Role of glutamate transporters in corticostriatal synaptic transmission

Neuroscience 158 (2009) 1608 –1615

ROLE OF GLUTAMATE TRANSPORTERS IN CORTICOSTRIATAL SYNAPTIC TRANSMISSION C. BEURRIER,a G. BONVENTO,b L. KERKERIAN-LE GOFFa AND P. GUBELLINIa*

Gubellini et al., 2004; Ferraguti and Shigemoto, 2006). It is involved in all aspects of brain development and function from the early stages of neurogenesis to normal ageing, through cognition, learning and motor functions (Headley and Grillner, 1990). Since glutamate is not degraded in the synaptic cleft, the most efficient way for removing this neurotransmitter from the extracellular space is via cellular uptake through glutamate transporters (GTs; for a review, see Danbolt, 2001). GTs constitute a structurally related family of Na⫹- and voltage-dependent excitatory amino acid transporters (EAATs), and five have been cloned from human tissues (EAAT1 to EAAT5; Danbolt, 2001; Schousboe et al., 2004). In rodents’ CNS, five GTs have been identified so far: GLAST and GLT-1 (homologous to EAAT1 and EAAT2, respectively), expressed by glial cells, EAAC1 (homologous to EAAT3) and EAAT4, expressed by neurons, and EAAT5, expressed by retinal rod photoreceptors (Pow and Barnett, 2000; Danbolt, 2001). GTs are crucial to keep the extracellular levels of glutamate low in physiological conditions, and this function can be also relevant to neuropathological situations characterized by dysfunctions of glutamatergic systems, such as amyotrophic lateral sclerosis, Alzheimer’s disease, epilepsy and Parkinson’s disease (PD) (Carlsson and Carlsson, 1990; Choi, 1992; Rothstein et al., 1992; Chapman, 2000; Danysz et al., 2000; Gubellini et al., 2006). In particular, regarding PD, the expression of GLT-1 mRNA is increased in the striatum and in the output structures of the basal ganglia of parkinsonian rats treated with L-DOPA inducing dyskinesia (Liévens et al., 2001; Robelet et al., 2004). Such increase could represent a compensatory mechanism to counter corticostriatal glutamatergic hyperactivity observed in this model (Gubellini et al., 2006). The striatum of the adult rat expresses GLT-1, GLAST and EAAC1 (Danbolt, 2001). In this structure, GLT-1 and GLAST are localized on astrocyte membrane processes surrounding virtually all neuronal cell bodies and synaptic complexes (Rothstein et al., 1994). It has been show in other brain structures that their localization can change with the type of structure neighboring the astrocyte (neuropil, cell bodies, pia mater or capillary endothelium; Chaudhry et al., 1995; Lehre et al., 1995), but no such information is available for the striatum. It has also been shown that striatal levels of GLT-1 are very high, while those of GLAST are low. Concerning EAAC1, it is localized in post-synaptic elements (dendritic shafts and spines) but not within presynaptic axon terminals (Rothstein et al., 1994). The striatum is the major input station of the basal ganglia, receives massive excitatory afferences from cortical fibers releasing glutamate and is involved in motor control and learning (Graybiel, 1990;

a Institut de Biologie du Développement de Marseille-Luminy, UMR 6216 (CNRS-Université de la Méditerranée Aix-Marseille), Marseille, France b CEA, Institute of Biomedical Imaging (I2BM), Molecular Imaging Research Center (MIRCen), F-92265 Fontenay-aux-Roses, France

Abstract—High-affinity glutamate transporters (GTs) play a major role in controlling the extracellular level of this excitatory neurotransmitter in the CNS. Here we have characterized, by means of in vitro patch-clamp recordings from medium spiny neurons (MSNs), the role of GTs in regulating corticostriatal glutamatergic synaptic transmission in the adult rat. Charge transfer and decay-time, but not amplitude, of excitatory postsynaptic currents (EPSCs) were enhanced by DL-threo-␤-benzyloxyaspartate (TBOA), a broad inhibitor of GTs. Moreover, TBOA also potentiated currents induced by high-frequency stimulation (HFS) protocols. Interestingly, the effect of TBOA on EPSCs was lost when MSNs were clamped at ⴙ40 mV, a condition in which neuronal GTs, that are voltage-dependent, are blocked. However, in this condition TBOA was still able to enhance HFS-induced currents, suggesting that glial GT’s role is to regulate synaptic transmission when glutamate release is massive. These data suggest that neuronal GTs, rather than glial, shape EPSCs’ kinetics and modulate glutamate transmission at corticostriatal synapse. Moreover, the control of glutamate concentration in the synaptic cleft by GTs may play a role in a number of degenerative disorders characterized by the hyperactivity of corticostriatal pathway, as well as in synaptic plasticity. © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: glutamate transporters, striatum, mGlu receptors, TBOA, electrophysiology.

Glutamate is the main excitatory neurotransmitter in the mammalian brain, exerting its action via three classes of ionotropic glutamate receptors, i.e. AMPA, kainate and Nmethyl-D-aspartate (NMDA), and three groups of metabotropic glutamate receptors (mGluR) (Pin and Acher, 2002; *Correspondence to: P. Gubellini, Equipe IC2N-IBDML UMR6216; case 907, Parc Scientifique de Luminy, 13288 Marseille cedex 9, France. Tel: ⫹33-0-491269248; fax: ⫹33-0-491269244. E-mail address: [email protected] (P. Gubellini). Abbreviations: ACSF, artificial cerebrospinal fluid; AP-5, D,L-2-amino-5phosphonopentanoic acid; cPPT, central polypurine tract; DHK, dihydrokainic acid; EAATs, excitatory amino acid transporters; EPSCs, excitatory postsynaptic currents; GFP, green fluorescent protein; GTs, glutamate transporters; HFS, high-frequency stimulation; mGluR, metabotropic glutamate receptors; MSNs, medium spiny neurons; NMDA, N-methyl-Daspartate; PD, Parkinson’s disease; PDC, L-trans-pyrrolyidine-2,4-dicarboxylate; PGK, phosphoglycerate kinase I promoter; PPR, paired-pulse ratio; (RS)-CPPG, ␣-cyclopropyl-4-phosphonophenylglycine; (S)-MCPG, (S)-alpha-methyl-4-carboxyphenylglycine; TBOA, DL-threo-␤-benzyloxyaspartate; WPRE, woodchuck post-regulatory element. 0306-4522/09 © 2009 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2008.11.018

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Levy et al., 1997). Although glutamate is the main excitatory neurotransmitter in the striatum, and although corticostriatal hyperactivity is an hallmark of PD, little is known, currently, regarding the role of GTs in modulating synaptic transmission in this structure, while this function has been already characterized in other brain regions, such as the hippocampus (Hestrin et al., 1990; Isaacson and Nicoll, 1993; Sarantis et al., 1993; Tsukada et al., 2005), the cortex (Kidd and Isaac, 2000; Campbell and Hablitz, 2004), the cerebellum (Marcaggi et al., 2003; Takatsuru et al., 2006) and the avian nucleus magnocellularis (Otis et al., 1996; Turecˇek and Trussel, 2000). Thus, the goal of the present work was to characterize whether and how GTs regulate corticostriatal synaptic transmission. To address this issue, we used the broad spectrum blocker DL-threo-␤-benzyloxyaspartate (TBOA) and we performed in vitro patchclamp recordings from striatal medium spiny neurons (MSNs) in adult rat slices. We measured the effects of TBOA on evoked glutamatergic excitatory postsynaptic currents (EPSCs) and on currents triggered by high-frequency stimulation (HFS). In addition, we tested whether mGluRs modulate the effects of GT inhibition. Here we show, for the first time in the striatum, that neuronal GTs shape the kinetic of EPSCs, while glial GTs appear to be recruited when a massive release of glutamate is triggered by HFS at corticostriatal synapses.

EXPERIMENTAL PROCEDURES Animals and slices preparation All animal experiments have been carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and conformed to the ethical guidelines of the French Ministry of Agriculture and Forests (Animal Health and Protection Veterinary Service). Lewis male rats weighing 250 – 300 g (10 –12 weeks) were utilized and all efforts were made to minimize their number and suffering. To obtain rats with striatal astrocyte expressing green fluorescent protein (GFP), we used a lentiviral vector pseudotyped with Mokola with a strong tropism toward astrocytes (Colin et al., 2008; Pertusa et al., 2008) and expressing GFP, which was injected in the striatum. More precisely, we used a self-inactivated (SIN-W) lentiviral vector (Déglon et al., 2000) containing the central polypurine tract (cPPT) sequence, the mouse phosphoglycerate kinase I promoter (PGK), the woodchuck post-regulatory element (WPRE) sequence and encoding GFP (SIN-W-cPPT-PGK-GFP-WPRE). This vector was diluted in PBS-BSA to a final concentration of 200 ng p24/␮l. Rats were anesthetized with a mixture of ketamine (15 mg/kg) and xylazine (1.5 mg/kg). Suspensions of lentiviral vector were injected into the striatum using a 34-gauge blunt-tip needle linked to a Hamilton (Reno, NV, USA) syringe by a polyethylene catheter. The stereotaxic coordinates were: anteroposterior (AP) ⫹0.5 mm; lateral (L) ⫹3.0 mm from bregma; and ventral (V) ⫺4.5 mm from the dura. Rats received a total volume of 3 ␮l per striatum at a rate of 0.2 ␮l/min. The brains were cut in coronal slices (250 ␮m) in ice-cold solution (in mM: 110 choline, 2.5 KCl, 1.25 NaH2PO4, 7 MgCl2, 0.5 CaCl2, 25 NaHCO3, 7 glucose, pH⫽7.4) bubbled with O2/CO2 (95/5%). Slices were stored in artificial cerebrospinal fluid (ACSF), 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, gassed with O2/CO2 (95/5%), at room temperature, containing 250 ␮M kynurenic acid and 1 mM sodium pyruvate. Re-

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cordings were performed at 35 °C in standard ACSF solution (without kynurenic acid and sodium pyruvate).

Electrophysiological recordings and data analysis In order to record striatal MSNs, whole-cell patch-clamp microelectrodes (4 –5 M⍀) were filled with a CsCl solution, whose composition was (in mM): 140 CsCl, 10 NaCl, 0.1 CaCl, 10 Hepes, 1 EGTA, 2 Mg-ATP and 0.5 Na-GTP, pH⫽7.3, containing 0.5% biocytin. MSNs of the dorsal striatum were visualized by infrared videomicroscopy (Nikon and Olympus, Japan) before patching, and were identified a posteriori by histochemistry. For astrocyte recordings, the microelectrode solution was (in mM): KCl 130, MgCl2 2, Hepes 10, EGTA 5, CaCl2 2, Na-ATP 2, pH⫽7.3. Astrocytes were identified prior recordings due to their green fluorescence, and by their electrophysiological properties (Adermark and Lovinger, 2006). Electrophysiological recordings were performed by an AxoPatch 200B and a MultiClamp 700B amplifier with pClamp software (Molecular Devices, Sunnyvale, CA, USA). Picrotoxin at 50 ␮M was always added to the bath solution to block GABAA receptor-mediated synaptic activity. Series and input resistance was continuously monitored by sending a 5 mV pulse. Neurons showing ⱖ20% change in series resistance were discarded from the analysis. Synaptic stimulation for activating corticostriatal fibers and triggering EPSCs was delivered at 0.1 Hz by a bipolar electrode placed close to the recording pipette. More precisely, electrode tips were positioned in the external capsule, near the cingulum: for example, at 9.7 mm rostro-caudal level (interaural), the electrode tips were placed between coordinates 3– 4 mm lateral and 6 –7 mm dorsoventral (Paxinos and Watson, 1986). Data were analyzed offline by Clampfit 10.2 (Molecular Devices), Origin 7.5 (Originlab Corporation, Northampton, MA, USA) and MiniAnalysis 6.0 (Synaptosoft, Decatur, GA, USA). EPSC charge transfer was calculated starting just after the stimulus artifact to 100 ms after. Charge transfer of large currents induced by HFS trains was measured from the peak of the last EPSC in the train to baseline recovery. EPSC amplitude was measured by averaging a 0.3 or 4 ms time interval centered on the maximum amplitude value at ⫺60 mV and ⫹40 mV respectively. To quantify EPSC kinetics, its decay-time (from 90% to 10% of peak amplitude) was fitted with the sum of two exponentials, which allows calculating an amplitude-weighted decay-time constant (␶w) and provides a better fit than one exponential; ␶w was calculated as:

␶w⫽(A1 ␶1⫹A2 ␶2) ⁄ (A1⫹A2) where A1 and A2 are the amplitudes of the components with time constants ␶1 and ␶2 (see also Marcaggi et al., 2003). Drugs utilized were from Tocris-Cookson (Bristol, UK). Puff applications of glutamate were done by using a two-channel TooheySpritzer (Toohey Company, Fairfield, NJ, USA). Statistical analysis was performed by Wilcoxon non-parametrical test for matched pairs or by Mann-Whitney non-parametrical test for unmatched pairs (as specified in the figures’ legends). Data are presented as mean⫾ S.E.M.

Histochemistry Following electrophysiological recordings, slices were fixed in Antigenfix (DiaPath, Rome, Italy), cryoprotected and quickly frozen for storage at ⫺80 °C. The detection of biocytin-filled neurons was directly performed on slices with fluorescent streptavidin-Alexa 488 (Invitrogen, Paisley, UK) and a confocal microscope (Olympus Fluoview, Japan).

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Fig. 1. Morphological characterization of striatal MSNs and modulation of EPSC kinetics by GTs blockade. (A) A striatal MSN injected with biocytin, revealed by Alexa 488 fluorescence and confocal microscopy (scale bar⫽20 ␮m). Note the small soma and the dendritic tree covered with spines (inset). (B) The histogram shows the effect of the application of TBOA (30 ␮M) on charge transfer (c.t.), ␶w and amplitude (ampli) of corticostriatal EPSC recorded at ⫺60 mV. TBOA significantly increased c.t. and ␶w (* P⬍0.05 and ** P⬍0.01 compared with control, Wilcoxon test) but had no effect on ampli. Traces depict the effect of TBOA on the EPSC kinetics (stimulation artifacts are cut and the traces at are normalized to peak ampli).

RESULTS GTs shape EPSC kinetics Striatal MSNs were recorded both at ⫺60 mV for measuring EPSCs in physiological conditions, and at ⫹40 mV in order to block neuronal GTs due to their voltage-dependence (see also below, and Wadiche et al., 1995; Diamond, 2001). Since the CsCl pipette solution required for such recordings prevented the electrophysiological identification of striatal MSNs, all the recorded neurons were morphologically identified a posteriori by injecting them with biocytin through the patch pipette. Only cells corresponding to MSNs, i.e. showing spines on their dendrites and with a soma diameter around 10 –20 ␮m (Fig. 1A), were kept for data analysis. In order to study the role of GTs in modulating corticostriatal synaptic transmission, three parameters of corticostriatal EPSC were measured before and during the application (5–10 min) of the GTs blocker TBOA (30 ␮M): charge transfer, decay-time (␶w) and amplitude (Fig. 1B). Interestingly, this GT blocker significantly increased EPSC charge transfer (n⫽8, P⬍0.05) and ␶w (from 7.84⫾1.67– 14.22⫾3.72 ms; n⫽8, P⬍0.01), but had no effect on amplitude (from 204.7⫾16.1 pA to 219.9⫾37.5 pA; n⫽8, P⬎0.05). The effect of TBOA was completely reversible after 20 –30 min washout (not shown). These data suggest that GTs shape the kinetic of corticostriatal EPSC and regulate the amount of charge transferred through AMPA receptors following synaptic activation. Since ␶w is extremely sensitive to changes in series resistance, we have performed a correlation analysis between these two parameters. No correlation was found (r⫽⫺0.08, n⫽12, Spearman test), confirming that our results were not biased by seal quality.

The lack of effect of TBOA on EPSC amplitude could rely on AMPA receptor saturation, triggered by an excess of glutamate due to GTs blockade. To test this hypothesis we performed a series of experiments in the presence of kynurenic acid, a low-affinity and competitive antagonist of excitatory amino acid receptors. This drug (200 ␮M) inhibited per se corticostriatal AMPA EPSC amplitude by 36.4⫾8.2% (n⫽5), which is in agreement with previous

Fig. 2. AMPA receptors are not saturated by excess glutamate in the presence of TBOA. The histogram shows that, in the presence of 200 ␮M kynurenic acid, 30 ␮M TBOA still had no significant effect on the amplitude of EPSCs recorded at ⫺60 mV (§ P⬎0.05, Wilcoxon test). The traces depict the lack of effect on EPSC amplitude in the presence of kynurenic acid.

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Fig. 3. Glial transporters do not shape corticostriatal EPSC. (A) The histogram shows that TBOA (30 ␮M) did not significantly affect charge transfer (c.t.), ␶w and amplitude (ampli) of corticostriatal EPSC recorded at ⫹40 mV, a condition in which the GTs of the recorded neuron are blocked by depolarization. Traces depict this lack of effect of TBOA on the EPSC (stimulation artifacts are cut and the traces at are normalized to peak amplitude). (B) The graph shows the effect of TBOA on ␶w in five representative neurons, measured in each cell at ⫺60 mV and ⫹40 mV (in the presence of 40 ␮M AP-5). Note the ␶w increase at ⫺60 mV, and the lack of effect at ⫹40 mV (** P⬍0.01, Mann-Whitney test). The traces (stimulation artifacts are cut and the traces at are normalized to peak ampli) depict one of these recorded neurons in which TBOA increased ␶w at ⫺60 mV, but not at ⫹40 mV.

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amino-5-phosphonopentanoic acid (AP-5, 40 ␮M), in order to have a pure AMPA EPSC also at ⫹40 mV. We measured TBOA effect at both ⫺60 and ⫹40 mV in the same neuron, as shown in Figure 3B. Interestingly, in each recorded cell TBOA increased ␶w at ⫺60 mV (from 9.22⫾0.84 ms to 12.25⫾1.15 ms; n⫽5, P⬍0.01), but not at ⫹40 mV (from 57.77⫾8.97 ms to 58.38⫾9.22 ms; n⫽5). These data further support that glial GTs do not modulate EPSC kinetics. We then tested TBOA in a condition in which more glutamate is released from the presynaptic terminals, i.e. by delivering HFS at 50 Hz (lasting 100 or 500 ms). In this condition, a massive release of glutamate can occur, thus extra- and perisynaptic glial GTs can become involved due to glutamate diffusion (Marcaggi et al., 2003; Tzingounis et al., 2007). Longer or repeated protocols, as well as lower or higher frequency trains, were avoided because they could induce synaptic plasticity phenomena (Partridge et al., 2000; Gubellini et al., 2004; Fino et al., 2005). Interestingly, the application of TBOA (30 ␮M, 5–10 min) significantly increased HFS-induced currents recorded at ⫹40 mV (Fig. 4A) by 136.1⫾38.7% (n⫽9, P⬍0.01) and 246.1⫾85.2% (n⫽9, P⬍0.05) for 100 and 500 ms trains, respectively, and this effect was reversible after 20 –30 min washout. To test whether such large HFS-induced currents could be enhanced by TBOA also in more physiological conditions, we repeated the same experiment at a holding potential of ⫺60 mV. As expected, in this conditions TBOA enhanced by 317.7⫾81.3% (n⫽8, P⬍0.01) and

data in the striatum (Cherubini et al., 1988). However, also in the presence of kynurenic acid, TBOA had no significant effect on the amplitude of corticostriatal EPSC (Fig. 2), which was 214.0⫾8.6 pA before and 194.4⫾9.2 pA during the application of this GT blocker (n⫽8, P⬎0.05). Thus, we discarded the hypothesis that AMPA receptor saturation occurs in the presence of TBOA. Role of neuronal vs. glial GTs To test whether glial GTs (GLAST and GLT-1) are specifically involved in modulating corticostriatal EPSCs, we took advantage of the fact that GTs are voltage-dependent: it has been shown, in fact, that at depolarized potentials (from 0 to ⫹40 mV), glutamate uptake is inhibited (Wadiche et al., 1995). Thus, by clamping a MSN at ⫹40 mV it is possible to block its own neuronal GTs, and any effect produced by TBOA will be due to its action on glial GTs, since its effect on neuronal GTs will be occluded (Diamond, 2001). Interestingly, the effect of TBOA on charge transfer and ␶w lost its significance at ⫹40 mV (Fig. 3A), suggesting that glial GTs do not play a major role in shaping EPSC kinetics (changes of EPSC ␶w and amplitude were, respectively, from 226.3⫾26 ms to 281.3⫾37.8 ms, and from 203⫾25.6 pA to 182.0⫾29.8 pA; n⫽11, P⬎0.05 for all). In another set of experiments, we performed our recording in the presence of the NMDA antagonist D,L-2-

Fig. 4. GTs limit glutamate diffusion during HFS. (A) The histogram and the traces show that the large currents triggered by 100 and 500 ms HFS (50 Hz), recorded at ⫹40 mV, were significantly enhanced by blocking glial GTs with TBOA. (B) At ⫺60 mV, similar large currents could be induced by HFS trains lasting 100 and 500 ms and were also potentiated by TBOA (* P⬍0.05, ** P⬍0.01, compared with control, Wilcoxon test).

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by 794.0⫾187.6% (n⫽7, P⬍0.05) the currents induced by 100 and 500 ms trains, respectively (Fig. 4B). These data suggest that the role of glial GTs at corticostriatal synapse should be, rather than shaping EPSCs, limiting glutamate diffusion when the release of this neurotransmitter is high. The lack of effect of TBOA at ⫹40 mV could be due to the fact that its concentration (30 ␮M) was not high enough to block glial GTs, although it has been shown that Ki of this drug is around 1–10 ␮M (Bridges and Esslinger, 2005), and the IC50 for EAAT1, EAAT2 and EAAT3 is, respectively, 33⫾3.7, 6.2⫾0.49 and 15⫾1.3 ␮M (Shimamoto et al., 2004). Thus, in our conditions TBOA should be active on both glial and neuronal GTs at the doses we used. It is worth mentioning that higher TBOA doses (ⱖ50 ␮M) were tested, but were successively avoided

since they induced recording loss (not shown). However, to test whether TBOA could effectively block glial GTs in our conditions, we measured the currents associated to glutamate uptake recorded from striatal astrocytes that were identified by their fluorescence due to GFP expression (Fig. 5A). In order to isolate GT currents, these experiments were performed in 10 ␮M 6-cyano-7-nitroquinoxaline-2,3-dione and at a holding potential of ⫺60 to ⫺70 mV. We could characterize two types of striatal astrocytes on the basis of their current/voltage relationship, i.e. “passive” and “complex,” as previously described (Fig. 5B; Adermark and Lovinger, 2006). As expected, 1 mM glutamate puffs (10 –30 ms) triggered slow and reversible inward currents (32.3⫾10.5 pA) that were totally blocked after the application of 30 ␮M TBOA for 5–10 min (n⫽5), suggesting that in our conditions glial GTs were completely and efficiently blocked by this inhibitor (Fig. 5C). In order to further address the role of glial GTs in the striatum, we also used the classical GLT-1 inhibitor dihydrokainic acid (DHK, 300 ␮M) at ⫺60 mV. Surprisingly, this drug reduced significantly and reversibly EPSC amplitude by 35.3⫾9.5% (n⫽7, P⬍0.05), and had no significant effect on charge transfer (⫺27.6⫾20.8%; n⫽6, P⬎0.05) and ␶w (34.8⫾41.5% of control; n⫽6; P⬎0.05). Moreover, paired-pulse ratio (PPR), delivered with an inter-pulse interval of 30 ms, was significantly increased by DHK (153.1⫾14.5%; n⫽7, P⬍0.05). Similar effects were observed with 50 ␮M DHK (not shown). This opens the possibility that the observed inhibitory effect of this drug may also rely on a presynaptic mechanism, not related to its action as a GLT-1 blocker. Mechanisms of action of GTs blockade

Fig. 5. TBOA blocks glutamate-induced currents recorded from striatal astrocytes. (A) Visualization of a striatal astrocyte following intrastriatal infection of a lentiviral vector expressing GFP (scale bar⫽10 ␮m). (B) Electrophysiological traces showing a typical current/voltage relationship of a “passive” (upper sweeps) and “complex” (middle sweeps) astrocyte in response to hyper- and depolarizing voltage pulses lasting 200 ms and given in 10 mV increments (lower sweeps) from a holding potential of ⫺70 mV. (C) Lower traces, recorded from the same “complex” astrocyte in B, depict the current associated to glutamate transport triggered by a puff application of 1 mM glutamate for 30 ms (arrow) in control condition (black trace). After 10 min incubation in 30 ␮M TBOA (gray trace), glutamate failed to trigger any current.

GTs blockade can increase the synaptic concentration of glutamate, which could thus act presynaptically, for example on group II and III mGluRs, and affect the release probability, as well postsynaptically on group I mGluRs that are known to increase neuronal excitability (Pin and Acher, 2002; Gubellini et al., 2006). To test the involvement of mGluRs in TBOA effect, we blocked these receptors by perfusing a combination of 100 ␮M (S)-alpha-methyl-4carboxyphenylglycine [(S)-MCPG] and 30 ␮M (RS)-␣-cyclopropyl-4-phosphonophenylglycine (CPPG), 5 min prior and during TBOA application. In the presence of mGluRs antagonists (Fig. 6A–C), charge transfer and ␶w of AMPA EPSC were still significantly increased by TBOA, respectively by 29.7⫾15.1% and 64.3⫾21.2% (n⫽8, P⬍0.05 and P⬍0.01 respectively), while amplitude was again not affected (⫺1.5⫾8.1%; n⫽8, P⬎0.05). Similarly, 100 ms HFS-induced currents were still significantly increased by 352.6⫾121.8% (n⫽7, P⬍0.05) also in the presence of mGluRs antagonists (Fig. 6D). These effects were not significantly different to those obtained without mGluRs antagonists (P⬎0.05 for all). Thus, it seems likely that mGluRs do not modulate the synaptic changes induced by GT blockade.

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Fig. 6. The effects of TBOA are not mediated by mGluRs. (A–C) The histograms show that the effect of 30 ␮M TBOA on the three parameters of EPSC was unchanged in the presence of mGluRs antagonists (TBOA⫹Ant; 100 ␮M (S)-MCPG⫹30 ␮M CPPG): this GTs inhibitor was still able to significantly increase EPSC charge transfer and ␶w even when mGluRs were blocked (while it still had no significant effect on amplitude). (D) Similarly, the increase in charge transfer of HFS currents (100 ms at 50 Hz) induced by 30 ␮M TBOA was similar in the presence of mGluRs antagonists (* P⬍0.05, ** P⬍0.01 compared with pre-TBOA application, Wilcoxon test; § P⬎0.05 when comparing the effect of TBOA in absence and in presence of mGluRs antagonists; Mann-Whitney test).

DISCUSSION Our data in the striatum of adult rats clearly show that blocking GTs enhances the kinetic of glutamatergic EPSC. These effects are presumably due to decreased glutamate removal and/or longer permanence of this neurotransmitter in the synaptic cleft. We also show that only neuronal GTs play a role in shaping EPSC kinetics, while glial GTs are presumably involved in limiting glutamate diffusion during tetanic synaptic activation. Known effects of GTs blockade in the brain Previous electrophysiological slice studies in several CNS structures have provided heterogeneous results following GTs blockade. For example, in the hippocampus Hestrin et al. (1990) reported that DHK increases NMDA EPSC amplitude, while Isaacson and Nicoll (1993) showed no effect on glutamatergic EPSCs of the large GTs inhibitor L-transpyrrolyidine-2,4-dicarboxylate (PDC), but again Tsukada et al. (2005) reported that a selective glial GT blocker (derivative of TBOA) increases epileptiform activity and prolonged the decay-time of EPSCs, but not their amplitude. Moreover, Asztely et al. (1997) suggested that GTs play a critical temperature-dependent role in limiting synaptic spillover of glutamate. In the cerebellum, the effects of GT blockade seem to depend on the number of stimulated fibers and their spatial organization (Marcaggi et al., 2003), and affect the kinetics of EPSCs (Takahashi et al., 1995; Takatsuru et

al., 2006). However, Sarantis et al. (1993) showed that PDC does not increase non-NMDA synaptic currents, but it rather inhibited them. In the barrel cortex, Kidd and Isaac (2000) demonstrated that the combined application of the large GTs blocker D,L-threo-beta-hydroxyaspartate (THA) and DHK decreases EPSC amplitude, while if co-applied with cyclothiazide (a compound decreasing AMPA receptor desensitization) they enhanced this parameter. The use of different GT inhibitors, the anatomofunctional differences between these structures, and the heterogeneity of experimental conditions and approaches, might in part explain such variability. For this reason, it was difficult for us to make any prevision on the results we could obtain in the striatum and to compare our data with those already present in the literature. However, we believe we obtained enough evidence that GTs play a role in corticostriatal synaptic transmission. Mechanisms of GT-mediated regulation of striatal synaptic transmission The present work in the striatum clearly shows that GT blockade significantly enhances EPSCs kinetics. This is in line with the abovementioned results obtained in the hippocampus (Tsukada et al., 2005) and in the cerebellum (Takahashi et al., 1995; Takatsuru et al., 2006). Regarding its lack of effect on EPSC amplitude, a possible mechanism due to receptor saturation can be excluded because TBOA was ineffective on this parameter also in the presence of kynurenic acid. However, it should be considered

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that Campbell and Hablitz (2004) also found no effect of TBOA on EPSC amplitude in the rat neocortex. In light of these reports, our lack of effect on EPSC amplitude is not surprising and is in line with literature data. It can thus be assumed that the blockade of both neuronal and glial GTs, by increasing the permanence and/or concentration of glutamate at corticostriatal synapses, enhances the charge transfer and kinetic (␶w) of the EPSC, but not its amplitude. It has been shown that GTs are voltage-dependent and that at depolarized potentials they are inhibited (Wadiche et al., 1995). This property can be used as an experimental tool for selectively blocking neuronal GTs, by voltageclamping the recorded neuron at depolarized potentials (Diamond, 2001). Thus, we can assume that the effects of TBOA we measured at ⫹40 mV were mainly due to the blockade of glial GTs, since the effect of this drug on neuronal GTs was occluded by depolarization/blockade. Our data suggest that glial GTs, i.e. GLAST and GLT-1, do not play a significant role in shaping EPSC kinetics at corticostriatal synapses, although blocking glial GTs has been reported to affect both AMPA and NMDA EPSC in the hippocampus (Tsukada et al., 2005). An alternative hypothesis could be that blocking only glial GTs is not sufficient to significantly affect EPSC kinetics in the striatum, i.e. the blockade of both neuronal and glial GTs is required to obtain a significant effect. However, we show here that glial GTs may play a role in limiting the diffusion of glutamate when corticostriatal synapses are engaged in tetanic activity by HFS, a condition in which synaptic spillover may occur (Marcaggi et al., 2003; Tzingounis et al., 2007). In these conditions, however, it cannot be excluded that neuronal GTs located in the neurons surrounding the recorded one (both at ⫺60 and ⫹40 mV) may participate in glutamate uptake during HFS. The EPSC inhibition obtained by DHK shown here was quite puzzling, although already described by Kidd and Isaac (2000). It could be due to AMPA receptor desensitization, as suggested by these authors. However, the fact that we did not observe any AMPA receptor desensitization with TBOA does not support this hypothesis. Moreover, the increase in PPR induced by DHK suggests that a presynaptic mechanism can be implicated in the inhibitory action of this drug. As suggested in previous papers, this inhibitory effect of DHK could be related to its action as a ligand of ionotropic glutamate receptors (Sarantis et al., 1993; Kidd and Isaac, 2000). For example, it might act on kainate receptors, which are known to indirectly down-regulate striatal synaptic transmission with a possible presynaptic mechanism (Chergui et al., 2000). Although this hypothesis is supported by the fact that these receptors are localized presynaptically (in the monkey striatum, see Kieval et al., 2001), more studies are required to fully address these issues. However, what our data clearly suggest is that DHK might have an effect on glutamate transmission other than its role as GLT-1 blocker, but elucidating these mechanisms goes far beyond the goal of this work. Finally, since GT inhibition likely increases glutamate concentration at corticostriatal synapses, this can result in the activation of mGluRs that can, in turn, modulate the

effect of TBOA by affecting glutamate release and neuronal excitability (Gubellini et al., 2004). For example, such mGluRs-GTs interaction has been described in the supraoptic nucleus (Oliet et al., 2001). However, in conditions in which all mGluRs were blocked, we observed no significant changes in TBOA effects, ruling out that such interaction might modulate the effects of TBOA in the striatum.

CONCLUSION What can be concluded from our work is that EAAC1, rather than GLAST and GLT-1, plays a significant role in shaping the kinetic of corticostriatal EPSCs recorded from striatal MSNs in the adult rat. Conversely, GLAST and GLT-1 seem more involved in limiting glutamate diffusion during HFS. These results open new perspectives in the study of striatal synaptic plasticity, and provide novel insights for studying the role of GTs in neuropathologies associated to excitotoxicity and/or hyperactivity of striatal glutamate transmission, such as PD and amyotrophic lateral sclerosis (Carlsson and Carlsson, 1990; Choi, 1992; Rothstein et al., 1992; Chapman, 2000; Danysz et al., 2000; Gubellini et al., 2006). Acknowledgments—P.G. wishes to thank Dr. F. Tell and Dr. M. Crest (ITIS, UMR6150 CNRS, IFR Jean Roche, Faculté de Médecine de Marseille) for their kind support, and Dr. M. F. Pozza (Novartis Neuroscience, NIBR, Basel, Switzerland) for his kind help in putting together part of the experimental setup. This work was financed by grants from: the CNRS, the CEA, the Fondation de France (project “Pathogenie de la maladie de Parkinson,” Engt. 2005-013853 attributed to P.G.) and the French Ministry of Education and Research (project ANR-05-NEUR-021 “mGluRs Park”).

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(Accepted 11 November 2008) (Available online 17 November 2008)