Effect of 6-OHDA injection on the AMPA glutamate receptor subunits in the substantia nigra of Sprague–Dawley rats

Neuroscience Letters 241 (1998) 1–4

Effect of 6-OHDA injection on the AMPA glutamate receptor subunits in the substantia nigra of Sprague–Dawley rats Y. He a, T. Lee a ,*, S.K. Leong b a

Department of Surgery, National University of Singapore, Lower Kent Ridge Road, Singapore 119260, Singapore Department of Anatomy, National University of Singapore, Lower Kent Ridge Road, Singapore 119260, Singapore

b

Received 20 August 1997; received in revised form 19 November 1997; accepted 25 November 1997

Abstract Parkinson’s disease (PD) is characterized by the destruction of dopaminergic cells in the substantia nigra (SN). The cause of the cell death and the development of Parkinsonism is however unknown. There are increasing evidences to suggest the involvement of glutamate mediated by its receptors. Using immunohistochemistry and cell counting, the present study investigated whether the numbers of neurons immunostained with glutamate a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor subunits GluR1, GluR2/3 and GluR4 in the SN of rats would change after the injection of 6-hydroxydopamine (6-OHDA) into the SN. The results showed that the numbers of GluR1 positive cells were significantly decreased in the substantia nigra pars compacta (SNc), pars reticulata (SNr) and pars lateralis (SNl) from 3 days (13.7%) to 3 months (40.3%) and of GluR2/3 cells, from 1 week (17.6%) to 3 months (19.1%) after 6-OHDA injection, compared to those in the contralateral non-injected side. There was, however, no significant difference in the number of GluR4 positive cells between the injected and non-injected SN. The results were discussed.  1998 Published by Elsevier Science Ireland Ltd.

Keywords: Parkinson’s disease; Excitotoxicity; Glutamate receptor; a-Amino-3-hydroxy-5-methyl-4-isoxazole propionic acid; Subunits; 6-Hydroxydopamine

The progressive loss of dopaminergic nigrostriatal neurons is the most prominent feature of Parkinson’s disease (PD) which presents with the clinical triads of dyskinesia, rigidity and resting tremor. Recent experimental studies have implicated the excitatory amino acid, glutamate, in the pathogenesis of Parkinson’s disease and the development of Parkinsonian symptoms [8–10]. Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system (CNS). Its action is mediated by its receptors which can be broadly classified into the ionotropic and the metabotropic types. Ionotropic receptors have been further divided into three main subtypes: N-methyl-D-aspartate (NMDA), kainate (KA), and a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors [14]. Furthermore, molecular cloning has recently revealed a family of genes encoding highly related subunits for the ionotropic AMPA (GluR1, GluR2/ * Corresponding author. Tel.: +65 7724241; fax: +65 7778427.

3, GluR4), KA (GluR5, GluR6 and possibly KA-1), and NMDA (NMDAR1 and NMDAR2) receptors [6,7]. Klockgether et al. [8] demonstrated that AMPA antagonist 6-nitro-7-sulfamoyl-benzo [f] quinoxaline-2,3-dione (NBQX) produced an apparent improvement in the symptoms of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induced Parkinsonian monkey, and microinjection of NBQX into the substantia nigra reduced the muscular rigidity of monoamine-depleted rats. In addition, NBQX potentiated the antiparkinsonian effects of L-dopa in both Parkinsonian rats and monkeys. These results were later confirmed by Lo¨schmann et al. [10], and further reinforced by the finding that the injection of AMPA into the substantia nigra (SN) would activate the AMPA receptors in the SN, causing Parkinsonian rigidity [9]. In the present study, we investigated whether there was any change in the number of SN neurons expressing AMPA receptor subunits GluR1, GluR2/3 and GluR4 in Parkinsonian rats produced by 6-hydroxydopamine (6-OHDA) injec-

0304-3940/98/$19.00  1998 Published by Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(97) 00979- 8

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Y. He et al. / Neuroscience Letters 241 (1998) 1–4

tion into the SN of rats in order to understand the precise relationship between glutamate and Parkinson’s disease. Thirty five male Sprague–Dawley rats (190–210 g) were anaesthetized by intraperitoneal injection of 7% chloral hydrate at a dosage of 0.4 mg/g. Four microlitres of 0.2% 6-hydroxydopamine (6-OHDA; Sigma) dissolved in 0.02% ascorbic acid solution (pH 4–5) was then stereotaxically injected into the right SN at AP: −4mm, ML: −1mm, and DV: −7.5mm from the bregma, according to the atlas of Paxinos and Watson [15]. Five control rats received 4 ml of 0.02% ascorbic acid instead of 6-OHDA. The 6-OHDA lesioned rats were sacrificed by decapitation at 1 day, 3 days, 1 week, 2 weeks, 1 month, 2 months and 3 months later, with five animals used for each survival period. Control rats were sacrificed in the same manner 2 weeks after vehicle injection as preliminary studies showed that the changes to be described later were definite at that time. After decapitation, the brains were quickly removed and frozen on dry ice and subsequently kept in a freezer at −70°C for immunohistochemical staining. Coronal sections of the midbrain were cut at 20 mm thickness in a cryostat and picked up randomly on slides. The sections were then fixed immediately with 4% paraformaldehyde for 20 min, washed three times for 5 min in phosphate-buffered saline containing 0.1% Triton X-100 (PBSTX). They were then incubated with a monoclonal mouse anti-rat TH antibodies (Boehringer Mannheim Biochimica) at a dilution of 1:100, or with polyclonal rabbit anti-rat GluR1, GluR2/3, GluR4 antibodies (UBI, USA) respectively at a dilution of 1:50 in PBS-TX, and processed for the immunohistochemical demonstration of the respective

Fig. 1. (A–F) Photomicrographs showing TH (A,B) and GluR1 (C–F) immunostaining in the 6-OHDA lesioned (A,C,E) and the contralateral (B,D,F) SN. (A) Shows a pronounced loss of TH positive cells in the lesioned SN 2 weeks after injection, compared to the contralateral (B) side. Correspondingly, GluR1 positive cells are lost in the lesioned SN (C,E), compared to the contralateral (D,F) side. (E,F) Higher magnification of the squares in (C) and (D), respectively. The immunopositive cells are fusiform or trigonal in shape, bearing long processes. Scale bar, 100 mm.

Fig. 2. (A–D) GluR2/3 positive cells are much reduced in the SN (A,C) at 2 weeks after 6-OHDA injection, compared to the contralateral SN (B,D). (C,D) Enlargement of the boxed areas in (A) and (B), respectively showing the fusiform GluR2/3 cells in the SN. Scale bar, 100 mm.

immunoreactive products according to the procedure of Martin [11]. After visualisation with the peroxidase anti-peroxidase method, eight sections of the SN in each rat were examined and the numbers of immunopositive cells were counted. Eight sections were taken in order to meet the requirements for evaluating statistical significance [4]. These sections were taken from different levels of the SN at random, and contained both the right and left SN. Five rats were included in each group of Parkinsonian and control rats. TH, GluR1, GluR2/3 or GluR4 immunopositive cells with clearly visible cell bodies and processes in both the injected and contralateral substantia nigra pars compacta (SNc), pars lateralis (SNl) and par reticulata (SNr) were counted at 40 × magnification as previously described [4]. The data were analyzed by paired t-tests. TH positive cells in the non-lesioned rat were concentrated in the SNc, and less densely in SNl, and SNr. There was no obvious difference in TH staining between the vehicle injected and the contralateral non-injected SN. However, there was a significant loss of TH positive cells in the SNc, SNr and SNl of 6-OHDA lesioned SN, and the cell loss became significant from 1 week to 3 months after lesioning, compared to the contralateral SN (Table 1 and Fig. 1A,B). In the non-lesioned SN the majority of GluR1 positive cells were located in the SNc. Some of them were scattered in the SNr and SNl. These cells were fusiform or trigonal with long processes. There was a significant reduction in the number of GluR1 stained cells in the lesioned SNc, SNr and SNl at 3 days to 3 months after 6-OHDA lesioning, compared to the contralateral SN (Table 1 and Fig. 1C–F). There was, however, no significant difference in the localisation and the number of GluR1 positive cells between the vehicle injected and non-injected SN. GluR2/3 positive cells were located mainly in the SNc and SNl of the non-lesioned SN, with a few of them scattered in the SNr. Most of these cells were fusiform. After 6OHDA injection, there was a significant reduction in the number of labelled GluR2/3 cells in the ipsilateral SNc, SNl and SNr at 1 week to 3 months after the lesion (Table

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Y. He et al. / Neuroscience Letters 241 (1998) 1–4 Table 1 Numbers of TH, GluR1, GluR2/3 and GluR4 positive cells in the SN of 6-OHDA induced Parkinsonian rats Survival periods

1 day 3 days 1 week 2 weeks 1 month 2 months 3 months

TH stained cells

GluR1 stained cells

GluR2/3 stained cells

GluR4 stained cells

SN (lesioned)

SN SN (contralateral) (lesioned)

SN SN (contralateral) (lesioned)

SN SN (contralateral) (lesioned)

SN (contralateral)

110.17 ±17.83 110.67 ±14.01 60.42 ±14.37 32.49 ±12.70 24.92 ±15.75 15.67 ±6.01 16.58 ±12.66

112.74 ±20.45 116.49 ±15.36 107.52 ±10.27* 121.83 ±19.66* 101.37 ±18.31* 99.18 ±22.66* 111.04 ±19.31*

112.72 ±12.15 92.89 ±8.11* 98.17 ±11.49* 101.72 ±7.47* 78.53 ±17.39* 70.43 ±11.75* 89.79 ±16.77*

98.17 ±18.17 110.51 ±19.74 103.97 ±14.47* 98.20 ±8.11* 101.60 ±16.19* 111.89 ±19.43* 99.67 ±10.63*

117.37 ±25.37 115.91 ±11.48 151.09 ±30.69 141.66 ±20.00 114.12 ±23.55 119.85 ±12.09 102.36 ±12.36

111.47 ±10.92 80.16 ±4.31 77.22 ±11.03 60.47 ±10.17 48.07 ±16.22 53.17 ±7.15 53.61 ±4.85

96.69 ±14.12 106.25 ±16.02 85.76 ±15.11 84.94 ±7.63 88.39 ±12.21 92.17 ±17.32 80.63 ±10.56

115.56 ±20.51 119.26 ±17.04 144.53 ±33.91 136.14 ±19.21 110.79 ±19.60 120.47 ±14.19 99.50 ±16.47

Number of rats

5 5 5 5 5 5 5

Data are the mean±SD. *P , 0.05, paired t-test.

1 and Fig. 2A–D). The expression of GluR2/3 was not changed in the vehicle injected SN, compared to the noninjected side. In the non-lesioned SN, GluR4 positive cells were located predominantly in the SNl, with some of them scattered in SNr. These cells were large, spherical or fusiform with long processes. The number of GluR4 positive cells in the 6OHDA lesioned SNl and SNr was not significantly different from that in the contralateral SN from 1 day to 3 months after the lesion (Table 1 and Fig. 3). Previous studies had demonstrated a reduction in the AMPA binding sites in the SN of Parkinsonian human brains [5] or of MPTP-treated mice [17]. The results of the present investigation also showed a decrease in the expression of AMPA receptors in the rat SN after the 6OHDA injection, involving GluR1 and GluR2/3, but not GluR4. This suggests that glutamate receptor subunits may play different roles in the pathogenesis of Parkinson’s disease and the development of Parkinsonian symptoms.

Fig. 3. (A–D) There is no significant difference between the numbers of GluR4 positive cells in the 6-OHDA lesioned SN (A,C), compared to the contralateral (B,D) side, at 2 weeks after injection. (C,D) The immunostained cells are small, spherical, or fusiform in shape, with long processes visible in the enlarged rectangles in (A) and (B), respectively. Scale bar, 100 mm.

Increased activity in the subthalamic nucleus (STN) has been thought to play a critical role in producing the Parkinsonian symptoms [12,13]. The STN receives glutamatergic input from the cortex [16]; its output to the globus pallidus and SN is also glutamatergic [1]. Furthermore, the excitatory synaptic transmission in the STN and the target areas of the STN appeared to be mediated by AMPA receptors [2,16]. Bergman showed that lesion of the STN in MPTPtreated monkeys reversed akinesia, rigidity and tremor [3]. Later Klockgether [9] demonstrated that activation of AMPA receptors in the rat SN led to Parkinsonian rigidity. According to the results of the present study, GluR1 and GluR2/3 positive cells in the SN were reduced in number. How such can contribute to Parkinsonism is not clear. It would be interesting to further study the functions of different glutamate receptor subunits in the development of Parkinsonism and the antiparkinsonic actions of the specific types of glutamate receptor antagonists. Since some GluR1 positive cells were found to be colocalised with TH [11], and there was a reduction of TH positive cells after 6-OHDA treatment, it would not be unreasonable to conclude that the decreased expression of GluR1 in the lesioned SN could be attributed partly to the degeneration of TH positive dopaminergic neurons. It was, however, noted that GluR1 positive cells were significantly reduced at 3 days after 6-OHDA lesioning, while TH positive cells became markedly decreased only at 1 week after lesioning. This implies that GluR1 positive dopaminergic cells may be more vulnerable to the neurotoxin. [1] Albin, R.L., Young, A.B. and Penney, J.B., The functional anatomy of basal ganglia disorders, Trends Neurosci., 12 (1989) 366–375. [2] Albin, R.L., Makowiec, R.L., Hollingsworth, Z., Dure, L.S., Penney, J.B. and Young, A.B., Excitatory amino acid binding

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Y. He et al. / Neuroscience Letters 241 (1998) 1–4

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10]

sites in the basal ganglia of the rat: a quantitative autoradiographic study, Neuroscience, 46 (1992) 35–48. Bergman, H., Wichmann, T. and DeLong, M.R., Reversal of experimental parkinsonism by lesions of the subthalamic nucleus, Science, 249 (1990) 1436–1438. Burke, R.E., Macaya, A., DeVivo, D., Kenyon, N. and Janec, E.M., Neonatal hypoxic-ischemic or excitotoxic striatal injury results in a decreased adult number of substantia nigra neurons, Neuroscience, 50 (1992) 559–569. Difazio, M.C., Hollingsworth, Z.R., Penney, J.B. and Young, A.B., Glutamate receptors in the substantia nigra of Parkinson’s disease brains, Neurology, 42 (1992) 402–406. Gasic, G.P. and Heinemann, S., Receptors coupled to ionic channels: the glutamate receptor family, Curr. Opin. Neurobiol., 1 (1991) 20–26. Keina¨nen, K., Wisden, W., Sommer, B., Werner, P., Herb, A., Berdoorn, T.A., Sakmann, B. and Seeburg, P.H., A family of AMPA-selective glutamate receptors, Science, 249 (1990) 556–560. Klockgether, T., Turski, L., Hononre´, T., Zhang, Z., Gash, D.M., Kurlan, R. and Greenamyre, T., The AMPA receptor antagonist NBQX has antiparkinsonian effects in monoamine- depleted rats and MPTP-treated monkeys, Ann. Neurol., 30 (1991) 717–723. Klockgether, T. and Turski, L., Toward an understanding of the role of glutamate in experimental parkinsonism: agonist-sensitive site in the basal ganglia, Ann. Neurol., 34 (1993) 585– 593. Lo¨chmann, P.A., Lange, K.W., Kumow, M., Rettig, K.J., Ja¨hnig,

[11]

[12]

[13]

[14]

[15] [16]

[17]

P., Honore´, T., Turski, T., Wachtel, H., Jenner, P. and Marsden, C.D., Synergism of the AMPA-antagonist NBQX and the NMDA-antagonist CPP with L-Dopa in models of Parkinson’s disease, J. Neural Transm. (PD-Sect), 3 (1991) 203–213. Martin, L.J., Blackstone, C.D., Levey, A.I., Huganir, R.L. and Price, D.L., AMPA glutamate receptor subunits are differentially distributed in rat brain, Neuroscience, 53 (1993) 327–358. Miller, W.C. and Delong, M.R., Altered tonic activity of neurons in the globus pallidus and subthalamic nucleus in the primate MPTP model of parkinsonism. In M.B. Carpenter and A. Jayaraman (Eds.), The Basal Ganglia II, Plenum, New York, 1987, pp. 414–427. Mitchell, I.J., Clarke, C.E. and Boyce, S., Neural mechanisms underlying Parkinsonian symptoms based upon regional uptake of 2-deoxyglucose in monkeys exposed to MPTP, Neuroscience, 32 (1989) 213–226. Monaghan, D.T., Bridges, R.J. and Cotman, C.W., The excitatory amino acid receptors: their classes, pharmacology, and distinct properties in the function of the central nervous system, Annu. Rev. Pharmacol. Toxicol., 29 (1989) 365–402. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1986. Rouzaire-Dubois, B. and Scarnati, E., Pharmacological study of the cortical-induced excitation of subthalamic nucleus neurons in the rat: evidence for amino acids as putative neurotransmitters, Neuroscience, 21 (1987) 429–440. Wu¨llner, U., Brouillet, E., Isacson, O., Young, A.B. and Penney, J.B., Glutamate receptor binding sites in MPTP-treated mice, Exp. Neurol., 121 (1993) 284–287.