These authors have contributed equally to the work.
Metabotropic glutamate receptor subtype 4 selectively modulates both glutamate and GABA transmission in the striatum: implications for Parkinson’s disease treatment
Article first published online: 14 MAR 2009
Journal Compilation © 2009 International Society for Neurochemistry. No claim to original US government works
Journal of Neurochemistry
Volume 109, Issue 4, pages 1096–1105, May 2009
How to Cite
Cuomo, D., Martella, G., Barabino, E., Platania, P., Vita, D., Madeo, G., Selvam, C., Goudet, C., Oueslati, N., Pin, J.-P., Acher, F., Pisani, A., Beurrier, C., Melon, C., Kerkerian-Le Goff, L. and Gubellini, P. (2009), Metabotropic glutamate receptor subtype 4 selectively modulates both glutamate and GABA transmission in the striatum: implications for Parkinson’s disease treatment. Journal of Neurochemistry, 109: 1096–1105. doi: 10.1111/j.1471-4159.2009.06036.x
- Issue published online: 14 APR 2009
- Article first published online: 14 MAR 2009
- Received November 7, 2008; revised manuscript received March 3, 2009; accepted March 4, 2009.
- γ-aminobutyric acid;
- basal ganglia;
- metabotropic glutamate receptor
- Top of page
- Materials and methods
- Supporting Information
Alterations of striatal synaptic transmission have been associated with several motor disorders involving the basal ganglia, such as Parkinson’s disease. For this reason, we investigated the role of group-III metabotropic glutamate (mGlu) receptors in regulating synaptic transmission in the striatum by electrophysiological recordings and by using our novel orthosteric agonist (3S)-3-[(3-amino-3-carboxypropyl(hydroxy)phosphinyl)-hydroxymethyl]-5-nitrothiophene (LSP1-3081) and l-2-amino-4-phosphonobutanoate (L-AP4). Here, we show that both drugs dose-dependently reduced glutamate- and GABA-mediated post-synaptic potentials, and increased the paired-pulse ratio. Moreover, they decreased the frequency, but not the amplitude, of glutamate and GABA spontaneous and miniature post-synaptic currents. Their inhibitory effect was abolished by (RS)-α-cyclopropyl-4-phosphonophenylglycine and was lost in slices from mGlu4 knock-out mice. Furthermore, (S)-3,4-dicarboxyphenylglycine did not affect glutamate and GABA transmission. Finally, intrastriatal LSP1-3081 or L-AP4 injection improved akinesia measured by the cylinder test. These results demonstrate that mGlu4 receptor selectively modulates striatal glutamate and GABA synaptic transmission, suggesting that it could represent an interesting target for selective pharmacological intervention in movement disorders involving basal ganglia circuitry.
excitatory post-synaptic current
excitatory post-synaptic potential
inhibitory post-synaptic current
inhibitory post-synaptic potential
miniature spontaneous excitatory post-synaptic current
- mGlu receptor
metabotropic glutamate receptor
medium spiny neuron
spontaneous excitatory post-synaptic current
spontaneous inhibitory post-synaptic current
substantia nigra pars compacta/reticulata
The basal ganglia (BG) are a network of interconnected subcortical structures involved in the control of extrapyramidal motor function and in the regulation of limbic and cognitive processes. The main input nucleus of this network is the striatum, which projects directly to the output BG nuclei, the internal globus pallidus (GP) [or entopeduncular nucleus in rodents] and substantia nigra pars reticulata (SNr), and indirectly via the external GP (or GP in rodents) and subthalamic nucleus (Graybiel 1990; Groves 1983; Smith and Bolam 1990). The balance between excitatory (glutamatergic) and inhibitory (GABAergic) afferences to internal GP and SNr is crucial for the physiological functioning of the BG and, accordingly, its disruption is believed to underlie several movement disorders, such as Parkinson’s disease (PD) and Huntington’s chorea (Albin et al. 1989; Wichmann and DeLong 1996, 1998).
The striatum is mainly constituted by GABAergic projection medium spiny neurons (MSNs) and by GABAergic and cholinergic interneurons, and its cortical and thalamic glutamatergic input is regulated by various receptors, including dopamine, acetylcholine and metabotropic glutamate (mGlu) receptors (Calabresi et al. 2000; Gubellini et al. 2004). Three groups of mGlu receptors have been described (Conn and Pin 1997; Pin and Acher 2002; Ferraguti and Shigemoto 2006) and are expressed in the BG, where they play a key role in regulating synaptic transmission and plasticity in both physiological and pathological conditions (Rouse et al. 2000; Gubellini et al. 2004; Conn et al. 2005). In particular group-III (mGlu4, 7, and 8) receptors, which are mainly pre-synaptic and negatively coupled to adenylyl cyclase, and whose activation inhibits L, N and P/Q voltage-dependent Ca2+ channels, resulting in decreased excitability and neurotransmitter release. Group-III mGlu receptors may thus represent possible pharmacological targets for neuropathologies characterized by unbalance of synaptic transmission in the BG, such as PD. In line with this hypothesis, it has been shown that injecting group-III agonists into the GP fully reverses akinetic deficits in a rat PD model (Lopez et al. 2007). Similarly, Marino et al. (2003) reversed reserpine-induced akinesia in rats by intracerebroventricular injection of a selective mGlu4 receptor allosteric potentiator, presumably acting at GP level (Valenti et al. 2003). Interestingly, Lopez et al. (2007) also showed that injection of group-III agonists in the SNr increases akinesia: this could be due to the differential expression of group-III mGlu receptors subtypes between SNr and GP, and supports the use of subtype-selective agonists as antiparkinsonian strategy.
The loss of dopamine following the lesion of substantia nigra pars compacta (SNc) in PD models leads to increased glutamate transmission in the striatum, recorded both in vitro (Calabresi et al. 1993; Schwartig and Huston 1996; Greenamyre 2001; Gubellini et al. 2002, 2006; Picconi et al. 2004) and in vivo (Orr et al. 1987; Chen et al. 2001; Tseng et al. 2001), which is presumed to play a key role in the expression of PD symptoms (Blandini et al. 1996). Such increased glutamate release could possibly result in increased striatal GABAergic activity or levels (Mora et al. 2007). In the striatum, activation of group-III mGlu receptors inhibits glutamatergic transmission (Pisani et al. 1997; Gubellini et al. 2004), but the specific subtype(s) involved in such regulation remains to be determined, and it is still unknown whether these receptors also modulate GABAergic transmission. It is thus of great importance to address these questions, as this information could be of interest for developing specific pharmacological therapies for PD. Here we demonstrate, by means of electrophysiological recordings, and by using a novel group-III orthosteric agonist and l-2-amino-4-phosphonobutyric acid (l-AP4), that mGlu4 receptor is involved in the pre-synaptic modulation of both glutamatergic and GABAergic transmission in the striatum. Moreover, we show that stimulation of striatal mGlu4 receptor improves motor deficit in a rat PD model.
Materials and methods
- Top of page
- Materials and methods
- Supporting Information
Slice preparation and maintenance
All animal experiments have been carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and all efforts were made to minimize their number and sufferance. Male Wistar rats, mGlu4 receptor knock-out mice (mGlu4-KO; Pekhletski et al. 1996) and wild-type (WT) CD-1™ (Pekhletski et al. 1996) mice aged 4–6 weeks were utilized. The brains were cut in coronal slices (200–250 μm) by a vibratome in ice-cold solution containing (in mM): 110 choline, 2.5 KCl, 1.25 NaH2PO4, 7 MgCl2, 0.5 CaCl2, 25 NaHCO3, 7 glucose, pH = 7.4, bubbled with a mix of 95% O2 and 5% CO2. Slices were kept in bubbled artificial CSF (ACSF) at around 20°C, whose composition was (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 2.4 CaCl2, 11 glucose, and 25 NaHCO3, pH = 7.4, and also containing 250 μM kynurenic acid and 1 mM sodium pyruvate to reduce glutamate-mediate toxicity and keep slices healthy. Both these drugs are rapidly and easily washed when the slice is put in the recording chamber. Recordings were done at 35°C in standard ACSF (without kynurenic acid and sodium pyruvate) flowing at ∼2.5 mL/min.
Intracellular sharp-microelectrodes (2 M KCl, 40–60 MΩ) electrophysiological recordings were performed by Axoclamp 2B amplifiers coupled to pClamp 10.2 digital acquisition data system (Molecular Devices, Palo Alto, CA, USA). A stimulating bipolar electrode was placed in the white matter between cortex and striatum to evoke glutamatergic excitatory post-synaptic potentials (EPSPs) in the presence of 3–10 μM bicuculline, while for GABAergic inhibitory post-synaptic potentials (IPSPs) it was placed into the striatum close to the recording electrode in the presence of 10 μM 6-cyano-7-nitroquinoxaline-2,3-dione, an α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor antagonist, and 40 μM DL-(−)-2-Amino-5-phosphonopentanoic acid, a NMDA receptor antagonist. Synaptic stimulation was delivered at 0.1 Hz. Paired-pulse ratio was calculated between the second and the first EPSP triggered by twin pulses (40–60 ms interval).
Whole-cell patch-clamp recordings were performed by borosilicate micropipettes (4–5 MΩ) filled with a Cs-gluconate solution containing (in mM): 120 Cs-gluconate, 13.6 CsCl, 10 HEPES, 1.1 EGTA, 0.1 CaCl2, 2.5 Mg-ATP, and 0.3 Na-GTP, pH 7.3. MSNs were localized by infrared videomicroscopy and recorded by an AxoPatch 200B amplifier coupled to pClamp 10.2 software (Molecular Devices). Series and input resistance were continuously monitored by sending 5 mV pulses and cells showing ≥ 20% change in series resistance during the experiment were discarded from the analysis. Synaptic stimulation to evoke post-synaptic responses was delivered at 0.1 Hz by a bipolar metal electrode placed in the white matter between cortex and striatum.
To record evoked glutamatergic excitatory post-synaptic currents (EPSCs) and spontaneous EPSC (sEPSC), MSNs were clamped at a holding potential of −60 mV, corresponding to the reversal potential of currents mediated by GABAA receptors with our Cs-gluconate micropipette solution. In these conditions, EPSCs and sEPSCs were completely blocked by bath application of 6-cyano-7-nitroquinoxaline-2,3-dione (not shown). Conversely, evoked and spontaneous GABAergic inhibitory post-synaptic currents (IPSCs and sIPSCs, respectively) were recorded at a holding potential of +10 mV, the reversal potential of currents mediated by ionotropic glutamate receptors. In these conditions, IPSCs and sIPSCs were completely blocked by bath application of the GABAA receptor antagonists bicuculline and picrotixin (not shown). This method (Cossart et al. 2000) allowed us recording both glutamatergic and GABAergic activity from the same MSN with no need of pharmacological blockade of GABAA or α-amino-3-hydroxy-5-methylisoxazole-4-propionate receptors, respectively.
Drugs were from Tocris-Cookson (Bristol, UK), with the exception of (3S)-3-[(3-amino-3-carboxypropyl(hydroxy)phosphinyl)-hydroxymethyl]-5-nitrothiophene (LSP1-3081), which was produced as described in Acher et al. (2007) and Selvam et al. (2007). All the tested compounds were dissolved in standard ACSF, were applied for 10–15 min, and their effect was evaluated during the last period of the perfusion time.
Data analysis and statistical test
Data were analyzed offline by Clampfit 10.2 (Molecular Devices), Origin 7.5 (Originlab Corp., Northampton, MA, USA) and MiniAnalysis 6.0 (Synaptosoft, Decatur, GA, USA) software. For analyzing spontaneous and miniature post-synaptic currents, the detection threshold (3–5 pA) was set to twice the noise after trace filtering (traces in Figures are not filtered). Only cells exhibiting stable activity and baseline were taken into account, and each cell was analyzed by the same person to avoid bias in the identification of spontaneous currents. Percentage values are calculated for each single experiment and all data are presented as mean ± SEM for each condition. For dose/response experiments, sigmoid curve fits and EC50 were calculated by Prism software (GraphPad, San Diego, CA, USA). Statistical analysis for electrophysiological data was performed with a non-parametrical matched-pairs test (Wilcoxon) by comparing the measured values before (control) and during the application of the tested compounds. Cylinder test data were analyzed by Kruskal–Wallis test followed by Dunn’s post-test. Significance values are reported in the Results and in the Figure legends.
- Top of page
- Materials and methods
- Supporting Information
LSP1-3081 is a potent and selective agonist of group-III mGlu receptors
By using in silico screening procedures, we recently identified a new series of group-III mGlu receptors agonists (Triballeau et al. 2005; Acher et al. 2007). The hit-to-lead process led us to identify LSP1-3081 (Fig. S1) as a new potent group-III orthosteric agonist. LSP1-3081 selectively and dose-dependently activated all group-III receptors transiently expressed in HEK293 cells, with a potency equivalent to that of l-AP4 (Table S1). LSP1-3081 was found to display a higher potency on mGlu4 receptor, followed by mGlu8, and very low on mGlu7 (Table S1). Of note, LSP1-3081 is also devoid of non-specific agonist or antagonist activity on group-I or II mGlu receptors (not shown). In addition, the larger molecular structure of LSP1-3081 should diminish the non-specific effects of glutamate derivatives, such as l-AP4, on targets other than mGlu receptors, i.e. Na+-independent glutamate transporter (Schulte et al. 1992; Chase et al. 2001) or glutamine synthetase (Gill and Eisenberg 2001;Krajewski et al. 2008).
Group-III mGlu receptors inhibit striatal glutamate transmission by a pre-synaptic mechanism
We first tested our novel agonist LSP1-3081, in comparison with l-AP4, by sharp-microelectrode intracellular recordings obtained from electrophysiologically identified striatal MSNs. These cells had a resting membrane potential around −85 mV that was not adjusted by current injection, absence of spontaneous action potential discharge, and tonic firing activity during current-induced membrane depolarization (not shown), as described previously (Calabresi et al. 1990). None of the drugs utilized altered significantly the intrinsic membrane properties of the recorded MSNs. We found that bath-applied LSP1-3081 (0.5–100 μM) depressed corticostriatal glutamatergic EPSPs in a dose-dependent manner, with an EC50 = 8.14 μM and a maximal inhibition of 33.25 ± 2.31% at 30 μM (Fig. 1a). As a control, we also tested the effect of l-AP4, which exerted a dose-dependent inhibition on EPSPs similar to that previously observed (Pisani et al. 1997), with a calculated EC50 = 9.32 μM and a maximal inhibition of 43.44 ± 2.69% at 60 μM (Fig. 1a). The effect of both drugs was reversible after washout (not shown).
We then verified whether the observed effects of LSP1-3081 and l-AP4 are actually mediated by a pre-synaptic mechanism involving decreased glutamate release. In agreement with this hypothesis, we found that paired-pulse ratio was significantly increased by 30 μM LSP1-3081 as well as 30 μM l-AP4 (Fig. 1b). Accordingly, whole-cell patch-clamp recordings (Fig. S2) showed that the frequency of sEPSCs recorded at −60 mV was significantly reduced by 10 μM LSP1-3081 and 10 μM l-AP4 to 59.7 ± 9.3% and 64.2 ± 8.5% of control, respectively (p < 0.01, n = 11 for both). Conversely, sEPSC amplitude was affected neither by LSP1-3081 nor by l-AP4 (94.0 ± 5.0% and 94.9 ± 4.5% of control, respectively; p > 0.05, n = 11 for both), further suggesting that both drugs have a pre-synaptic site of action (Fig. S2). In order to study whether action-potential independent spontaneous activity is modulated by group-III mGlu receptors, we measured the frequency and the amplitude of miniature spontaneous EPSC (mEPSC) recorded in the presence of the Na+ channel blocker tetrodotoxin (1 μM). LSP1-3081 (10 μM) significantly inhibited mEPSCs frequency to 70.0 ± 8.0% of control, but amplitude was unaffected (100.0 ± 2.0% of control; p < 0.01 and p > 0.05, respectively; n = 6 for both; Fig. 2). Similarly, 10 μM l-AP4 significantly inhibited mEPSCs frequency, but not amplitude (respectively 40.4 ± 6.6% and 94.0 ± 21.5% of control; p < 0.001 and p > 0.05, respectively; n = 6 for both; Fig. 2).
In a set of control experiments, we verified whether our agonists act specifically on group-III mGlu receptors. For this purpose, we pre-incubated the slices with the group-II/III antagonist (RS)-α-cyclopropyl-4-phosphonophenylglycine (CPPG, 100 μM), which had no significant effect per se on the amplitude of EPSC evoked by cortical stimulation (not shown). In the presence of CPPG, 10 μM LSP1-3081 failed to significantly inhibit EPSC amplitude (88.3 ± 17.1% of control; p > 0.05, n = 6), while in the absence of this antagonist it significantly reduced this parameter to 70.0 ± 4.2% of control (p < 0.05, n = 6). We obtained similar results with l-AP4, which in control conditions significantly reduced EPSC amplitude to 62.0 ± 8.1% of control (p < 0.05, n = 6), but in the presence of CPPG it was ineffective (94.2 ± 10.4% of control; p > 0.05, n = 6).
Activation of group-III mGlu receptors pre-synaptically inhibits striatal GABAergic transmission
We then studied whether group-III mGlu receptors are able to affect also GABAergic inputs to striatal MSNs. In a first set of experiments, we tested LSP1-3081 (1 nM to 30 μM) on GABAergic IPSPs. As shown in Fig. 3, this agonist effectively inhibited IPSPs in a dose-dependent manner (EC50 = 0.32 μM; maximal inhibition 95.78 ± 0.33% at 3 μM), as well as l-AP4 (Fig. 3; EC50 = 1.49 μM; maximal inhibition 62.28 ± 2.49% at 10 μM). These inhibitory effects were reversible after drug washout (not shown).
We then tested the effect of 10 μM LSP1-3081 and 10 μM l-AP4 on spontaneous GABAergic synaptic activity. We found that the two agonists exerted a significant inhibition on sIPSCs frequency recorded at +10 mV, which was reduced to 82.2 ± 7.0% of control by LSP1-3081 (p < 0.05, n = 11), and to 57.0 ± 4.5% of control by l-AP4 (p < 0.001, n = 11; Fig. S3). Conversely, sIPSCs amplitude was not significantly affected by LSP1-3081 or l-AP4 (respectively, 97.9 ± 5.2% and 90.1 ± 8.1% of control; p > 0.05, n = 11 for both), suggesting an inhibitory action on GABA release. Accordingly, LSP1-3081 and l-AP4 significantly reduced the frequency miniature sIPSCs recorded in 1 μM tetrodotoxin (Fig. 4), respectively to 79.9 ± 3.0% and 81.6 ± 6.4% of control (p < 0.05, n = 6 for both), without affecting their amplitude (95.1 ± 4.0% and 89.9 ± 8.0% (p > 0.05, n = 6 for both).
Striatal glutamatergic and GABAergic transmission are specifically regulated by mGlu4 receptor
The EC50 of LSP1-3081 and l-AP4 for group-III mGlu receptors (Table S1) and the concentration utilized here (10–30 μM) suggest that their inhibitory effect on striatal glutamate and GABA synaptic transmission is presumably mediated by the activation of mGlu4 or mGlu8, rather than mGlu7 receptor. However, considering the low distribution of mGlu8 receptor in this structure (Saugstad et al. 1997; Corti et al. 1998; Messenger et al. 2002), the best candidate for the effects of l-AP4 and LSP1-3081 is mGlu4. In order to test this hypothesis, we performed an additional set of control experiments.
First, we used the selective mGlu8 receptor agonist (S)-3,4-dicarboxyphenylglycine [(S)-DCPG]. Interestingly, the amplitude of evoked EPSC and IPSC was not significantly affected by 1–3 μM (S)-DCPG (Fig. 5a), doses at which this drug is active and selective for mGlu8 (Thomas et al. 2001; Valenti et al. 2003; Ayala et al. 2008). The average amplitude of evoked corticostriatal EPSC and IPSC was, respectively, 94.9 ± 5.9% and 95.6 ± 10.1% of control in the presence of (S)-DCPG (p > 0.05, n = 6 for both). Conversely, EPSCs were significantly inhibited by LSP1-3081 and l-AP4 (10 μM) to 70.0 ± 4.2% and 62.0 ± 8.1% of control, respectively (Fig. 5a; p < 0.05, n = 6 for both). Also IPSCs were significantly inhibited by these two agonists, respectively to 73.6 ± 10.5% and 58.2 ± 10.3% of control (Fig. 5a; p < 0.05, n = 6 for both). Moreover, sEPSCs and sIPSCs frequency was not affected by (S)-DCPG (not shown). Interestingly, our negative results obtained with (S)-DCPG are similar to those shown before in the hippocampus of adult rats, where mGlu8 receptor is absent (Ayala et al. 2008). These data strongly suggest that mGlu8 is lacking in the striatum, at least functionally, and are in agreement with literature data that do not demonstrate the presence of this receptor in this structure.
We performed a second set of experiments in slices from mGlu4-KO mice to further and unequivocally test whether mGlu4 receptor is involved in the inhibitory effect of LSP1-3081 and l-AP4. Interestingly, this effect was lost in mGlu4-KO animals (Fig. 5b): the average EPSC and IPSC amplitude during the application of 10 μM LSP1-3081 was, respectively, 102.3 ± 12.4% and 108.9 ± 7.2% of control (p > 0.05, n = 6 for both). Similarly, 10 μM l-AP4 failed to alter EPSC and IPSC amplitude that was, respectively, 95.8 ± 6.0% and 105.3 ± 12.5% of control during the application of this compound (p > 0.05, n = 6 for both). Accordingly, sEPSCs and sIPSCs were not affected by these two agonists (not shown). On the other hand, in WT mice, LSP1-3081 significantly inhibited the amplitude evoked EPSC and IPSC, respectively to 75.9 ± 7.3% and 71.3 ± 9.1% of control (p < 0.05, n = 6 for both). Similarly, l-AP4 reduced EPSC and IPSC amplitude to 56.3 ± 13.5% and 56.2 ± 13.0% of control (p < 0.05, n = 6 for both). These results were similar to those obtained in rats in the same conditions (p > 0.05 when comparing the inhibitory effects of both drugs obtained in WT mice with those obtained in rats, Mann–Whitney test).
Intrastriatal l-AP4 injection improves akinesia in 6-OHDA-lesioned rats
Unilateral 6-hydroxydopamine (6-OHDA) lesion of the SNc resulted in massive ipsilateral striatal dopamine depletion (Fig. S4) and disrupted the function of the contralateral forepaw. Such motor impairment can be correlated with akinesia, one of the hallmarks of PD (Albin et al. 1989; Wichmann and DeLong 1996, 1998). Here we show that, while control rats (n = 4) mostly used both forepaws to touch the cylinder wall (57.1 ± 9.2% of double contacts and ∼21% of single contacts for each forepaw), 6-OHDA-lesioned rats (n = 12) significantly reduced the number of double contacts (28.4 ± 4.4%, p < 0.05) because of contralateral forepaw akinesia (Fig. 6). Interestingly, intrastriatal injection of 10 nmol LSP1-3081 (n = 6) or l-AP4 (n = 6) greatly improved akinesia (Fig. 6), resulting in a significant recover of double contacts (respectively, 49.6 ± 4.5% and 46.8 ± 12.1%, p < 0.05 for both), while the injection of ACSF (n = 12) had no effect (34.5 ± 6.5%).
- Top of page
- Materials and methods
- Supporting Information
Here we report a new specific functional role of mGlu4 receptor in the striatum, whose activation by l-AP4 and the new group-III orthosteric agonist LSP1-3081 inhibits not only glutamate but also GABA transmission with a pre-synaptic mechanism. Moreover, we show that intrastriatal injection of these agonists improves akinesia in a rat model of PD.
Expression and role of group-III mGlu receptors and mGlu4 in the striatum
It has long been known that activation of group-III mGlu receptors depresses corticostriatal glutamatergic synaptic transmission (Pisani et al. 1997). We show here that group-III mGlu receptors also inhibit intrastriatal GABAetgic transmission. In the striatum, both mGlu4 and mGlu7 receptors are expressed in synaptic terminals (Kinoshita et al. 1998; Bradley et al. 1999; Kosinski et al. 1999; Corti et al. 2002; Conn et al. 2005), but there is no evidence about mGlu8 and, accordingly, we observed no significant effect of (S)-DCPG. Moreover, high levels of mGlu4 and mGlu7 receptor mRNA have been detected in the striatum (Testa et al. 1994; Ghasemzadeh et al. 1996; Messenger et al. 2002), while mGlu8 mRNA was found at low (Messenger et al. 2002) or very low amount (Saugstad et al. 1997; Corti et al. 1998). Considering the potency of LSP1-3081 and l-AP4 on mGlu4 versus mGlu7 receptor, and the doses utilized, our effects were thus likely mediated by mGlu4 rather than mGlu7: negative data from mGlu4-KO mice further confirm this hypothesis. In the absence of selective mGlu7 agonists, a possible role of this receptor cannot be excluded but, considering the lower affinity of mGlu7 versus mGlu4 receptor for l-glutamate (Pin and Acher 2002), mGlu4 should be the main group-III mGlu receptor modulating glutamate and GABA synaptic transmission in the striatum, at least in physiological conditions.
Regulation of glutamate and GABA synaptic transmission in the striatum by mGlu4 receptor
Striatal MSNs receive not only a massive glutamatergic drive from cortical and thalamic fibres (Graybiel 1990; Levy et al. 1997), but also GABAergic inputs from MSN collaterals and GABAergic interneurons (Czubayko and Plenz 2002; Tunstall et al. 2002; Koos et al. 2004; Tepper and Bolam 2004; Tepper et al. 2004; Venance et al. 2004; Bracci and Panzeri 2006; Gustafson et al. 2006; Taverna et al. 2008). Group-III mGlu receptors have been shown to modulate GABAergic synaptic transmission in the GP (Matsui and Kita 2003; Valenti et al. 2003), SNc (Valenti et al. 2005) and SNr (Wittmann et al. 2001), but we provide here the first evidence that they play such a role also in the striatum through mGlu4 receptor. Accordingly, mGlu4 is expressed in the striatum in synaptic terminals forming both type I (asymmetrical) and type II (symmetrical) synapses (Corti et al. 2002), giving further support to our findings. Our data also suggest that mGlu4 receptor activation depresses action potential-independent neurotransmitter release, in line with previous data obtained with l-AP4 in other BG structures (Wittmann et al. 2001;Matsui and Kita 2003; Valenti et al. 2003), likely by negatively modulating voltage-dependent Ca2+ channels and Ca2+-dependent neurotransmitter release (Conn and Pin 1997).
Functional implications of mGlu4 receptor-modulated release of GABA in the striatum
The traditional view of the functional organization of the striatum proposes that mGlu receptors act as autoreceptors for regulating pre-synaptically cortical and thalamic inputs, and thus indirectly modulate striatal output by limiting ‘upstream’ the excitation mediated by glutamate. Our findings suggest the presence of a ‘downstream’ modulatory mechanism operated by mGlu4 acting as a heteroreceptor inhibiting intrinsic striatal GABA release. Such double regulation appears critical for the functioning of BG network, both in physiological and pathological conditions. In fact, striatal glutamatergic hyperactivity in PD models is believed to be paralleled by GABAergic hyperactivity as suggested, for example, by increased striatal GABA levels (Bruet et al. 2003) and glutamate decarboxylase expression in striatopallidal MSNs (Soghomonian and Laprade 1997), or by the enlargement of striatopallidal MSNs terminals (Ingham et al. 1997). Thus, the functional consequence could be an increased GABAergic striatal output and, accordingly, Lopez et al. (2007) showed that injection of (1S,3R,4S)-1-Aminocyclopentane-1,3,4-tricarboxylic acid (ACPT-I) in the GP could reverse akinetic deficits in rats. Interestingly, GABAergic transmission is regulated by mGlu4 receptor both in the rat striatum (present work) and the GP (Valenti et al. 2003), and this receptor is little or not expressed in the SN and entopeduncular nucleus, where mGlu7 is predominant (Bradley et al. 1999; Corti et al. 2002). This suggests that a selective mGlu4 agonist administered systemically would preferentially inhibit striatopallidal neurons, rather than those of the direct pathway, as well as striatal GABAergic neurons. This, however, would be paralleled by the inhibition of corticostriatal glutamatergic synapses on both MSNs and interneurons. It is difficult to predict what would be the net effect on the overall output of the striatum, as we do not know, at this stage, which one of these effects would prevail. It would be thus interesting to gain more precise knowledge on the localization of mGlu4 receptor in the brain, in particular in the BG, in order to predict which synapses will be inhibited by systemic administration of selective mGlu4 agonists. Interestingly, intracerebroventricular injection of l-AP4 provides motor benefits in rat PD models (Valenti et al. 2003), and intracerebroventricular injection of a mGlu4 receptor positive allosteric modulator decreases haloperidol-induced catalepsy and reserpine-induced akinesia (Niswender et al. 2008), supporting the use of systemic administration of these drugs. To further address this point, we tested the effect of intrastriatal injection of LSP1-3081 or l-AP4 in the rat 6-OHDA model of PD, and we show that this treatment significantly improves forepaw akinesia. The more likely explanation of this effect could be that activating striatal mGlu4 receptors reduces both glutamatergic and GABAergic hyperactivity due to dopaminergic lesion.
In conclusion, this work further extends our knowledge on group-III mGlu receptors role in modulating striatal synaptic transmission: here we provide pharmacological and functional evidence that mGlu4 receptor regulates negatively both glutamate and GABA synaptic release in the striatum. Moreover, stimulation of these receptors improves akinesia in a rat model of PD, opening new perspectives for selective pharmacological treatments for PD targeting specific brain structures thanks to their differential expression of mGlu receptors subtypes.
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- Materials and methods
- Supporting Information
This work was financed by grants from the CNRS, the Université de la Méditerranée, the French Ministry of Education and Research (project ANR-05-NEUR-021 ‘mGluRs Park’), and the Italian Ministry of Education (PRIN 2006). EB was supported by a scholarship of the ‘Leonardo da Vinci’ Programme Unipharma-Graduates-3 (http://www.unipharmagraduates.it) coordinated by the University of Rome ‘La Sapienza’. AP and PG were partially supported by a French/Italian ‘Galilée/Galileo’ grant.
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- Supporting Information
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- Top of page
- Materials and methods
- Supporting Information
Supplementary Materials and Methods
Table S1. Activity of LSP1-3081 and l-AP4 on different group-III mGlu receptors rat clones expressed in HEK293 cells. The activity of mGlu receptors was assessed by measuring IP production (Gomeza et al. 1996). Experiments were performed in 96-well microplates as already described (Goudet et al. 2004). All data are mean ± SEM of n experiments (n indicated within brackets).
Figure S1. Molecular structure of LSP1-3081 compared to l-AP4. Note the larger molecular structure of LSP1-3081, which should diminish the non-specific effects of glutamate derivatives, such as l-AP4.
Figure S2. Examples of spontaneous glutamatergic activity recorded from two MSNs [holding potential (HP) = −60 mV] in control condition and in the presence of 10 μM of LSP1-3081 (a) and l-AP4 (b). The parts of the traces included in the boxes are expanded below. The graph (c) shows that the frequency, but not the amplitude, of sEPSCs was significantly reduced by each drug, suggesting a pre-synaptic site of action (**p < 0.01, n = 11 for each condition).
Figure S3. Examples of spontaneous GABAergic activity recorded from two MSNs [holding potential (HP) = +10 mV] in control condition and in the presence of 10 μM of LSP1-3081 (a) and l-AP4 (b). The parts of the traces included in the boxes are expanded below. The graph (c) shows that the frequency, but not the amplitude, of sIPSCs was significantly inhibited by each drug, suggesting a pre-synaptic site of action (*p < 0.05, ***p < 0.001, n = 11 for each condition).
Figure S4. Photomicrograph of a frontal brain section of a 6-OHDA-lesioned rat showing the loss of autoradiographic binding for 3H-mazyndol in the striatum on the ipsilateral side (left), compared to the contralateral (right).
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