Gene expression of glutamate receptors GluR1 and NR1 is differentially modulated in striatal neurons in rats after 6-hydroxydopamine lesion

Neurochemistry International 43 (2003) 639–653

Gene expression of glutamate receptors GluR1 and NR1 is differentially modulated in striatal neurons in rats after 6-hydroxydopamine lesion S.K. Lai a,b , Y.C. Tse a,b , M.S. Yang a , C.K.C. Wong a , Y.S. Chan b , K.K.L. Yung a,∗ b

a Department of Biology, Hong Kong Baptist University, Kowloon Tong, Hong Kong, PR China Department of Physiology, Faculty of Medicine, The University of Hong Kong, Sassoon Road, Hong Kong, PR China

Received 2 October 2002; accepted 28 January 2003

Abstract In the present study, we attempted to address the modulation of the gene expression of N-methyl-d-aspartate (NMDA) and ␣-amino-3hydroxy-5-methyl-4-isoxazole-propionate (AMPA) glutamate receptors in the neostriatum of the 6-hydroxydopamine (6-OHDA)-lesioned rat, an animal model of Parkinson’s disease. After 2 weeks of lesion, reverse transcriptase-polymerase chain reactions (RT-PCRs) revealed significant reduction in GluR1 mRNA expression but a significant enhancement of NR1 mRNA expression in the striatal tissues of the lesioned side. No modulation in the mRNA expression of GluR2, GluR3, GluR4 and NR2B were found. Immunofluorescence with digital imaging analysis also demonstrated a significant reduction in GluR1 immunoreactivity in the lesioned neostriatum. Interestingly, the reduction in GluR1 immunoreactivity was primarily observed in presumed striatal medium spiny neurons but not in parvalbumin-labeled striatal GABAergic interneurons. Immunoreactivity for GluR2, GluR2/3, GluR4, NR1 and NR2B was unchanged in neurons of the neostriatum of the lesioned side. The present results indicate that there is an opposite trend in modulation in the gene expressions of GluR1 and NR1 in the neostriatum of 6-OHDA-lesioned rats after dopamine denervation. Modulation of GluR1 mRNA and immunoreactivity is likely to be limited in the striatal projection neurons. These findings have implications for the use of NMDA and AMPA receptor antagonists in the treatment of Parkinson’s disease. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Rat model of Parkinson’s disease; Etiology; Basal ganglia; Medium spiny neurons; Striatal interneurons; Ionotropic glutamate receptors; Motor symptoms

1. Introduction Parkinson’s disease is a hypokinetic motor disorder caused by a specific and progressive loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) of the basal ganglia (Yurek and Sladek, 1990; Alvord and Forno, 1992; Albin et al., 1995; Obeso et al., 2000). The loss of dopaminergic neurons results in a loss of dopaminergic synaptic inputs from the SNc to neurons in the neostriatum, the principal region of the basal ganglia. This results in an imbalance of the major output pathways of the basal ganglia and thus manifests the motor symptoms of Parkinson’s disease (DeLong, 1990; Albin et al., 1995; Obeso et al., 2000). Glutamatergic systems are known to play significant roles in neuropathology of Parkinson’s disease. During the course of Parkinson’s disease, over-activity of glutamatergic pathways to and within the basal ganglia, notably the corticostriatal, and subthalamopallidal and subtha∗

Corresponding author. Tel.: +852-34117060; fax: +852-34115995. E-mail address: [email protected] (K.K.L. Yung).

lamonigral pathways, are noted (Calabresi et al., 1993; Greenamyre, 1993, 2000; Blandini et al., 1996a,b, 1997; Chase et al., 1998; Rodriguez et al., 1998; Chase and Oh, 2000). In addition, one of the possible causes of the specific cell death of the dopaminergic neurons in the SNc is suggested to be glutamate-mediated excitotoxicity (Olney et al., 1990; Rodriguez et al., 1998; Beal, 1998). Blockade of glutamate receptors by selective antagonists, in particular using the ionotropic glutamate receptor antagonists, ameliorates the Parkinsonian motor symptoms in animal models (Greenamyre and O’Brien, 1991; Greenamyre, 1993, 2000; Stauch et al., 1995; Blandini et al., 1996a,b, 2001; Blanchet et al., 1997, 1999; Starr et al., 1997; Blandini and Greenamyre, 1998; Chase et al., 1998; Nash et al., 1999; Chase and Oh, 2000; Konitsiotis et al., 2000; Steece-Collier et al., 2000). Functions of glutamate are mediated by a diversified family of ionotropic and metabotropic glutamate receptors. Nmethyl-d-aspartate (NMDA) receptors (NR1, NR2A, NR2B, NR2C, NR2D and NR3) and ␣-amino-3-hydroxy-5-methyl4-isoxazole-propionate (AMPA) receptors (GluR1, GluR2,

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GluR3 and GluR4) are two major groups of ionotropic glutamate receptors (Gasic and Hollmann, 1992; Nakanishi, 1992; Hollmann and Heinemann, 1994; Nakanishi et al., 1998). Functional properties of NMDA and AMPA depend on their subunit compositions (Moriyoshi et al., 1991; Meguro et al., 1992; Monyer et al., 1992; Buller et al., 1994; Nakanishi et al., 1998). Modulations in gene expression of NMDA and AMPA receptors are found in animal models of Parkinson’s disease by various techniques. However, there is still no general consensus about the patterns of changes of the glutamate receptors in the neostriatum after dopamine denervation. Most of the previous ligand binding studies indicated that there were decreases in both NMDA and AMPA receptor binding sites in the neostriatum of the 6-hydroxydopamine (6-OHDA)lesioned rats (Wullner et al., 1994; Zavitsanou et al., 1996; O’Dell and Marshall, 1996; Tarazi et al., 1998). However, other authors reported only a reduction in NMDA binding sites in the same model (Porter et al., 1994). In contrast, previous in situ hybridization experiments found significantly higher levels of NR1 expression (Tremblay et al., 1995; Andres et al., 1996), NR2A (Ulas and Cotman, 1996; Ganguly and Keefe, 2001), and GluR1 and GluR2 (Tremblay et al., 1995) mRNA expression in the lesioned neostriatum of 6-OHDA-lesioned rats. Increases in NR2A mRNA were found in both enkephalin-positive (presumed striatonigral medium spiny neurons) and enkephalin-negative striatal neurons (Ganguly and Keefe, 2001). Other studies reported no change in the expression of NR2B mRNA (Tremblay et al., 1995; Kayadjanian et al., 1996) and AMPA receptor mRNAs in the neostriatum of 6-OHDA-lesioned rats and in Parkinsonian patients (Bernard et al., 1996). Last but not the least, GluR1 immunoreactivity was seen to be markedly increased in the caudate and putamen of 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned monkeys (Betarbet et al., 2000). However, there was no change in the overall abundance of NR1, NR2A and NR2B proteins in the striatal tissues of 6-OHDA-lesioned rats, whereas the expression of NR1 and NR2B proteins in the membrane fractions of the striatal tissues was decreased by Western blotting experiments (Dunah et al., 2000). Despite the above findings, less information is available so far about the changes in NMDA and AMPA receptor subunits in subpopulations of striatal neurons after the 6-OHDA lesion in rats. No previous study has attempted to document the changes of mRNA and immunoreactivity of glutamate receptors in the rat model in a single study. The objectives of the present chapter were to address this old but still controversial question by a combination of techniques, namely reverse transcriptase-polymerase chain reaction (RT-RCR) and immunofluorescence. Changes in the levels of NMDA and AMPA subunit mRNAs and immunoreactivity in the neostriatum of 6-OHDA-lesioned rats were documented. In addition, changes in glutamate receptor immunoreactivity at the cellular level were investigated by double immunofluoresncence.

Preliminary reports of the present data have been published in abstract form (Tse and Yung, 1998, 1999a,b; Lai and Yung, 2000).

2. Materials and methods 2.1. Animals Twenty adult rats (Sprague Dawley females, 200–250 g) were used in the present study. The animals were obtained from the University of Hong Kong. The handling of rats and all procedures involving the use of animals were approved in accordance with the Animals (Control of Experiments) Ordinance, Hong Kong Special Administrative Region, China. All efforts were made to ensure that both animal numbers and suffering were minimal in all the experiments. 2.2. Unilateral 6-hydroxydopamine lesion in rats Rats were deeply anesthetized with sodium pentobarbital (60 mg/kg, i.p., Saggital). The rats then received unilateral stereotaxic injections of 6-OHDA (4 ␮l of 3 mg/ml in 0.9% saline containing 0.2 mg/ml ascorbic acid) in the right medial forebrain bundle. The co-ordinates used were in accordance to a rat brain atlas (bregma, 0.45 cm; lateral 0.09 cm; dura 0.75 cm) (Paxinos and Watson, 1986). The injection was administered within a period of 6 min and the needle of the Hamilton syringe was kept in position for a further 10 min following the deposit in order to prevent back filling along the injection tract. The animals were allowed to recover after surgery and kept in the animal house, Hong Kong Baptist University. 2.3. Rat rotation tests Rat rotation tests were performed in order to determine the success of the 6-OHDA lesion. Seven days after surgery, the lesioned rats were injected with apomorphine (1 mg/ml in saline, i.p., RBI). The numbers of rotations in a 30 min test were noted for each rat. Those rats that turned with an average of over seven turns per minute were regarded as successfully lesioned animals. 2.4. Reverse transcriptase-polymerase chain reaction Two weeks after surgery, 6-OHDA-lesioned and agematched normal rats were decapitated, and striatal tissues were dissected as described in previous reports (Lai et al., 2000; Sze et al., 2001). All labware was treated with diethyl pyrocarbonate and autoclaved prior to use. Striatal tissues were homogenized in TriZOL reagent (Gibco, RBL). After incubation for 5 min at room temperature, chloroform was added for phase separation. The upper aqueous phase was collected and total RNA was precipitated by mixing with isopropyl alcohol. The RNA pellet was washed once with

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75% ethanol and was air-dried. It was finally redissolved in RNase-free water. The A260 /A280 ratios were determined with readings between 1.6 and 1.8 using a spectrophotometer (UV-1601, Shimadzu). Semi-quantitative RT-PCR was conducted using the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (G3PDH) as an internal standard for PCR primers, i.e. a “primer-dropping” technique (see Wong et al., 1994), as described in our previous studies (Lai et al., 2000; Sze et al., 2001). RT-PCR was performed using G3PDH (200 bp: 5 -TTCCTACCCCCAATGTATCC-3, 5CCCCAGCATCAAAGGTG-3 ; 437 bp: 5 -ATGGTGAAGGTCGGTGTGAAC-3 , 5 -GCTGACAATCTTGAGGGAGT-3 ), NR1 (5 -AACCTGCAGAACCGCAAG-3 , 5 GCTTGATGAGCAGGTCTATGC-3 ), NR2B (5 -TGCACAATTACTCCTCGACG-3 , 5 -TCCGATTCTTCTTCTGAGCC-3 ), GluR1 (5 -AGGTTTGCTTTGTCACAA-3 , 5 CTTCTCCAGGTCCTGAAA-3 ), GluR2 (5 -CTATTTCCAAGGGGCGCTGAT-3 , 5 -CAGTCCAGGATTACACGCCG-3 ) and GluR3 (5 -TTCCGCTTTGCTGTGCAG-3 , 5 -AATGATGCGTCTGAATTC-3 ) primer pairs as described in previous studies (Yoshioka et al., 1996; Lai et al., 2000; Sze et al., 2001). In addition, RT-PCR was also performed using primer sets for GluR4 (5 -GAATGAACAAGGCCTCTTGG-3 , 5 -TTCATTCTCTTCGCCTCTGC-3 ; (Yoshioka et al., 1996). The number of cycles was varied to determine the optimal number that would allow detection of the amplified products, while keeping amplification for these genes in the log phase. Different amplification cycles were used (NMDA receptor subunits with G3PDH: NR1: 30 cycles; NR2B: 35 cycles; G3PDH: 30 cycles; AMPA receptor subunits with G3PDH: G3PDH: 33 cycles; GluR1: 35 cycles; GluR2: 35 cycles; GluR3: 35 cycles). Amplification cycle of GluR4 was 35 cycles. Total RNA was diluted to 1 ␮g/␮l in RNase-free water, mixed with 0.5 ␮g of pd(N)6 and 47 ␮l of RNase-free water to a final volume of 49 ␮l in a reaction tube containing RT-PCR beads. One of the above primer sets was added to give a final volume of 50 ␮l. The reaction was incubated at 42 ◦ C for 30 min, followed by 95 ◦ C for 5 min to inactivate the RT and to completely denature the template. Reactions were run for the optimal cycles with 55 ◦ C annealing cycle (1 min), 72 ◦ C extension cycle (1 min), and a 95 ◦ C denaturing cycle (50 s). The G3PDH primer set was added into the reaction according to its corresponding pre-calibrated cycle number. Control amplifications were done either without RT or without RNA. Amplification from primers, NR1, NR2B, GluR1, GluR2, GluR3 and GluR4, G3PDH produces fragments of 333, 222, 456, 539, 468, 219, 437 and 200 bp in length, respectively. The PCR products were loaded on agarose gels (1%) containing ethidiume bromide and electrophoresis was performed at 100 V for 30 min. Image of the DNA gels after RT-PCR analyses were digitally captured by a gel documentation system (Ultra Violet Products C-80). The optical density of each band was measured by an image analyzing

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software (Metamorph). As described in our previous study (Sze et al., 2001), the intensities of GluR1, GluR2, GluR3, NR1 and NR2B bands of the striatal tissues (from the lesioned side or the non-lesioned side) were normalized using the intensities obtained in the G3PDH bands and then expressed as a ratio. The relative intensities of GluR4 bands were also determined. Data obtained from six animals were averaged. Comparisons were made between the data from the non-lesioned side to those from the lesioned side of the same 6-OHDA-leisoned animals. Since the data were from the same animals, statistical analyses were made by paired Student’s t-tests (SPSS software). Changes were expressed as a percentage of that observed on the control non-lesioned side. 2.5. Immunofluorescence and double immunofluorescence Two weeks after surgery, lesioned and normal rats were deeply anaesthetized with an overdose of sodium pentobarbital (60 mg/kg, i.p., Saggital). They were perfused transcardially with 50–100 ml of normal saline followed by 200 ml fixative (3% para-formaldehyde and 0.01% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4). The brains were quickly removed from the skull after the perfusion and post-fixed in 3% para-formaldehyde (in 0.1 M PB, pH 7.4) for a few hours at 4 ◦ C. Sections of the neostriatum (70 ␮m) were cut on a vibrating microtome (Vibratome 1000, Technical Products International). The sections were collected in phosphate buffered saline (PBS; 0.01 M, pH7.4) and pre-incubated with normal goat serum (4% in PBS, Vector Labs, Burlingame, CA) for about 1 h at room temperature before immunofluorescence. As described in our previous studies (Lai et al., 2000; Sze et al., 2001), all the striatal sections for comparisons were incubated and reacted in a single reaction sequence to minimize variation in immunofluorescence. The striatal sections were incubated in primary antibody solutions against NR1 (rabbit polyclonal, 0.5 ␮g/ml in PBS supplemented with 2% normal goat serum and 0.1% Triton-X 100 [PBS-Triton]; Chemicon International, Temcula, CA, USA), NR2B (rabbit polyclonal, 0.5 ␮g/ml in PBS-Triton; Chemicon International), GluR1 (rabbit polyclonal, 0.5 ␮g/ml in PBS-Triton; Chemicon International), GluR2 (mouse monoclonal, 1.0 ␮g/ml in PBS-Triton; Chemicon International), GluR2/3 (rabbit polyclonal, 0.5 ␮g/ml in PBS-Triton; Chemicon International), and GluR4 (rabbit polyclonal, 1.0 ␮g/ml in PBS-Triton; Chemicon International) for 24 h at room temperature. After incubation, the sections were washed (3× PBS) and incubated in Alexa 488-conjugated secondary antibody solutions (goat anti-rabbit IgG or goat anti-mouse IgG; 1:200 in PBS-Triton; Molecular Probes) for 2 h at room temperature in dark. The sections were then washed (3× PBS), mounted in mounting medium (Vectashield, Vector Labs) and examined on a confocal microscope (LSM 510, Zeiss).

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In addition, double immunofluorescence was performed to reveal immunoreactivity for GluR1 together with parvalbumin (PV) or choline acetyltransferase (ChAT) immunoreactivity, i.e. specific neuronal markers for striatal interneurons. Sections were subjected to the above procedures with incubation in a mixture of the two different primary antibodies overnight at room temperature. The two primary antibodies in the mixture were from different species of origin, i.e. GluR1 (rabbit origin) with PV (mouse monoclonal, 1:500; Sigma) or GluR1 with ChAT (rat monoclonal, 1:500; Chemicon International). The sections were washed (3× PBS) and then incubated in a mixture of fluorochrome-conjugated secondary antibodies (Alexa 488- or Alexa 562-conjugated antibodies; Molecular Probes) for 2 h at room temperature in dark. The sections were also mounted and observed in the confocal microscope. In control experiments, the above reaction sequences were performed with the omission of either the primary or secondary antibody solutions, or each of the primary antibodies in turn, in the reaction sequence. 2.6. Semi-quantitative analysis of immunofluorescence and data analysis The levels of immunofluorescence were semi-quantified by digital image analysis as described in our previous study (Sze et al., 2001). Digital images of the neostriatum showing immunoreactivity for GluR1, GluR2, GluR2/3, GluR4, NR1 and NR2B (single labeling) or GluR1 with PV (double labeling) were captured under the same parameters in the confocal microscope at low (using a 20× lens) or high (using a 63× lens) magnifications, respectively. At least three sections of each side of each animal were used. The fluorescent intensity of the confocal microscope images was determined using image analyzing software (Metamorph). At low magnification, the level of immunofluorescence of at least three random views in the region of neostriatum, i.e. all the neuropilar elements, was measured. However, the fiber bundles that were immuno-negative were not included. At high magnification, the levels of immunofluorescence in the cytoplasm of the immunoreactive perikarya were determined. The immunofluorescence in the neuropilar elements and immuno-negative nuclei were excluded. In those sections that were double labeled to show GluR1 and PV immunoreactivity, only the intensity of GluR1 immunoreactivity in the PV-positive perikarya was determined. Intensity of GluR1 immunoreactivity in PV-negative neurons was excluded. Comparisons were made between the data obtained from the striatal sections of the lesioned side and those obtained from the non-lesioned side of the same 6-OHDA-lesioned animals. Similar to the RT-PCR analysis, statistical analyses were also made by paired Student’s t-tests (SPSS software). Changes were expressed as percentage of that observed on the control side.

3. Results 3.1. Effects of 6-OHDA lesion on the mRNA expression of AMPA- and NMDA-type glutamate receptor subunits in the neostriatum The present results demonstrated no difference in the expression of GluR1, GluR2, Glu3 and GluR4 mRNAs in the two sides of the striatal tissues in the normal non-lesioned rats (Fig. 1A). These indicate that there is no “side” variation of mRNA expression between the two sides of striatal tissues in rats. In contrast, 6-OHDA lesion resulted in differential regulations on the gene expression of glutamate receptor subunits in the striatal tissues. The results of RT-PCR indicated that among the four AMPA-type glutamate receptor subunits, there was a reduction in the levels of expression of GluR1 mRNA in the lesioned neostriatum of the 6-OHDA-lesioned rats (−15.41% when compared to the non-lesioned side, Fig. 1B; Table 1). There were no significant differences in the expression of the GluR2, GluR3 and GluR4 mRNAs between the lesioned and non-lesioned sides (Fig. 1B; Table 1). Similarly, there was no significant difference between the levels of NR1 and NR2B mRNAs in two sides of the striatal tissues in non-lesioned rats (Fig. 1C). In 6-OHDA-lesioned rats, the level of NR1 mRNA was found to be increased in the striatal tissues of the lesioned side (+27.97%, Fig. 1D; Table 1). In contrast, the levels of NR2B mRNA between the lesioned striatal and the non-lesioned striatal tissues were found to remain the same (Fig. 1D; Table 1). 3.2. Effects of 6-OHDA lesion on immunoreactivity for glutamate receptor subunits in the neostriatum 3.2.1. GluR1 In the control sections that were processed without the primary antibody, no immunoreactivity was seen (Fig. 2A). Using confocal microscopy, immunoreactivity for GluR1 was found in the rat neostriatum (Fig. 2B and C). The fiber bundles were immuno-negative (Fig. 2B and C). At low magnification, GluR1 immunoreactivity was found to decrease in the lesioned side (Fig. 2C) when compared to the non-lesioned side (Fig. 2C). Image analyses of the intensity of the sections revealed that there was a significant decrease in immunofluorescence intensity in the lesioned side (−23.35%, Table 2) when compared to the non-lesioned side. At the higher magnification, robust GluR1 immunoreactivity was found in the perikarya of striatal neurons that displayed indented nuclei, a characteristic of striatal interneurons as indicated by our previous study (Fig. 2D) (Kwok et al., 1997). In addition, low intensity GluR1 labeling was also found in the cytoplasm of neurons that were oval in shape (Fig. 2D) and these neurons were presumed to be medium spiny neurons (Bernard et al.,

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Fig. 1. Amplification of GluR1, GluR2, GluR3, NR1 and NR2B subunits with G3PDH and GluR4 in the striatal tissues obtained from normal and 6-OHDA-lesioned rats. (A) Bands that are approximately 456 bp (GluR1), 539 bp (GluR2), 468 bp (GluR3) and 219 bp (GluR4) are observed respectively in two sides of striatal tissues of normal rats. In addition, a band approximately 200 bp (G3PDH) is also observed in the cases of GluR1, GluR2 and GluR3 PCR product. No difference is found in the PCR products between the two sides of striatal tissues of normal rats. (B) Bands with similar molecular weights are observed in striatal tissues of 6-OHDA-lesioned rats showing the AMPA receptor subunit PCR products. Comparisons between the PCR of the non-lesioned side (N) and the lesioned side (L) are shown. There is a depletion in the level of GluR1 PCR product in the lesioned side (indicated by asterisk) when compared with the non-lesioned side. In contrast, similar levels of GluR2, GluR3 and GluR4 PCR products are found in between the non-lesioned and lesioned sides. (C) Bands that are approximately 333 bp (NR1) and 222 bp (NR2B) are seen respectively in the two sides of striatal tissues of normal rats. Bands of approximately 437 bp (G3PDH) are also seen. No difference was seen in the levels of NR1 and NR2B PCR products in the two sides of neostriatum of normal rats. (D) In the comparison between the NR1 and NR2B PCR products of the non-lesion side (N) to those of the lesioned side (L) of neostriatum, there is an increase in the expression of NR1 PCR product in the lesioned side (indicated by an arrow). In contrast, no difference is observed in the levels of the NR2B PCR products between the lesioned and non-lesioned sides.

1997). In the lesioned side of neostriatum, many presumed spiny neurons still found displayed GluR1 immunoreactivity but the level of intensity was found to be lower than that found in the same type of neurons in the non-lesioned side (compared Fig. 2D–E). Image analysis revealed significant decrease of GluR1 immunoreactivity in the presumed medium spiny neurons of the lesioned side when compared to the non-lesioned side (−16.49%, Table 2). Some sections were subjected to double labeling of GluR1 and one of the two specific neurochemical markers for stri-

atal interneurons, namely PV or ChAT, no double-labeled GluR1- and ChAT-immunoreactive perikarya were seen (data not shown). In contrast, all of the intensely labeled GluR1-immunoreactive neurons were found to contain PV immunoreactivity (Fig. 3A–F). Slightly higher levels of GluR1 labeling were observed in the PV-positive neurons in the non-lesioned than in the lesioned sides (Fig. 3A–F). However, statistical analysis revealed that no significant difference in GluR1 immunoreactivity was found in the PV-immunoreactive interneurons between the lesioned and non-lesioned side of the neostriatum (Table 2).

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Table 1 Semi-quantitative analysis of RT-PCR results in the neostriatum of 6-OHDA-lesioned rats Non-lesioned side GluR1 (ratio between GluR1 and G3PDH bands) GluR2 (ratio between GluR2 and G3PDH bands) GluR3 (ratio between GluR3 and G3PDH bands) NR1 (ratio between NR1 and G3PDH bands) NR2B (ratio between NR2B and G3PDH bands) GluR4 (relative intensity)

1.39 2.49 1.09 1.18 1.22 93.17

± ± ± ± ± ±

0.03 0.96 0.67 0.11 0.17 5.53

Leisoned side 1.17 2.11 1.03 1.5 1.40 92.26

± ± ± ± ± ±

0.03∗∗ [−15.41%] 0.73 0.01 0.01∗ [+27.97%] 0.16 4.70

Average values that are obtained from six animals are shown. The relative intensity of fluorescence of each band in the gel is measured by an image analyzing software (Metamorph). There are two different sets of analysis. In cases of the PCR products of GluR1, GluR2 and GluR3, NR1 and NR2B, a “primer-dropping” technique is employed. The values shown are ratios of the band intensities observed for the glutamate receptor PCR products and the internal control of G3PDH PCR product. The ratios are expressed as the mean ± S.E.M. Comparison was made among the striatal tissues from the non-lesioned side and the lesioned side of the same 6-OHDA-lesioned rats. Statistical analyses were performed by paired Student’s t-test. The percentage of change in respect to the values of the non-lesioned side is indicated by ‘±’ signs. In case of the PCR products of GluR4, the averaged relative intensities are expressed as the mean ± S.E.M. Statistical comparisons between the two sides are made between the mean intensities. ∗ P < 0.05. ∗∗ P < 0.01.

3.2.2. GluR2 Immunoreactivity for GluR2 was also observed in the neostriatum (Fig. 4A–D). At low magnification, immunoreactivity for GluR2 was primarily found in the neuropil as well as in cell bodies of the neostriatum (Fig. 4A and B). Image analyses revealed that the intensity of GluR2 im-

munoreactivity in the non-lesioned side was the same as the lesioned side (Table 2). At higher magnification, many GluR2-immunoreactive neurons were found in both sides of the neostriatum. The morphology of the neurons resembled medium spiny neurons as previously described (Kwok et al., 2000). There was

Table 2 Semi-quantitative analysis of intensity of immunofluorescence in the neostriatum of 6-OHDA-lesioned rats Relative intensity

Non-lesioned side

Lesioned side

GluR1 (region of neostriatum) GluR1 (striatal neurons) GluR1 (striatal PV-positive neurons)

2107.13 ± 263.63 (12 sections from four rats) 813.47 ± 46.72 (n = 203) 1305.53 ± 264.02 (n = 59)

1615.07 ± 172.70∗ [−23.35%] (12 sections from four rats) 679.33 ± 42.29∗ [−16.49%] (n = 201) 1011.6 ± 185.02 (n = 66)

GluR2 (region of neostriatum) GluR2 (striatal neurons)

1921.27 ± 229.93 (12 sections from four rats) 701.94 ± 175.39 (n = 204)

1648.03 ± 224.97 (12 sections from four rats) 734.82 ± 174.72 (n = 205)

GluR2/3 (region of neostriatum) GluR2/3 (striatal neurons)

1154.96 ± 156.24 (12 sections from four rats) 1917.28 ± 265.75 (n = 201)

941.17 ± 166.29 (12 sections from four rats) 1793.28 ± 332.78 (n = 207)

GluR4 (region of neostriatum) GluR4 (striatal neurons)

2243.17 ± 110.83 (12 sections from four rats) 972.8 ± 41.44 (n = 48)

2206.17 ± 62.27 (12 sections from four rats) 899.0 ± 76.14 (n = 39)

NR1 (region of neostriatum) NR1 (striatal neurons)

2310.67 ± 55.38 (12 sections from four rats) 803.0 ± 59.25 (n = 253)

2125.67 ± 94.50 (12 sections from four rats) 660.67 ± 24.83 (n = 262)

NR2B (region of neostriatum) NR2B (striatal neurons)

1937.50 ± 23.50 (12 sections from four rats) 763.67 ± 63.68 (n = 234)

1880.75 ± 53.75 (12 sections from four rats) 693.0 ± 6.35 (n = 223)

Semi-quantitative analysis of levels of immunofluorescence of GluR1, GluR2, GluR2/3, GluR4, NR1 and NR2B obtained from 6-OHDA-lesioned rats is shown. Comparisons were made between the data obtained in the striatal sections of the lesioned and non-lesioned sides. Digital images of the neostriatum showing immunoreactivity for GluR1, GluR2, GluR2/3, GluR4, NR1 and NR2B (single labeling) or GluR1 with PV (double labeling) were captured under the same parameters in the confocal microscope at low (using a 20× lens) or high (using a 63× lens) magnifications, respectively. The fluorescent intensity of the confocal microscope images were determined by the using an image analyzing software (Metamorph). At low magnification, the level of immunofluorescence of at least three random views in the region of neostriatum, i.e. all the neuropilar elements, was measured but the fiber bundles that were immuno-negative were not included (indicated as “region of neostriatum” in the table). At high magnification, the levels of immunofluorescence in the cytoplasm of the immunoreactive perikarya were determined. The immunofluorescence in the neuropilar elements and immuno-negative nuclei were excluded (indicated as “striatal neurons” in the table). In those sections that were double labeled to show GluR1 and PV immunoreactivity, only the intensity of GluR1 immunoreactivity in the PV-positive perikarya was determined. Intensity of GluR1 immunoreactivity in PV-negative neurons was excluded (indicated as “striatal PV-positive neurons” in the table). The numbers of sections and animal studied, or the numbers of neurons studies [n] are given in parentheses. Data are presented as mean ± S.E.M. Statistical analyses were performed by paired Student’s t-test. The percentage of decrease in number in respect to the values of the non-lesioned side are indicated by the minus sign. ∗ Significance at P < 0.05.

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Fig. 2. Immunofluorescent micrographs showing the neostriatum of the non-lesioned side and the lesioned side of the 6-OHDA-lesioned rats immunostained to reveal immunoreactivity for GluR1. Sections were obtained from the same animals. (A) In the control section that was processed without the primary antibody (control), no immunoreactivity was found. (B) Immunoreactivity for GluR1 is abundant in the non-lesioned side of striatum. Fiber bundles are immunonegative (two are indicated by white stars). A few intensely labeled perikarya are seen (arrows). (C) Immunoreactivity for GluR1 is observed in the lesioned side of striatum. Similar to (A), the fiber bundles are immunonegative (two are indicated by white stars). A few intensely labeled perikarya are also seen (arrows). There is a decrease of fluorescent intensity of GluR1 labeling in the region when compared to the non-lesioned side. (D) In the non-lesioned side, GluR1-immunoreactive neurons are observed at higher magnification. Some of the GluR1-immunoreactive neurons are medium-sized, oval in shape, and posses a large unstained circular nucleus (some are indicated by arrowheads). They are presumed medium spiny neurons. In addition, some interneuron-like GluR1-immunoreactive perikarya that are more intensely labeled are found (open arrow). (E) In the lesioned side, the oval-shaped GluR1-immunoreactive perikarya are still observed (some are indicated by arrowheads). The intensity of GluR1 immunofluorescence in this kind of neurons is seen to be reduced (arrowheads). In contrast, in those intensely labeled GluR1-positive interneuron-like neurons, similar level of fluorescence intensity is observed (open arrows). Scale bars: 200 ␮m (in (C), for (A) and (B)), and 20 ␮m (in (D), also for (C)).

no obvious difference in patterns of distribution nor morphological difference between the GluR2-immunoreactive neurons found in the both sides (Fig. 4C and D). Image analysis revealed that the intensities of GluR2 immunoreactivity between the lesioned and non-lesioned sides were not significantly different (Table 2).

3.2.3. GluR2/3 Similar to GluR2 immunoreactivity, GluR2/3 immunoreactivity was primarily found in the neuropil and in cell bodies in both sides of neostriatum (Fig. 4E–H). At low magnification, image analysis revealed that the intensities of GluR2/3 immunoreactivity between the non-lesioned side (Fig. 4E)

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Fig. 3. Color immunofluorescent micrographs showing the neostriatum of the non-lesioned side and the lesioned side of the 6-OHDA-lesioned rats immunostained to reveal immunoreactivity for GluR1 together with immunoreactivity for parvalbumin (PV). Sections were obtained from the same animals. Immunoreactivity for GluR1 (A and B) is identified by Alexa 488 (green) signal and immunoreactivity for PV (C and D) is identified by Alexa 562 (red) signal. (A) At higher magnification, a intensely labeled GluR1-positive neuron is seen in the non-lesioned side. (B) Similarly, in the lesioned side, intensely labeled GluR1-immunoreactive neuron is seen. The level of GluR1 labeling is similar to that found in the non-lesioned side. (C) The same neuron in (A) is found to be PV-positive. (D) The neuron shown in B is also PV immunoreactive. The superimposed images of (A + C) and (B + D) are shown in (E) and (F), respectively. Scale bar: 20 ␮m (in (F), for all micrographs).

䉴 Fig. 4. Immunofluorescent micrographs showing the neostriatum of the non-lesioned side and the lesioned side of the 6-OHDA-lesioned rats immunostained to reveal immunoreactivity for GluR2 (A–D) or GluR2/3 (E–H). Sections were obtained from the same animals. (A) In the non-lesioned neostriatum, immunoreactivity for GluR2 is seen. The fiber bundles are immunonegative (two are indicated by white stars; also in (B), (E) and (F)). (B) In the lesioned side, GluR2 immunoreactivity is also found. The levels of immunolabeling are similar between two sides. (C) At higher magnification, GluR2-immunoreactive perikarya are seen in the non-lesioned side (some are indicated by asterisks). The cytoplasm is densely labeled and a large, circular and unstained nucleus is often seen. (D) Similarly, GluR2-positive perikarya are also found in the lesioned side (some are indicated by asterisks). The GluR2-positive neurons appeared to display similar levels of labeling and morphological characteristics as those found in the non-lesioned side. (E) In the non-lesioned side of neostriatum, immunoreactivity for GluR2/3 is observed in the neuropil and cell bodies of neurons (some are indicated by arrows). (F) In the lesioned side, similar level of GluR2/3 labeling is observed. GluR2/3-positive neurons are also seen (some are indicated by arrows). (G) At higher magnification, in the non-lesioned side, GluR2/3-immunoreactive neurons are found to be medium in size with a large unstained circular nucleus. The cytoplasm is densely labeled (some are indicated by asterisks). (H) In the lesioned side, GluR2/3-labeled neurons are found to be similar to those observed in the non-lesioned side. Similar level of GluR2/3 labeling is observed. Scale bars: 200 ␮m (in (F), also for (A), (B) and (E)), and 20 ␮m (in (H), also for (C), (D) and (G)).

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and the lesioned side (Fig. 4F) were not statistically different (Table 2). At higher magnification, GluR2/3-containing neurons were also found to be very similar in appearance to the GluR2-containing neurons (Fig. 4G and H). All of them were medium-sized with densely labeled cytoplasm and they were presumed medium spiny neurons (see Kwok et al., 1997, 2000). Image analysis showed that there was no significant difference in the immunofluorescence intensities between the non-lesioned side (Fig. 4G) and the lesioned side (Fig. 4H; Table 2). 3.2.4. GluR4 At low magnification, GluR4 immunoreactivity was found in the neuropil and in cell bodies of the non-lesioned and lesioned neostriatum (Fig. 5A and B). The GluR4-immunoreactive neurons were less than those observed in GluR2 and GluR2/3 immunolabeling. Image analysis revealed that intensities of GluR4 labeling were not significantly different in both sides (Table 2). At higher magnification, similar to GluR1 immunolabeling, all of the GluR4-containing neurons were intensely labeled and they resembled striatal interneurons in their morphology (see Kwok et al., 1997). In contrast, no

GluR4-immunoreactive medium spiny neuron-like neurons were seen (Fig. 5C and D). Image analysis showed no significant difference in intensities of cytoplasmic GluR4 immunoreactivity between the non-lesioned and lesioned sides (Table 2). 3.2.5. NR1 and NR2B At low magnification, immunoreactivity for NR1 and NR2B was also found to be abundant in both sides of neostriatum (Fig. 6A and B for NR1; Fig. 6E and F for NR2B). NR1 and NR2B immunoreactivity was primarily observed in the neuropil and also in a large number of perikarya in the noestriatum (Fig. 6A and B for NR1; Fig. 6E and F for NR2B). Image analyses revealed that in both cases, no significant difference in fluorescence intensities was observed between the non-lesioned and lesioned sides (Table 2). At higher magnification, both NR1 and NR2B immunoreactivity was primarily found in the cytoplasm of neurons that resembled medium spiny neurons (Fig. 6E and F for NR1; Fig 6G and H for NR2B) as described by our previous studies (Lai et al., 2000; Sze et al., 2001). Results of the image analysis revealed that levels of cytoplasmic NR1 or NR2B immunofluorescence intensities were not different in the lesioned and non-lesioned sides (Table 2).

Fig. 5. Immunofluorescent micrographs showing the neostriatum of the non-lesioned side and the lesioned side of the 6-OHDA-lesioned rats immunostained to reveal immunoreactivity for GluR4. Sections were obtained from the same animals. (A) In the non-lesioned side, immunoreactivity for GluR4 is seen in the neuropil. The fiber bundles are immunonegative (two are indicated by white stars; also in (B)). (B) In the lesioned side, immunoreactivity for GluR4 is observed. Similar levels of GluR4 labeling are found in the lesioned side when compared to that in the non-lesioned side. (C) At higher magnification, in the non-lesioned side, GluR4-immunoreactive perikarya are seen (one is indicated by open arrow). In addition, GluR4-positive neuropilar elements are also seen. (D) In the lesioned side, GluR4-immunoreactive perikarya are seen (one is indicated by open arrow). The GluR4-immunoreactive perikarya display similar level of labeling as in the non-lesioned side. Scale bars: 200 ␮m (in (B), for (A)), and 20 ␮m (in (D), for (C)).

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Fig. 6. Immunofluorescent micrographs showing the neostriatum of the non-lesioned side and the lesioned side of the 6-OHDA-lesioned rats immunostained to reveal immunoreactivity for NR1 (A–D) or NR2B (E–H). Sections were obtained from the same animals. (A) In the non-lesioned side, immunoreactivity for NR1 is found to be abundant. The fiber bundles are immunonegative (two are indicated by white stars; also in (B), (E) and (F)). A large of number of NR1-immunoreactive perikarya are seen (two are indicated by arrows). (B) In the lesioned side, immunoreactivity for NR1 is also seen to be abundant. Similar to the non-lesioned side, a large number of NR1-positive perikarya are found (two are indicated by arrows). (C) At higher magnification, many NR1-immunoreactive perikarya are observed. Most of them are found to be medium in size, and display a large circular and unstained nucleus (some are indicated by asterisks). (D) In the lesioned side, similar observation as in (C) is seen. Some NR1-positive neurons are also indicated by asterisks. (E) In the non-lesioned side, immunoreactivity for NR2B is seen in the region. Fiber bundles are negative (indicated by stars). (F) In the lesioned side, similar levels of NR2B labeling are seen. (G) At higher magnification, NR2B-immunoreactive perikarya are seen in the non-lesioned side (some are indicated by asterisks). They displayed similar morphological characteristics of NR1-positive neurons (C). (H) In the lesioned side, NR2B-immunoreactive perikarya are also seen. They also display similar morphology and level of NR2B labeling to the neurons in the non-lesioned side. Scale bars: 200 ␮m (in (F), also for (A), (B) and (E)), and 20 ␮m (in (H), also for (C), (D) and (G)).

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4. Discussion 4.1. Down-regulation of GluR1 glutamate receptor in the neostriatum after 6-hydroxydopamine lesion The present results have demonstrated that 2 weeks after dopamine denervation, differential effects of the AMPA subunit compositions in neurons of the neostriatum are seen. There is no modulation in the expression of GluR2, GluR3 and GluR4 mRNAs in striatal tissues. Moreover, no modulation is also observed in the expression of GluR2, GluR2/3 and GluR4 immunoreactivity in neurons of the neostriatum. The present findings are in general consistent to previous ligand binding studies, which have revealed that no change (Porter et al., 1994) or decrease (Wullner et al., 1994; Zavitsanou et al., 1996; O’Dell and Marshall, 1996; Tarazi et al., 1998) in AMPA receptor binding sites in the neostriatum after dopamine denervation. However, previous findings by in situ hybridization have indicated that there is no change of GluR1, GluR2, GluR3 and GluR4 mRNA expression in the neostriatum of 6-OHDA-lesioned rats (Tremblay et al., 1995) or in the caudate putamen of Parkinsonian patients (Bernard et al., 1996). A previous immunocytochemical study (Betarbet et al., 2000) has also found that there are increases in GluR1 mRNA and protein expression in MPTP-lesioned monkeys. The findings of Betarbet et al. (2000) are made in primate model of Parkinson’s disease and species variation may need to be taken into account for the difference in results seen in the present study. In addition, no analysis has been made at the cellular level in this previous study and therefore there is no additional information about the modulation of GluR1 immunoreactivity in different striatal neuronal subpopulations (Betarbet et al., 2000). 4.2. Dopamine denervation has differentially modulated GluR1 gene expression in striatal neurons The decrease in GluR1 proteins is seen primarily in neurons that bear characteristics of striatal medium spiny neurons (reviewed by Bolam and Bennett, 1995; Gerfen and Wilson, 1996; Smith et al., 1998). GluR1 immunoreactivity has been localized to medium spiny neurons in a previous study (Bernard et al., 1997). In contrast, the intensity of GluR1 immunoreactivity in PV-positive striatal GABAergic interneurons (Gerfen and Wilson, 1996) has been found to be unchanged (reviewed by Bolam and Bennett, 1995). Expression of GluR4 mRNA as well as GluR4 immunoreactivity in striatal neurons is also found to be unchanged. This is a new piece of information as PV-positive interneurons are known to be the sole subpopulation of striatal interneurons that express high levels of GluR1 and GluR4 immunoreactivity (Chen et al., 1996; Kwok et al., 1997). The present findings thus indicate that there are differential effects of dopamine denervation on GluR1 expression in different subpopulations of striatal neurons.

Medium spiny neurons are the principal output neurons of the neostriatum that form the two major pathways of the basal ganglia, i.e. the direct and indirect pathways (reviewed by Gerfen and Wilson, 1996; Smith et al., 1998; Bolam et al., 2000). These two major pathways mediate excitatory signals that primarily arise from the cortex and the thalamus and glutamate is utilized as the neurotransmitter (Gerfen and Wilson, 1996; Smith et al., 1998; Bolam et al., 2000). The reduction in GluR1 receptor expression in medium spiny neurons may therefore affect glutamate neurotransmission through the two major pathways in the basal ganglia. It is well established that the spiny neurons are the major targets of the dopaminergic synaptic inputs from the SNc (reviewed by Smith and Bolam, 1990; Gerfen and Wilson, 1996; Smith et al., 1998; Bolam et al., 2000). The dopaminergic inputs primarily terminate on the neck of spines in the striatal spiny neurons (Smith and Bolam, 1990; Gerfen and Wilson, 1996; Smith et al., 1998; Bolam et al., 2000). Previous studies have indicated that the number of spines of presumed medium spiny neurons as well as the number of asymmetrical synapses are significantly reduced in the neostriatum after dopamine denervation (Ingham et al., 1993, 1998; Arbuthnott et al., 2000). A large proportion of GluR1 receptor immunoreactivity is found in spines and also localized in the postsynaptic membrane of asymmetrical synapses at the subcellular level (Bernard and Bolam, 1998). Reduction in spines after dopamine denervation in medium spiny neurons may account for the reduction of GluR1 immunoreactivity found in spiny neurons. However, it is unknown why the other AMPA receptor subunits such as GluR2, GluR2/3 and NR1 are presumably localized also in spines (Bernard et al., 1997; Bernard and Bolam, 1998) are not affected after the dopamine denervation. In contrast, no modulation of GluR1 expression is found in PV-positive GABAergic interneurons. The PV-positive GABAergic interneurons are now thought to be one of the key groups of neurons that maintain the balance of signals between the direct and indirect pathways (reviewed by Bolam et al., 2000). The fact that AMPA subunit composition remains stable in the PV-positive neurons may indicate that the PV-positive neurons may be less affected by the dopamine denervation than the medium spiny neurons. It is therefore logical that there is less reduction of GluR1 immunoreactivity in PV-positive neurons as they are aspiny neurons (reviewed by Bolam and Bennett, 1995; Gerfen and Wilson, 1996; Bolam et al., 2000). 4.3. Dopamine denervation may selectively affect different groups of striatal neurons The differential modulation of GluR1 expression in spiny neurons and PV-positive neurons, respectively may also reflect an indirect influence other than the direct effect of dopamine denervation. Glutamatergic pathways are known to be over-active in Parkinsonian condition and these pathways include the corticostriatal pathway and the pathway

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originated from the subthalamic nucleus (Calabresi et al., 1993; Greenamyre, 1993, 2000; Blandini et al., 1996a,b, 1997; Chase et al., 1998; Rodriguez et al., 1998; Chase and Oh, 2000). The over-activity of glutamatergic pathways may therefore also contribute in the modulation of the GluR1 expression in the spiny neurons. In addition to spiny neurons, cortical synaptic inputs to the neostriatum also terminate on PV-positive GABAergic interneurons and these neurons are suggested to exert a feed-forward inhibition on spiny neurons (reviewed by Bolam et al., 2000). The present results may indicate that the effects of over-activity in cortical inputs to spiny neurons and PV-positive GABAergic neurons may be different. In addition to that, one may even speculate that there may be heterogeneous origins of the cortical inputs to the two subpopulations of striatal neurons within the cortex that is differentially affected by dopamine denervation. Native AMPA channels are believed to be composed of heteromeric subunits (Gasic and Hollmann, 1992; Nakanishi, 1992; Nakanishi et al., 1998) and the functional properties of the AMPA receptors, i.e. Ca2+ permeability, are closely related to the expression of GluR2 subunit in the AMPA channel (Bennett et al., 1996; Pellegrini-Giampietro et al., 1997). The functions of other AMPA subunits in terms of modulation of the AMPA channel activities are less understood. The reduction of GluR1 receptor expression may be a response to the over-activity of glutamatergic pathways. The alteration in AMPA subunit composition may in turn modulate the overall glutamate neurotransmission in medium spiny neurons but not in PV-positive GABAergic striatal interneurons after dopamine denervation. The physiological significance of this differential effect of modulation of GluR1 in spiny neurons and in GABAergic interneurons however, remains to be established. 4.4. Up-regulation of NR1 mRNA in the neostriatum after 6-OHDA lesion Another interesting finding of the present study is that there is an up-regulation of NR1 mRNA only in the striatal tissues after the 6-OHDA lesion. However, no modulation of NR1 immunoreactivity is seen in striatal neurons, i.e. in presumed medium spiny neurons. The present findings do provide a clue that an increase in mRNA expression of NR1 may not affect the overall availability of NR1 proteins in striatal neurons. The present results match previous in situ hybridization results (Tremblay et al., 1995) and Western blotting analysis (Dunah et al., 2000). In contrast, the present result does not match most of the previous ligand binding studies (Wullner et al., 1994; Zavitsanou et al., 1996; O’Dell and Marshall, 1996; Tarazi et al., 1998) in which reductions of NMDA binding sites have been found in the neostriatum after dopamine denervation. These differences can be accounted for in the context of the known distinct turnover rate of NR1 from NR2 subunits (Stephenson, 2001) and the subunit specificity of ligand binding (Grimwood et al., 2000;

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Lynch and Guttmann, 2001; Stephenson, 2001). However, it is not known how an increase in NR1 mRNA expression only could affect the NMDA channel properties in striatal neurons after dopamine denervation (Tremblay et al., 1995). In addition, no modulation of NR2B mRNA (as seen in a previous study, Kayadjanian et al., 1996) as well as immunoreactivity is found in striatal neurons after the 6-OHDA lesions. Interestingly, a previous report has indicated that although the overall abundance of NR subunits does not change in striatal tissues, NR1 and NR2B proteins are found to be decreased in the membrane fractions of striatal tissues and these results indicate a change in turnover of NR subunits (Dunah et al., 2000). In addition, the phosphorylated NR1 and tyrosine phosphorylated NR2B are found to decrease after dopamine denervation (Dunah et al., 2000). The phosphorylation of NR subunits are also known to lead to modulation of the ion channel conductance and the opening properties of the NMDA channels (Grosshans and Browning, 2001; Scott et al., 2001; Vissel et al., 2001). These indicate that the physiological states of NMDA receptors may change after dopamine denervation. As mentioned as above, dopamine denervation has resulted in significant reduction of spines as well as asymmetrical synapses in the neostriatum (Ingham et al., 1993, 1998; Arbuthnott et al., 2000) and NMDA receptor immunoreactivity in the neostriatum are also localized primarily on spines of presumed medium spiny neurons (Bernard and Bolam, 1998). Thus, the increase in NR1 mRNA expression may be a compensation mechanism to combat the effect of spine reductions after dopamine denervation. Since NR1 and NR2B subunit specific antisense oligonucleotides specifically reduce expression of NR1 and NR2B mRNAs and immunoreactivity/proteins in normal rats (Lai et al., 2000; Sze et al., 2001), the present finding may have implications in using NMDA receptor antagonists in treatment of Parkinson’s disease. Acknowledgements The present work was supported by Faculty Research Grants, Hong Kong Baptist University and Research Grant Council, Hong Kong (to K.K.L. Yung). S.K. Lai and Y.C. Tse were supported by Postgraduate Studentships, Hong Kong Baptist University during the course of the work. The authors would also like to thank Ms. L.Y. Man for technical assistance. References Albin, R.L., Young, A.B., Penney, J.B., 1995. The functional anatomy of disorders of the basal ganglia. Trends Neurosci. 18, 63–64. Alvord, E.C., Forno, L.S., 1992. Pathology. In: Koller, W.C. (Ed.). Handbook of Parkinson’s Disease. Marcel Dekker, New York, pp. 255–284. Andres, M.E., Gysling, K., Araneda, S., Venegas, A., Bustos, G., 1996. NMDA-NR1 receptor subunit mRNA expression in rat brain after

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