein and these were the bands analyzed in this study. Asterisks indicate significant differences between the lanes marked by bars.

Effect of Chronic L-dopa Treatment on Abundance, Subunit Composition, and Phosphorylation of NMDA Receptors in Striatal Membranes. Treatment of unilateral 6-OHDA-lesioned rats with L-dopa or saline was initiated 2 days after behavioral testing with apomorphine, and continued for 21 days. Consequently, the rats were studied 38 days after unilateral 6-OHDA lesion, in contrast to the 16-day postlesion period used in the above-mentioned experiments. In lesioned rats treated with saline, the reductions in NR1 and NR2B observed at 16 days persisted (NR1, 69 ± 3.6%; NR2B, 64 ± 4.2%) (Fig. 7,A and B). However, chronic treatment of lesioned rats with L-dopa normalized the abundance of NR1 (98 ± 3.3%) and NR2B (103 ± 4.3%) in the lesioned striatum (Fig. 7, A and B). The abundance of NR2A GluR2/3, -2-Actinin and PSD-95 were not altered by treatment with L-dopa or saline (Fig. 7, A and B). To control for the possibility that L-dopa treatment might produce bilateral effects, we compared the unlesioned striatum of L-dopa treated rats to the unlesioned striatum of the saline treated rats, and found that the subunit abundance in these sets of samples were not different (data not shown).

View larger version (66K): [in this window] [in a new window]   Fig. 7.   Chronic treatment of unilateral nigrostriatal-lesioned rats with L-dopa normalized the subunit expression of NR1 and NR2B, and increased the tyrosine phosphorylation of NR2A and NR2B in striatal membranes. Abundance: A and B, membrane protein extracts from the striatum of 6-OHDA-lesioned rats treated with saline (lanes 1 and 2) and L-dopa (lanes 3 and 4) were separated on SDS-PAGE by loading 5 µg of protein per lane. The blots were probed for NR1, NR2A, NR2B, GluR2/3, -Actinin-2, and PSD-95. B, densitometric analysis of the relative amounts of NR1, NR2A, NR2B, GluR2/3, -Actinin-2, and PSD-95 from the lesioned striatum of saline- and L-dopa-treated rats. Values on the ordinate represent the amount of each protein as percentage of unlesioned striatum. Data are means ± S.E. obtained from five rats. Subunit composition: C and D, striatal membrane protein extracts from 6-OHDA-lesioned rats treated with saline (C) and L-dopa (D) were precipitated with NR1 antibody. Samples from input (I; lanes 1 and 6; 5 µg), immunoprecipitation pellet (P and p; 5 and 2.5 µg of protein, respectively), and supernatant (S and s; 5 and 2.5 µg of protein, respectively) were subjected to electrophoresis. The blots were probed with anti-NR1, anti-NR2A, and anti-NR2B antibodies. Phosphorylation. E and F, membrane proteins from the striatum of 6-OHDA-lesioned rats treated with saline (E) and L-dopa (F) were precipitated with anti-phosphotyrosine antibody. The blots were probed with anti-NR2A, anti-NR2B, and anti-phosphotyrosine (PY) antibodies. Lanes 1 and 6 contain 2.5 µg of the input. Lanes 2 to 5 contain samples from immunoprecipitation pellet (P and p; 20 and 10 µg of protein, respectively). Asterisks indicate significant differences between the lanes marked by bars.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

In rats with unilateral 6-OHDA lesions of the nigrostriatal pathway, we have found that there is a reduction in the abundance of NR1 and NR2B in the membrane fractions of lesioned striatum, whereas the abundance of NR2A is not altered. These alterations appear to arise from a selective decrease in the number of NMDA receptors composed of NR1/NR2B. The phosphorylation state of the NMDA subunits also is modified: there is a decrease in the serine phosphorylation of NR1 at residues 890 and 896, whereas the tyrosine phosphorylation of NR2B but not NR2A is altered. Interestingly, these changes are seen only in NMDA receptors present on striatal membranes and not receptors found in total striatal homogenate. This finding implies a redistribution of receptor subunits from the membrane to the cytoplasmic compartment. Finally, chronic treatment with L-dopa results in normalization of the abundance and composition of striatal NMDA receptors in the membrane fraction, but produces marked increases in the phosphorylation of NR1 at serine 890, serine 896, and serine 897, and tyrosine phosphorylation of NR2A and NR2B.

Alterations in Subunit Expression and Composition of NMDA Receptors in 6-OHDA-Lesioned Rat. The abundance and composition of NMDA receptors present in total striatal extracts were not changed in the 6-OHDA-lesioned rats. These data are in accord with previous study of Menegoz et al. (1996) but differ from the study by Oh et al. (1998) in that the latter study reported a modest (25%) increase in NR2A with no change in NR2B in total striatal extract after 6-OHDA lesions. These differences may be related to the duration of survival because both our study and that of Menegoz et al. (1996) examined rats 14 days after lesioning, whereas Oh et al. (1998) studied rats at 21 days after lesioning.

Alterations in Phosphorylation of NMDA Subunits in 6-OHDA-Lesioned Rat. Tyrosine phosphorylation is a post-translational mechanism that plays an important role in modifying the properties of NMDA channels. The NR2A, NR2B, and NR2D but not NR1 have been reported to be tyrosine phosphorylated in vivo (Lau and Huganir, 1995; Dunah et al., 1998b). Two previous studies have investigated the tyrosine phosphorylation of NR2A and NR2B in total striatal protein extracts of 6-OHDA-lesioned rats (Menegoz et al., 1996; Oh et al., 1998). Both studies reported a modest increase in tyrosine phosphorylated NR2B (17-18%), with no change in the tyrosine phosphorylation of NR2A. However, we found only an ~4% increase in tyrosine-phosphorylated NR2B from total striatal extracts. Such disparities may arise either from differences in the duration of the lesions, as noted above, or from differences in the efficiencies of extraction of NMDA receptors. In our study, we used 2% SDS as extraction buffer, whereas the other investigators used 1% SDS as solubilization buffer.

Effects of Chronic L-dopa Treatment on NMDA Subunits in 6-OHDA-Lesioned Rats. Unilateral 6-OHDA-lesioned rats have been used as experimental models for investigating the effects of chronic L-dopa treatment in human Parkinson's disease. The treatment paradigm used in this study is essentially the one developed by Chase and colleagues, consisting of L-dopa injections twice a day for 21 consecutive days. This treatment paradigm induces alterations in motor response, which include enhanced amplitude of rotation and shortened duration of action, thought to be analogous to the wearing off and fluctuations observed in patients (Papa et al., 1995). Previous work has revealed that low doses of NMDA receptor antagonists can prevent development of the behavioral alterations, and reduce severity once they have developed (Papa et al., 1995; Chase et al., 1998).

NMDA Receptor Properties in 6-OHDA-Lesioned Rats. The alterations in NMDA subunit abundance, composition, and phosphorylation observed in the lesioned striata may have important effects on the functional properties of striatal receptor channels. In particular, the reduction in the proportion of NMDA receptors composed of NR1 and NR2B, relative to receptors containing NR1 and NR2A would be expected to result in a population of receptors with high sensitivity for competitive NMDA antagonists, and reduced affinity for glutamate (Laurie and Seeburg, 1994; Lynch et al., 1994). The relative enrichment of NR1/NR2A receptors also would be predicted to lead to a corresponding increase in receptors with fast deactivation kinetics, and reduced affinity of the receptors for the noncompetitive polyamine antagonists such as ifenprodil and haloperidol (Williams et al., 1994; Lynch and Gallagher, 1996).