Brain, Vol. 125, No. 12, 2635-2645,
December 2002
© 2002 Oxford University Press
Striatal metabotropic glutamate receptor function following experimental parkinsonism and chronic levodopa treatment
1 Clinica Neurologica, Dipartimento di Neuroscienze, Università di Roma Tor Vergata, 2 I.R.C.C.S. Fondazione ‘Santa Lucia’, 3 Dipartimento di Fisiologia Umana e Farmacologia, Università di Roma La Sapienza, Roma and 4 I.N.M. Neuromed, Pozzilli, Isernia, Italy
Correspondence to: P. Calabresi, Clinica Neurologica, Dipartimento di Neuroscienze, Università di Roma ‘Tor Vergata’, Via Montpelier 1, Rome 00133, Italy E-mail: calabre{at}uniroma2.it
Received April 17, 2002. Revised June 19, 2002. Accepted June 22, 2002.
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Summary |
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Excessive activation of ionotropic glutamate receptors in the striatum contributes to the pathophysiology of motor symptoms in Parkinson’s disease. Metabotropic glutamate (mGlu) receptors regulate striatal excitatory synaptic transmission, and adaptive changes in their function might occur following dopaminergic denervation and chronic levodopa-treatment (L-DOPA). Corticostriatal glutamatergic transmission was examined in striatal slices obtained from rats unilaterally denervated with the dopaminergic neurotoxin, 6-hydroxy dopamine (6-OHDA), and from denervated rats chronically treated with L-DOPA plus benserazide (25 + 6.25 mg/kg, intraperitoneally, twice daily for 21 days). Selective agonists of mGlu2 and -3 receptor subtypes [compounds LY379268 and (2S,2'R,3'R)-2-(2',3'-[3H]-dicarboxycyclopropyl)glycine ([3H]DCG-IV)] exhibited a much greater potency in depressing excitatory transmission and corticostriatal synapses in slices prepared from 6-OHDA-lesioned animals. Dopaminergic denervation affected neither the ability of L-(+)-2-amino-4-phosphonobutyric acid (L-AP4; a selective agonist of mGlu4, -6, -7 and -8 receptors) to inhibit corticostriatal transmission, nor the ability of (S)-3,5-dihydroxyphenylglycine (3,5-DHPG; a selective agonist of mGlu1 and -5 receptors) to potentiate responses mediated by N-methyl-D-aspartate (NMDA) receptor activation in striatal neurones. The increased responsiveness to mGlu2/3 receptor agonists was no longer detected in slices from 6-OHDA-lesioned animals chronically treated with L-DOPA. 6-OHDA-induced denervation also led to an increased expression of striatal mGlu2/3 receptor proteins and to a >2-fold increase in the maximal density (Bmax) of [3H]DCG-IV binding sites. These increases were again reversed by chronic treatment with L-DOPA. No changes in the expression of mGlu4 receptors or the
i1 and i2 subunits of the Gi proteins were induced by any of the treatments. We suggest that an enhanced sensitivity of pre-synaptic inhibitory mGlu2/3 receptors might represent an adaptive change triggered by dopaminergic denervation, which can be reversed by L-DOPA treatment. Keywords: basal ganglia; dopamine; excitatory synaptic transmission; glutamate synaptic transmission
Abbreviations: DCG-IV = (2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine; 3,5-DHPG = (S)-3,5-dihydroxyphenylglycine; L-AP4 = L-(+)-2-amino-4-phosphonobutyric acid; L-DOPA = levodopa; EPSC = excitatory post-synaptic current; EPSP = excitatory post-synaptic potential; 6-OHDA = 6-hydroxydopamine; NMDA = N-methyl-D-aspartate; RMP = resting membrane potential
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Introduction |
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The main pathological feature of Parkinson’s disease is the degeneration of dopamine-containing nigrostriatal neurones. The irreversible loss of the dopamine-mediated control of striatal function leads to the motor symptoms observed in this disorder: bradykinesia, tremor and rigidity (Obeso et al., 2000
). The striatum also receives a massive glutamatergic innervation arising from most cortical areas (Graybiel, 1990; Smith and Bolam, 1990; Calabresi et al., 1996). The contribution of the descending glutamatergic corticostriatal pathway to the motor disorders associated with Parkinson’s disease and the dyskinesias observed following treatment with levodopa (L-DOPA) has been of increasing interest in recent years (Calabresi et al., 2000; Chase and Oh, 2000). An experimental lesion of the ascending nigrostriatal dopamine pathway by 6-hydroxydopamine (6-OHDA) mimics parkinsonian pathology (Papa et al., 1994) and results in an increased glutamatergic transmission in the striatum, as determined by electron microscopy, in vivo microdialysis or electrophysiology (Lindefors and Ungerstedt, 1990; Calabresi et al., 1993; Ingham et al., 1998; Meshul et al., 1999). Accordingly, blockade of ionotropic glutamate receptors ameliorates Parkinson’s disease symptoms and reverses the 6-OHDA-induced changes in striatal glutamate immunolabelling (Papa et al., 1995; Chase and Oh, 2000; Robinson et al., 2001).
Metabotropic glutamate (mGlu) receptors, which are coupled to G proteins, modulate corticostriatal transmission and control motor behaviour (reviewed by Pisani et al., 1998). To date, eight mGlu receptor subtypes (designated mGlu1 to mGlu8) have been cloned from mammalian brain. These mGlu receptors are classified into three main groups on the basis of sequence homology, pharmacological profile, and coupling to second messenger systems (Pin and Duvoisin, 1995; De Blasi et al., 2001). The recent availability of subtype-selective ligands (Schoepp et al., 1999) allows a detailed analysis of how individual mGlu receptor subtypes (or groups of subtypes) are involved in physiology and pathology. Interestingly, selective activation of different mGlu receptor subtypes differentially affects excitatory transmission at corticostriatal synapses (Lovinger and McCool, 1995; Pisani et al., 1997a, b, 2001). Thus, we decided to examine the action of these agonists on excitatory synaptic transmission in striatal slices obtained from 6-OHDA lesioned rats and from denervated animals receving a chronic L-DOPA treatment. To our knowledge, this study represents the first characterization of the adaptive changes of mGlu receptors following experimental parkinsonism and chronic L-DOPA treatment.
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Methods |
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Preparation and maintenance of brain slices
Corticostriatal slices were prepared from adult male Wistar rats (3–4 months old). The preparation and maintenance of coronal slices have been described previously (Calabresi et al., 1993
; Pisani et al., 1997a, b). Briefly, corticostriatal coronal slices (200–300 µm) were prepared from tissue blocks of the brain using a vibratome. A single slice was transferred to a recording chamber and submerged in a continuously flowing Krebs solution (35°C, 2–3 ml/min) gassed with 95% O2/5% CO2. The control solution comprised 126 mM NaCl, 2.5 mM KCl, 1.2 mM MgCl2, 1.2 mM NaH2PO4, 2.4 mM CaCl2, 11 mM glucose and 25 mM NaHCO3.
Intracellular recordings
Intracellular recording electrodes were filled with 2 M KCl (30–60 M
). Signals were recorded with the use of an Axoclamp 2A amplifier, displayed on a separate oscilloscope and stored on a digital system. The characteristics of action potentials and of current–voltage curves in different experimental conditions were studied using a fast chart recorder and a digital system (pClamp 8; Axon Instruments, Foster City, CA, USA). For synaptic stimulation, bipolar electrodes were used. These stimulating electrodes were located either in the cortical areas close to the recording electrode or in the white matter between the cortex and the striatum in order to activate corticostriatal fibres. To study paired-pulse facilitation, the intensity of stimulation was set to evoke corticostriatal excitatory post-synaptic potentials (EPSPs) of 5–8 mV amplitude, and the interstimulus interval was 60 ms. In addition, some experiments were performed in voltage-clamp in the whole-cell configuration. Electrodes (4–5 M) were filled with a solution containing 125 mM K+-gluconate, 10 mM NaCl, 1.0 mM CaCl2, 2.0 mM MgCl2, 0.5 mM 1,2-bis (2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA), 19 mM N-(2-hydroxyethyl)-piperazine-N-s-ethanesulfonic acid (HEPES), 0.3 mM guanosine triphosphate (GTP) and 1.0 mM Mg-adenosine triphosphate (Mg-ATP), adjusted to pH 7.3 with KOH. Striatal spiny neurones were clamped at –80 to –85 mV, close to their resting membrane potential (RMP), and were recorded using an Axopatch 1D amplifier and Clampex 8.1 software. To evoke excitatory post-synaptic currents (EPSCs), bipolar electrodes were placed on corticostriatal fibres. Stimuli were delivered at 0.1 Hz, with an interstimulus interval of 60 ms.
About 50% of the recordings were obtained in the presence of 50 mM picrotoxin in order to rule out a possible contamination of the EPSPs by depolarizing potentials mediated by 
-aminobutyric acid A-receptors. Since these experiments gave similar results to those obtained in the absence of this drug, all the data were pooled together.
Data analysis and drug applications
Quantitative data on modifications of EPSPs are expressed as a percentage of the controls, the latter representing the mean of responses recorded during a stable period (15–30 min) before the drug application. Values given in the text and figures are mean ± standard error of the mean (SEM) of changes in the respective cell populations. Wilcoxon’s test or Student’s t-test (for paired and unpaired observations) were used to compare the means, and ANOVA (analysis of variance) was used when multiple comparisons were made against a single control group. The concentration–response curves shown in the figures and the EC50 were obtained using Kaleidagraph 3.0 software running on a Power Macintosh. Drugs were applied by dissolving them in saline to the desired final concentration. LY379268 was kindly provided by Dr D.D. Schoepp and Dr A.E. Kingston (Eli Lilly, Indianapolis IN, USA). All other drugs were from Tocris.
Preparation of 6-OHDA denervated rats and chronic L-DOPA treatment
To obtain unilateral nigrostriatal lesions, rats (1–2 months old, anaesthetized with 45 mg/kg of pentobarbitone intraperitoneally) were injected with 6-OHDA (8 mg/4 ml of saline containing 0.1% ascorbic acid) via a Hamilton syringe through a cannula inserted just rostral to the substantia nigra using stereotaxic coordinates (Paxinos and Watson, 1986). Sham-operated (control) rats were injected with saline + 0.1% ascorbic acid alone. Twenty days later, the rats were tested with a 0.05 mg/kg subcutaneous injection of apomorphine, and contralateral turns were counted for 1 h. Only those rats that consistently made at least 400 contralateral turns were used for the electrophysiological recordings performed 2 months after the lesion. In some cases, 6-OHDA-lesioned rats were anaesthetized with diethyl ether and brain dissection confirmed that the nigrostriatal pathway was lesioned. This was established by the observation of a >95% loss of dopamine neurones in the substantia nigra pars compacta and the almost complete absence of dopamine terminals in the striatum. This was monitored by using a monoclonal antibody for tyrosine-hydroxylase. A group of 6-OHDA-lesioned rats received L-DOPA/benserazide (25/6.25 mg/kg) twice daily intraperitoneally for 3 weeks. Benserazide was given to prevent decarboxylation of L-DOPA in the periphery, as commonly used in clinical practice. As reported previously (Papa et al., 1994, 1995), the magnitude of the rotational response to L-DOPA more than doubled during the first week of treatment, but essentially remained constant thereafter.
Western blot analysis
Striata were removed and stored at –80°C for 20 days. On the day of the experiment, tissue was homogenized at 4°C in ice-cold sodium dodecyl sulfate (SDS) lysis buffer containing 1 mM phenylmethylsulfonyl fluoride, pH 7.4, with a motor-driven Teflon-glass homogenizer (1700 r.p.m.). Five microlitres were used for protein determinations. Thirty-five micrograms of proteins were resuspended in SDS–bromophenol blue reducing buffer containing 20 mM dithiothreitol. Western blot analysis was carried out using 8% SDS polyacrylamide gels; gels were run on a minigel apparatus (Mini Protean II Cell; Bio-Rad, Italy) and electroblotted on ImmunBlot PVDF Membrane (Bio-Rad) for 1 h using a semi-dry electroblotting system (Trans-blot system SD; Bio-Rad). Filters were then blocked overnight in TTBS buffer (100 mM Tris–HCl, 0.9% NaCl, 0.1% Tween 20, pH 7.4) containing 5% non-fat dry milk. Blots for mGlu receptors were incubated for 1 h at room temperature with primary polyclonal antibodies (1 µg/ml) against mGlu2/3 or mGlu4 receptors, or against the i1 and i2 subunits of the G proteins (Upstate Biotechnology, Lake Placid, USA). Blots were washed three times with TTBS buffer and then incubated for 1 h with secondary peroxidase-coupled anti-rabbit antibodies (diluted 1:5000 with TTBS). Immunostaining was revealed by enhanced chemiluminescence (Amersham, Milan, Italy).
Measurement of [3H]DCG-IV binding in striatal membranes
For binding studies, striata were stored at –80°C for 20 days and then (2S,2'R,3'R)-2-(2',3'-[3H]-dicarboxycyclopropyl)glycine ([3H]DCG-IV]) binding was measured in striatal homogenates, essentially as described by Mutel et al. (1998). In brief, tissue was homogenized in 25 volumes of 50 mM Tris–HCl (pH 7.1) and homogenates were centrifuged at 48 000 g for 10 min. The pellet was resuspended, incubated at 37°C for 10 min, and then centrifuged again at 48 000 g. The resulting pellet was stored frozen at –80°C. On the day of the experiment, membranes were thawed and washed three times in the assay buffer (50 mM Tris-HCl, pH 7.4, containing 2 mM MgCl2) by centrifugation at 48 000 g. The final pellet was resuspended in assay buffer. Aliquots of the final suspensions (100 µg of proteins) were transferred to test tubes containing 1 ml of 3–1000 nM [3H]DCG-IV (Amersham, specific activity 18.3 Ci/mmol). Samples were incubated for 1 h at room temperature. At the end of the incubation, membranes were filtered onto Whatmann GF/C glass fibre filters and washed three times with ice-cold binding buffer. Non-specific binding was defined as the binding left in the presence of 10 µM LY379268 (mGlu2/3 receptor agonist).
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Results |
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Intrinsic and synaptic properties of striatal neurones
Presumed striatal spiny neurones were recorded intracellularly in slices from control (sham-operated) rats (n = 31), 6-OHDA-lesioned rats (n = 33) and 6-OHDA-lesioned rats chronically treated with L-DOPA (25 + 6.25 mg/kg, twice daily for 21 days) (n = 32). Striatal spiny neurones recorded from the three experimental groups had similar intrinsic membrane properties: high RMP (sham: –85 ± 3 mV; 6-OHDA-lesioned: –84 ± 3 mV; 6-OHDA-lesioned plus L-DOPA: –84 ± 4 mV; P >0.05) and action potential discharge with little adaptation during depolarizing current pulses (data not shown). These electrophysiological properties were similar to those reported previously by our group and by others for medium spiny neurones of the striatum (Kita et al., 1984
; Jiang and North, 1991; Calabresi et al., 1993).
To evoke corticostriatal EPSPs, the stimulating electrode was placed close to the recording electrode in the white matter between the cortex and the striatum. At the RMP, these EPSPs were not affected by the N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801 [(5S,10R)-(+)-5-methyl-10,11-dehydro-5H-dibenzo[a,d]cyclohepta-5,10-imine maleate] (30 mM; n = 6 for each group), while they were fully abolished by the -amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) receptor antagonist, CNQX (6-cyano-7-nitroquinoxaline 2,3-dione) (10 mM; n = 6 for each group).
Effects of group II mGlu receptor activation on corticostriatal transmission in brain slices from control and treated rats
We examined the possible differential role of group II mGlu (mGlu2 and mGlu3) receptors in the modulation of synaptic transmission at corticostriatal synapses in control rats, 6-OHDA-lesioned rats, and 6-OHDA-lesioned rats treated with L-DOPA. In most of the experiments we used LY379268, a potent and selective mGlu 2/3 receptor agonist (Monn et al., 1999). As shown in Fig. 1A and C, this agonist produced a dose-dependent inhibition of EPSPs evoked by corticostriatal stimulation in all three experimental groups. However, the potency of this compound in inhibiting the EPSPs was much higher in 6-OHDA-lesioned slices (n = 13; EC50 0.042 µM) than in slices obtained from sham-operated rats (n = 12; EC50 0.37 µM; P < 0.001). Conversely, slices obtained from 6-OHDA-lesioned rats with chronic L-DOPA treatment showed a pharmacological sensitivity to LY379268 similar to that observed in sham-operated animals (n = 13; EC50 0.32 µM; P > 0.05). Similar results were obtained with DCG-IV, a drug that selectively activates mGlu2/3 receptors at concentrations lower than 2–5 mM (Schoepp et al., 1999) (Fig. 1B). In order to investigate whether the depression of EPSPs by group II mGlu receptor agonists was dependent on pre- or post-synaptic sites of action, we measured synaptic responses to a pair of stimuli before and during the applications of LY379268. In these experiments, the interstimulus interval was 60 ms. Paired-pulse modification of neurotransmission has been studied extensively and is attributed to a pre-synaptic change in release probability (Manabe et al., 1993; Schulz et al., 1994). An increase in the ratio of the second pulse response to the first pulse response (EPSP2/EPSP1) indicates a decrease in the release probability. The decrease in transmitter release is consistent with the observations that manipulations depressing transmitter release usually increase the magnitude of this ratio also at corticostriatal synapses (Calabresi et al., 1997). Interestingly, 0.1–1 µM LY379268 and 1 mM DCG-IV (n = 5 for each drug and experimental group in current–clamp experiments; n = 4 for each drug and experimental group in voltage–clamp experiments; P < 0.01 for all) induced a decrease in the amplitude of EPSCs and EPSPs that was coupled to a clear increase in the EPSP2:EPSP1 ratio (or EPSC2:EPSC1 ratio) in all the experimental groups (Fig. 1D and E). LY379268 and DCG-IV, at the concentrations tested in this study, did not affect RMP and input resistance of the recorded neurones (n = 12 for each experimental condition and each drug; P > 0.05; data not shown).
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Effects of group III mGlu receptor activation on corticostriatal transmission
We examined the action of l-(+)-2-amino-4-phosphorobutyric acid (L-AP4), a selective agonist acting at group III mGlu receptors (Pisani et al., 1997
a), on corticostriatal EPSPs in slices obtained from the three groups of animals. As shown in Fig. 2A and B, L-AP4 induced a dose-related inhibitory effect on the EPSP amplitude. The EC50 calculated for this effect was similar in the three groups of experiments (P > 0.05; n = 12 for each experimental condition): 11.8 µM in slices from sham-operated rats, 12 µM in slices from 6-OHDA-lesioned rats, and 11.4 µM in slices from 6-OHDA-lesioned rats treated with L-DOPA. Also, for L-AP4 (30 µM; n = 4 for each experimental condition), the reduction of the EPSP amplitude was associated with a significant (P < 0.01) increase in the paired-pulse facilitation, suggesting a pre-synaptic mechanism of action in all three experimental groups (Fig. 2C). L-AP4, at the concentration tested in this study, did not affect RMP and input resistance (n = 10 for each experimental condition; P > 0.05; data not shown).
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Effects of group I mGlu receptor activation on NMDA responses in striatal neurones
We also investigated the effect of 3,5-DHPG, a mGlu receptor agonist selectively acting at group I (mGlu1 and mGluR5) (Pisani et al., 1997
b, 2001) on the EPSP amplitude at the various concentrations used. As reported in Fig. 3A, this agonist failed to affect EPSP amplitude both in sham-operated rats and in 6-OHDA-lesioned animals (n = 5 for each experimental condition; P > 0.05). Accordingly, no effect on EPSP amplitude was detected in 6-OHDA-lesioned rats treated with L-DOPA either (n = 6; P > 0.05; data not shown). Since activation of group I mGlu receptors by 3,5-DHPG has been reported to enhance NMDA-mediated membrane depolarization in striatal spiny neurones (Pisani et al., 1997b, 2001), we also tested whether this facilitatory effect could express some pharmacological changes in 6-OHDA-lesioned rats compared with sham-operated animals. As shown in Fig. 3B, the EC50 measured for this effect was similar in the two groups: 44.8 µM in sham-operated rats (n = 10) and 40.2 µM in 6-OHDA-lesioned animals (n = 11; P > 0.05). Also, in 6-OHDA-lesioned rats treated with L-DOPA, the EC50 for the facilitatory action was similar (42.3 µM; n = 7; P > 0.05; data not shown). 3,5-DHPG, at the concentrations tested in this study, did not affect RMP and input resistance (n = 11 for each experimental condition; P > 0.05; data not shown).
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Expression of mGlu2/3 receptor protein, mGlu4 receptor protein, and G
i1 and Gi2 proteins in control and treated ratsImmunoblots with mGlu2/3 antibodies revealed two faint 100 kDa bands, corresponding to receptor monomers, and a higher molecular weight band, corresponding to receptor dimers. The higher band was more heavily labelled in the striatum of all groups of animals. Expression of mGlu2/3 receptors was up-regulated in the denervated striatum (left) of rats unilaterally infused with 6-OHDA. No changes were seen in the contralateral striatum as compared with the left or right striata of control rats. L-DOPA treatment had no effect on striatal mGlu2/3 receptor expression in control rats, but it brought the expression to control levels in the denervated striatum of 6-OHDA-treated rats (Fig. 4). No changes were detected in the expression levels of mGlu4 receptors, or the
i1 and i2 subunits of the G proteins in any group of animals (Fig. 4).
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[3H]DCG-IV binding in striatal membranes from control and treated rats
Saturation analysis of [3H]DCG-IV binding in striatal membranes of sham rats revealed a Bmax value of 1043 + 182 fmol/mg protein, and an apparent KD value of 212 + 58 nM, in agreement with values reported in rat cortical homogenates (Mutel et al., 1998
) (Fig. 5). The Bmax of [3H]DCG-IV binding was increased by >2-fold in the denervated striatum of 6-OHDA-treated rats, whereas no substantial changes were observed in the contralateral striatum. L-DOPA treatment in intact rats did not change the maximal density of [3H]DCG-IV binding sites, although it slightly increased the binding affinity. L-DOPA treatment in 6-OHDA-injected animals prevented the increase in the Bmax of [3H]DCG-IV binding in the denervated striatum (Table 1).
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Discussion |
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The corticostriatal projection represents the major excitatory glutamatergic input to the striatum (Smith and Bolam, 1990
). Glutamatergic fibres also arise from the thalamus (Lapper and Bolam, 1992). Interestingly, however, it has been reported that while thalamostriatal fibres preferentially target cholinergic interneurones, corticostriatal inputs mainly impinge on spiny neurones, which represent the neuronal subtype recorded for this study.
An enhanced glutamatergic transmission in the basal ganglia and, in particular, in the striatum is thought to be involved in the expression of motor deficits in Parkinson’s disease (Chase and Oh, 2000; Greenamyre, 2001). Thus, blocking glutamate receptors has been a major challenge in the treatment of this pathology (Papa et al., 1995; Rouse et al., 2000; Robinson et al., 2001). Recent studies have focused on mGlu receptors as targets for drugs of potential use in Parkinson’s disease (Rouse et al., 2000; Bruno et al., 2001). These receptors modulate excitatory synaptic transmission by at least two mechanisms: (i) by regulating the activity of voltage-sensitive ion channels or ionotropic glutamate receptors (particularly NMDA receptors) at post-synaptic sites; and (ii) by facilitating or inhibiting the release of glutamate from afferent fibres (reviewed by Pin and Duvoisin, 1995). In addition, mGlu receptors regulate processes of neurodegeneration and neuroprotection, and they have been implicated in the pathophysiology of neuronal degeneration in Parkinson’s disease (Bruno et al., 2001). Hence, appropriate drugs interacting with mGlu receptors might relieve motor symptoms and, at the same time, delay the ongoing neuronal degeneration of Parkinson’s disease.
mGlu receptors are coupled to G proteins and form a family of at least eight subtypes, subdivided into three groups (Pin and Duvoisin, 1995; De Blasi et al., 2001). Group I includes mGlu1 and mGlu5 receptors, which are coupled to polyphosphoinositide hydrolysis. Group II includes mGlu2 and mGlu3 receptors, which are negatively coupled to adenylyl-cyclase via a Gi protein. Group III mGlu receptors (mGlu4, -6, -7 and -8) are also coupled to a Gi protein in heterologous expression systems, although native mGlu6 receptors are coupled to a cGMP phosphodiesterase in the ‘ON’ bipolar cells of the retina (reviewed by De Blasi et al., 2001). Both group II and group III mGlu receptors are pre-synaptically localized and their activation inhibits glutamate release (reviewed by Pin and Duvoisin, 1995). Accordingly, we found that selective agonists of group II (LY379268, DCG-IV) and group III (L-AP4) mGlu receptors decrease excitatory synaptic transmission at the corticostriatal pathway via a pre-synaptic mechanism. In contrast, selective activation of group I mGlu receptors with 3,5-DHPG did not affect corticostriatal EPSPs, while it significantly enhanced post-synaptic responses to NMDA. We found that 6-OHDA-induced denervation of the nigrostriatal dopaminergic pathway selectively potentiated responses mediated by group II mGlu receptors without affecting responses mediated by group I or group III mGlu receptors.
Tang and colleagues found that paired-pulse facilitation is affected in denervated striata as a result of altered synaptic plasticity at glutamatergic synapses (Tang et al., 2001). Conversely, we did not find substantial modifications of paired-pulse facilitation following 6-OHDA treatment. Several reasons might account for this discrepancy, in particular the different age of the animals. Tang and colleagues, in fact, used young rats (up to 1 month old) to study paired-pulse facilitation under control conditions and following intrastriatal 6-OHDA injection, while our experiments were performed in adult rats (3–4 months), recorded 2 months after intranigral injection of 6-OHDA. It is therefore conceivable that the absence of paired-pulse facilitation described by Tang and colleagues in young 6-OHDA treated rats recovers with time after the lesion (Tang et al., 2001). In addition, it should be noted that the absent increase in paired-pulse facilitation described in young animals treated with 6-OHDA reflects the absence of corticostriatal long-term depression observed in these animals. Consistently, this form of synaptic plasticity is pre-synaptic in young animals (Choi and Lovinger, 1997), and therefore accounts for the increase in paired-pulse facilitation observed during development, but is post-synaptic in adults animals (Calabresi et al., 1999), and therefore it is not expected to affect paired-pulse facilitation.
Among other receptors present on corticostriatal terminals and coupled to Gi proteins, only the activity of D2 receptors is up-regulated in response to 6-OHDA denervation, whereas the activity of -aminobutyric acid B-receptors or m2/4 muscarinic receptors remains unchanged (Calabresi et al., 1993). This suggests that dopaminergic denervation induces changes in the expression and/or regulatory properties of mGlu2/3 receptors (see Alagarsamy et al., 2001; De Blasi et al., 2001) rather than changes in molecules that lie downstream in the signal propagation and are therefore common to all receptors coupled to Gi proteins. In our study, 6-OHDA lesions led to an increased expression of mGlu2/3 receptors without detectable changes in mGlu4 receptors. Although an increase in mGlu2/3 receptor density is in line with the enhanced responsiveness to LY379268 and DCG-IV, it is hard to explain why we detected an increase in the potency, but did not see the expected increase in efficacy, of mGlu2/3 receptor agonists. Possible explanations include a greater receptor reserve in denervated animals (as suggested by the lack of changes in the alpha subunits of Gi proteins) or the contribution of other pools of mGlu2/3 receptors (such as post-synaptic or glial receptors) to the overall increase in receptor number. The examination of mGlu2/3 receptor expression at the cellular or subcellular level may help to solve this caveat.
Remarkably, all changes induced by dopaminergic denervation were reversed by a chronic treatment with L-DOPA, which is standard drug in the treatment of Parkinson’s disease. Although L-DOPA does not protect or rescue nigrostriatal dopaminergic fibres (see Camp et al., 2000; Ishida et al., 2000 and references therein), it partially restores striatal dopaminergic transmission and exerts a trophic effect, repairing the ultrastructural changes in the corticostriatal pathway caused by dopaminergic denervation (Ingham et al., 1998). The normalization of the responsiveness of pre-synaptic mGlu2/3 receptors represents a novel example of the adaptive changes induced by L-DOPA in experimental animal models of parkinsonism (Chase and Oh, 2000).
The present findings have several implications. It has been hypothesized that selective agonists of group II mGlu receptors are beneficial in Parkinson’s disease because they reduce the hyperactivity of corticostriatal fibres and subthalamic excitatory fibres that develops in response to dopaminergic denervation (Rouse et al., 2000; Messenger and Duty, 2001). Accordingly, intracerebroventricular injection of mGlu2/3 receptor agonists reduces akinesia in reserpine-treated rats (Dawson et al., 2000; Messenger and Duty, 2001) The increased potency of LY379268 and DCG-IV found in 6-OHDA-lesioned rats may reflect the existence of a compensatory mechanism aimed at reducing the excessive activity of the corticostriatal pathway, and allows the prediction that, in parkinsonian patients, group II mGlu receptor agonists are effective at low doses. This may considerably improve the risk-to-benefit ratio associated with the use of these drugs in Parkinson’s disease.
The restorative effects of L-DOPA suggest that the activity of corticostriatal mGlu2/3 receptors is under the control of nigrostriatal dopaminergic transmission and that, for this reason, mGlu2/3 receptor agonists might have a lesser chance of success when given in combination with L-DOPA. One could predict an optimal response to mGlu2/3 receptor agonists either before the beginning of L-DOPA treatment or, alternatively, when L-DOPA loses its efficacy and patients spend most of their time in the ‘off’ phase. If this prediction is correct, an appropriate use of mGlu2/3 receptor agonists might help to resolve the motor fluctuations that are typical of the long-term L-DOPA syndrome. The potential use of mGlu2/3 receptor agonists in the treatment of Parkinson’s disease is further supported by the recent evidence that LY379268 protects nigral dopaminergic neurones against 6-OHDA toxicity (O’Neill et al., 2001).
It is worth noting that an alternative strategy in the treatment of Parkinson’s disease might be also represented by the use of group I selective antagonists. Accordingly, it has been reported that a mGlu5 receptor antagonist reverses the akinesia in 6-OHDA-lesioned rats (Amalric et al., 2001; Spooren et al., 2001). In line with this finding, we have demonstrated that activation of group I mGlu receptors (and in particular mGlu5 receptors) facilitates striatal NMDA responses in normal (Pisani et al., 1997b, 2001) as well as denervated animals (present study).
Future pharmacological studies are required to investigate the possibility that new drugs, combining both agonistic effects on group II mGlu receptors and antagonistic properties on group I mGlu receptors, may represent an ideal approach in the treatment of Parkinson’s disease.
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Acknowledgements |
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We wish to thank Dr A. E. Kingston (Eli Lilly, Indianapolis IN, USA) for providing LY379268. This work was supported by a Telethon (E. 729) grant and a CNR (Invecchiamento) and a CNR-MIUR (Neurobiotecnologie) grant to P.C. The study was also supported by a MIUR/CNR (Ministero dell’lstruzione, Università Ricerca/Consiglio Superiore delle Ricerche) (legge 95/95) grant to G.B., a MURST/CNR Neuroscience grant to P.C. and a grant from the National Research Council Biotechnology Project to P.C.
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