Neuroprotective potential of mGluR5 antagonist MTEP: effects on kainate-induced excitotoxicity in the rat hippocampus

Pharmacological Reports 2010, 62, 1051–1061 ISSN 1734-1140

Copyright © 2010 by Institute of Pharmacology Polish Academy of Sciences

Neuroprotective potential of mGluR5 antagonist MTEP: effects on kainate-induced excitotoxicity in the rat hippocampus Helena Domin1, Barbara Ziêba1, Krystyna Go³embiowska2, Magdalena Kowalska2, Anna Dziubina2, Maria Œmia³owska1 

Department of Neurobiology, Department of Pharmacology, Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12, PL 31-343 Kraków, Poland Correspondence: Helena Domin, e-mail: [email protected]

Abstract: Extensive research into glutamate receptors in the central nervous system has shown important role of metabotropic glutamate receptors (mGluR) as potential targets for neuroprotective drugs. The aim of the present study was to investigate neuroprotective potential of the highly selective mGlu5 antagonist 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (MTEP) against kainate (KA)-induced excitotoxicity in vivo. Our attention was focused mainly on the effectiveness of delayed treatment. In order to evoke neuronal injury, rats were unilaterally injected with kainic acid (KA; 2.5 nmol/1 μl) into the CA1 region of the hippocampus. MTEP (1, 5 or 10 nmol/1 μl) was administered into CA1 30 min, 1, 3 and 6 h after KA. Additionally, other rats were injected intraperitoneally (ip) with MTEP in a dose of 1 mg/kg, once daily for 7 days. The first injection of MTEP was 1 h after KA. Seven days after treatment, the brains were taken out and analyzed histologically to estimate the total number of neurons in CA region of dorsal hippocampus using stereological methods. The study was also aimed at determining a possible influence of MTEP on neuronal glutamate release induced by KA in the hippocampus, using microdialysis method. The obtained results showed that MTEP had neuroprotective effect after both intrahippocampal and intraperitoneal injection. It was found that MTEP could prevent excitotoxic neuronal damage even when it was applied 1–6 h after the toxin. Moreover, it was observed that MTEP significantly reduced the KA-induced glutamate release in the hippocampus. It seems to play a role in mediating neuroprotective effects of MTEP. Key words: 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (MTEP), mGlu5 antagonist, kainic acid, hippocampus, excitotoxicity, neuroprotection

Abbreviations: AMPA – a-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid, iGluRs – ionotropic glutamate receptors, KA – kainic acid, mGluRs – metabotropic glutamate receptors, MTEP – (3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine), NMDA – N-methyl-D-aspartate

Introduction It is generally assumed that excitotoxicity is the main mechanism of neurodegeneration in mammalian brain

[15, 47, 48]. Excitotoxicity occurs when excitatory neurotransmission is too high to be balanced by inhibitory transmission. The main excitatory neurotransmitter, glutamate, activates ionotropic glutamate receptors (iGluRs) (NMDA, AMPA and KA) and metabotropic glutamate receptors (mGluRs). Overactivation of iGluRs leads to neuronal death and may induce neurodegeneration, hence an inhibition of the toxic glutamatergic hyperactivity may induce neuroprotection. Indeed, it was found that NMDA or AMPA receptor antagonists had demonstrated neuroprotective Pharmacological Reports, 2010, 62, 1051–1061

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effects in vitro and in animal experiments; however, occurrence of many adverse actions such as ataxia, sedation, psychotic effects and memory loss makes them useless in the clinic [20, 30, 41]. Therefore, it seems that modulation of glutamatergic transmission may be more promising strategy of neuroprotection than a direct iGlu receptor blockade. Such modulation may be achieved by interaction with metabotropic glutamate receptors. The mGluRs constitute a family of eight subtypes, subdivided into three groups on the basis of sequence homology, pharmacological profile and transduction pathway. Group I mGluRs comprise mGluR1 and mGluR5 and are positively coupled with phosphoinositide hydrolysis, activation of phospholipase C, protein kinase C and calcium release [16]. Group II (containing mGluR2 and mGluR3) and group III mGluRs (including mGluR4, –6, –7 and –8) are negatively coupled to adenylate cyclase. Group II and III mGlu receptors are situated mainly presynaptically and their activation leads to the feedback inhibition of glutamate release through the inhibition of voltage-dependent calcium entry into the cell. Group I mGlu receptors are situated both presynaptically [39, 42] and postsynaptically [33, 59–61]. Some evidence indicates that stimulation of group I mGluRs potentiates neuronal excitation which in turn may enhance neurotoxicity [22, 55]. Contrariwise, pharmacological blockade of those receptors was found to produce neuroprotection [12, 25, 34, 45, 54]. However, the role of group I mGluRs in neuroprotection still remains controversial, as it has been reported that activation of these receptors may attenuate neuronal cell death [1, 6, 13, 44]. In our previous study, we found that the selective mGluR5 antagonist MTEP produced neuroprotective action against KA-induced excitotoxic damage in cortical and hippocampal neuronal cultures. Moreover, such neuroprotection was observed when MTEP was added not only before, but also 30 min to 6 h after starting intoxication [21]. Those findings seemed to be particularly valuable from the clinical viewpoint as they offered a chance of therapeutic use of MTEP or similar compounds in patients with excitotoxic damage, in whom neuroprotective treatment could be applied some time after damage. Therefore, in the present study we tried to determine whether MTEP may have neuroprotective effects in vivo, and particularly after delayed treatment. Similarly as in our earlier studies, we induced neurotoxicity using KA, which administration is generally

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assumed to be a good model of excitotoxicity, in which the secondary release of endogenous glutamate plays a significant role. Taking this into consideration, we additionally studied the effect of MTEP on KAinduced glutamate release in the hippocampus using microdialysis method in freely moving rats.

Materials and Methods In vivo studies: KA-induced excitotoxicity in rat hippocampus Animals

Male Wistar rats weighing about 250–300 g were used for the experiments. The rats were age-matched and were housed six to a cage on a 12:12 light-dark cycle, with free access to food and tap water. The rats after cannulae implantation were housed singly. During the experiment, all efforts were made to minimize animal suffering and to reduce the number of animals used, in accordance with the Local Bioethical Commission Guide for the Care and Use of Laboratory Animals. Cannulae implantation

The rats were anesthetized with equithesin [a mixture of chloral hydrate (POCH S.A. Poland), magnesium sulfate (POCH S.A. Poland) and pentobarbital sodium (Sigma)] and were stereotaxically, bilaterally implanted with chronic quide cannulae aimed at the dorsal hippocampus CA1 region. The guide cannulae (23-gauge stainless steel tubing), secured by dental cement, were anchored to the skull by three stainless steel screws. In order to prevent clogging, stainless steel stylets were placed in the guide cannulae and left until the animals were microinjected. Drug treatments

Seven days after cannulae implantation, the rats were unilaterally microinjected with KA (Tocris, USA) into the dorsal hippocampus CA1 region (coordinates: A = +5.7 mm, L = ±2.1 mm, H = +7.2 mm from the interaural line, according to the Paxinos and Watson stereotaxic atlas [53]. KA was freshly dissolved in 0.1 M phosphate buffer, pH 7.4, and was injected in a dose of

Neuroprotective effect of MTEP in rat hippocampus

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2.5 nmol/1 μl. The dose and place of KA microinjection was chosen on the basis of our earlier study as not producing behavioral seizures [66]. Some rats were additionally injected, through the same cannulae, with 3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]-pyridine (MTEP; Ascent Scientific, England), dissolved in redistilled water, in doses of 1, 5 or 10 nmol/1 μl into the CA1 region, 30 min, 1, 3 or 6 h after KA. The contralateral hippocampus of each rat was microinjected with a phosphate buffer and redistilled water as a control for KA and MTEP, respectively, and was used as a control side. The doses of MTEP were chosen on the grounds of our earlier pilot studies as well as previous studies of some authors using mGluR5 antagonist MPEP [10]. Another group of rats were administered intraperitoneally (ip) with MTEP in a dose of 1 mg/kg once daily for 7 days. The first injection was made 1 h after KA. Each day MTEP was suspended in a 1% aqueous solution of Tween 80. The dose and time schedule of treatment were chosen on the basis of our pilot studies and earlier studies of other authors [49, 52], in which authors showed antiparkinsonian-like and antidepressant-like effects of MTEP, respectively. Seven days after last ip injection of MTEP, the rats were sacrificed and their brains were histologically examined. Evaluation of damage and protection in CA region of hippocampus Tissue preparation and histology

Seven days after treatment, the rats were killed by an overdose of pentobarbital, their brains were removed, fixed in cold, buffered 4% paraformaldehyde for 7 days, and were then immersed in a buffered 20% sucrose solution for at least 5 days at 4°C. The brains were then frozen on dry ice, and 30 μm coronal sections were cut at levels containing the dorsal hippocampus (between bregma –2.12 to –4.30 mm, according to the Paxinos and Watson atlas [53]. The sections were mounted on glass slides, dried, stained with cresyl violet, cover-slipped with Permount, and were used for verification of the injection site and for a histological analysis of the lesion. Stereology

The total number of neurons in the pyramidal layer of the CA of the dorsal hippocampus was evaluated by

stereological counting as described previously [65]. The procedures were performed using a microscope (Leica, DMLB; Leica, Denmark) equipped with a projecting camera and a microscope stage connected to an xyz stepper (PRIOR ProScan) controlled by a computer using the Olympus Denmark CAST2 software, as described previously [50, 51, 65]. Systemic uniform random sampling was used to choose the sections. The first sampling item was randomly taken from the frontal part of the dorsal hippocampus, and all the following sampling items were taken at a fixed distance from the previous one. At least 10–12 sections through the entire length of the dorsal hippocampus were sampled. The total number of cells (N) in the pyramidal layer of the hippocampal CA region was estimated by measuring the reference volume (Vref; the area that contains the population of the cells) and the numerical density (Nv) of the cells within the Vref: N = VHAB × NL The pyramidal layer of the dorsal hippocampus CA region was outlined at a lower magnification (5×). CAST2 software provides templates of points in various arrays used in point counting for reference volume estimation. The VHAB value was determined by using point counting methods and applying Cavalieri’s principle [29] according to the formula: VHAB = Spi × A(pi) × t where Spi is the sum of the number of points (pi) counted, A(pi) is the area associated with each point, and t is the known distance between sections. The area of the counting frame was A(fr) = 3382 μm . For determination of the density of cells in the hippocampal CA region, the computer software generated a random selection of sites within the outlined area, from which the density was determined under higher magnification (63×). Cell density (Nv) was estimated by using the optical dissector method according to the formula: Nv = SQ / SP × v(dis) where SQ is the sum of cells counted from all the dissector frames, SP is the total number of all the dissector points, and v(dis) is the total volume of the dissector. Pharmacological Reports, 2010, 62, 1051–1061

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Statistical analysis

Statistical analysis was carried out using GraphPad Prism 4.00 software. Differences between the control (contralateral) and KA-lesioned hippocampi (ipsilateral) were compared by a paired two-tailed t-test. Differences between KA-lesioned and KA + MTEP treated hippocampi were compared by an unpaired two-tailed t-test; p-value less than 0.05 was considered statistically significant. In vivo microdialysis studies

The rats were anesthetized with ketamine (75 mg/kg, im) and xylazine (10 mg/kg im) and placed in a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA). The skull was exposed and small holes were drilled for the insertion of the vertical microdialysis probes in the dorsal hippocampus using the following coordinates: AP = –3.3 mm anterior from the bregma; L = +2.2 mm lateral from the sagittal suture; and H = –4.0 mm ventral from the dura surface according to the Paxinos and Watson stereotaxic atlas [53]. Microdialysis probes were constructed as described in detail elsewhere [26, 27]. One day after the surgery and probe implantation, the inlet of the dialysis probes was connected to a syringe pump (BAS, IN, USA) which delivered an artificial cerebrospinal fluid (aCSF) composed of (in mM): NaCl 147, KCl 4, CaCl 2.2; pH = 7.4 at a flow rate of 1.5 μl/min. After 2 h of washing period, when the extracellular level of neurotransmitters became stable, 4 baseline samples were collected every 20 min. Next, freshly prepared solutions of KA (50 μM) in aCSF or MTEP (50 μM) or KA + MTEP (MTEP was administered simultaneously with kainate) were perfused locally through a microdialysis probe for 20 min. Then, the perfusion fluids were switched back to aCSF for two additional collection periods (dialyzate fractions were collected every 20 min). At the end of the experiment the rats were killed and their brains were examined histologically to validate the correct probe placement. The samples were analyzed by HPLC with VIS detection. Analytical procedure

Glutamate was measured in dialyzates (20 μl) after derivatization with 4-dimethylaminoazobenzene-4´sulfonyl chloride (DABS-Cl) at 70°C for 12 min, according to Knecht and Chang [32]. Dabsylated amino

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acids were separated on an Ultrasphere ODS (4.6 × 150 mm, 3 μm) column (Supelco, Poland) by gradient elution, with solvent A (10 mM citric acid, 4% dimethylformamide) and solvent B (acetonitrile). Dabsylated compounds were detected by measuring an absorbance at 436 nm using Beckman Amino Acid System Gold with VIS detection. Statistical analysis

The statistical significance of microdialysis data was calculated using a one-way ANOVA for repeated measurements, followed by Tukey’s post-hoc test. The results were considered statistically significant when p < 0.05.

Results Effect of KA microinjection into the CA1

KA injected unilaterarally in a dose of 2.5 nmol into the CA1 region of the dorsal hippocampus induced extensive degeneration of CA pyramidal neurons (Fig. 2A). Stereological counting showed a strong, ca. 50% reduction in the number of neurons in the pyramidal layer of the ipsilateral dorsal hippocampus in comparison to the contralateral side [t(5) = 10.45, p = 0.0001, Fig. 1]. Behavioral observations did not show any generalized seizures after KA, only face twitching and sometimes movements of forelimbs were seen. Effect of MTEP after intrahippocampal injection

Cresyl violet staining showed that the extent of lesions in the CA pyramidal layer was significantly smaller in animals treated with the mGluR5 antagonist MTEP (Fig. 1 and Fig. 2C, D). The effects of MTEP were dose- and time-dependent. MTEP administered 30 min after KA at doses of 10 or 5 nmol/rat caused a significant increase in the number of neurons (an increase by 88% and 66%, respectively to dose) [t(9) = 5.866, p = 0.0002 and t(9) = 3.520, p = 0.00065, respectively] in comparison with KA-lesioned hippocampi (Fig. 1A). MTEP used in dose of 1 nmol/rat did not induce any significant protection effect (only a tendency towards an increase in the number of neu-

Neuroprotective effect of MTEP in rat hippocampus

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Fig. 1. Effects of intrahippocampal injections of KA (2.5 nmol/1 μl) and KA followed by MTEP after intrahippocampal injection on the number of neurons in the pyramidal layer of CA regions. The results of stereological counting showed that KA induced extensive neurodegeneration (50% loss) of CA pyramidal neurons. MTEP given 30 min (A) or 1 h (B) after KA caused a significant neuroprotection at doses of 5 and 10 nmol. MTEP administered 3 h (C) and 6 h (D) after KA significantly attenuated the neuronal damage only at dose of 10 nmol. Each bar represents the mean ± SEM of n = 6 per group. *** p < 0.001 contralateral side vs. KA (ipsilateral);  p < 0.05,  p < 0.01 and  p < 0.001 KA lesioned (ipsilateral) vs. KA + MTEP (ipsilateral) hippocampi

rons was observed) [t(9) = 2.262, p = 0.0500, Fig. 1A)]. A diminution of degeneration was also observed when MTEP at doses of 10 or 5 nmol/rat was injected intrahippocampally 1 h after KA. The results of stereological counting showed the significant increase in the number of living neurons in CA pyramidal layer of the ipsilateral dorsal hippocampus (increase by 55% and 33%, respectively to dose) [t(10) = 3.931, p = 0.0028 and t(10) = 2.303, p = 0.0441, respectively] in comparison to the side with KA alone (Fig. 1B). MTEP given 3 or 6 h after KA induced a neuroprotective effect only when given in a dose of 10 nmol/rat (the number of living neurons significantly increased by 49% and 37%, respectively) [t(9) = 2.801, p = 0.0207 and t(9) = 2.356, p = 0.0429, respectively] in comparison with KA-lesioned hippocampi (Fig. 1C, D).

pyramidal layer by 74% [t(9) = 2.924, p = 0.0169] in comparison with KA-lesioned hippocampi (Fig. 3). These results suggest a neuroprotective effects of mGluR5 antagonist MTEP.

A

C

B

KA KA ipsi ipsi

MTEP MTEP 1 1hhafter after KA KA ipsi ipsi

D

contra contra

MTEP MTEP 6 6hhafter after KAKA ipsi ipsi

Effect of MTEP after peripheral injections

Intraperitoneal administration of MTEP, for 7 days, at dose 1 mg/kg/daily, when the first injection was 1 h after KA, significantly attenuated the neuronal damage. The result of stereological counting showed a significant increase in the number of neurons in CA

Fig. 2. Microphotographs of coronal sections of rat brain hippocampi stained with cresyl violet. Arrows indicate a CA pyramidal layer where the neurons were counted. Loss of neurons and extensive gliosis can be seen in the hippocampus after KA microinjection (2.5 nmol/1 μl) (A) in comparison with the non-degenerated contralateral side (B), when only a small glial scar in the place of injection is seen. Neuroprotective effects of MTEP given 1 h after KA or even so late as 6 h after KA are shown in C and D, respectively. Calibration bars, 200 μm

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A

after MTEP i.p. ip . 1h 1 hafter KA ipsi

B

MTEP ip 1 h after KA

Fig. 3. Neuroprotective effects of MTEP injected intraperitoneally, 1 mg/kg, for 7 days, with the first injection given 1 h after KA. (A) Microphotograph of the coronal section of the rat brain hippocampus stained with cresyl violet. Arrows indicate a CA pyramidal layer where the neurons were counted. Diminution of the lesion in the CA pyramidal layer is clearly visible in comparison with the KA injected rat (Fig. 2A). (B) The results of stereological counting – MTEP induced a significant increase in the number of neurons in comparison with kainate-treated rats. Each bar represents the mean ± SEM of n = 6 per group. *** p < 0.001 contralateral side vs. KA (ipsilateral);  p < 0.05 KA lesioned (ipsilateral) vs. KA + MTEP (ipsilateral) hippocampi. Calibration bars, 200 μm

7.54, p < 0.02, respectively] (Fig. 4). MTEP (50 μM) infused throughout microdialysis probe together with KA for 20 min significantly decreased the extracellular glutamate level induced by KA to ca. 120% of basal level. The difference between both groups was statistically significant at 20, 40 and 60 min after administration [F(1,15) = 6.15, p < 0.02; F(1,15) = 5.34, p < 0.03; F(1,15) = 6.04, p < 0.03, respectively] (Fig. 4). Perfusion of MTEP (50 μM) alone had no effect on extracellular glutamate level in hippocampus. The difference between groups treated with MTEP alone and combination of KA and MTEP was not statistically significant. Fig. 4. Effect of MTEP on increase in extracellular glutamate level induced by KA in the rat hippocampus. KA (50 μM) and MTEP (50 μM) were given through microdialysis probe as indicated with an arrow. Duration of the treatment is shown by a horizontal line. Values express % of basal level and are the mean ± SEM of 6–11 animals per group. Basal extracellular glutamate levels were (in μM): 1.67 ± 0.21, 1.83 ± 0.08, 2.10 ± 0.14 in groups treated with KA, KA + MTEP and MTEP and control, respectively. + p < 0.05 shows difference between KA and KA plus MTEP; * p < 0.05, ** p < 0.003 KA vs. control level

Effect of MTEP on the glutamate release induced by kainate in rat hippocampus: in vivo microdialysis study

KA (50 μM) applied locally through a microdialysis probe for 20 min induced a significant increase in extracellular glutamate level in hippocampal dialyzates, reaching values of 159, 158 and 140% of the baseline at 20, 40 and 60 min after administration [F(1,13) = 13.46, p < 0.003; F(1,13) = 13.16, p < 0.003; F(1,13) = 1056

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Discussion The present results demonstrate that MTEP, a selective mGluR5 antagonist has neuroprotective effect in vivo against excitotoxicity induced by intrahippocampal administration of KA. The neuroprotection was found after both intrahippocampal MTEP microinjection and intraperitoneal injections. Our earlier results showed neuroprotective effect of MTEP against KA-induced excitotoxic damage in primary cultures of mouse cortical and hippocampal neurons [21]. Therefore, our previous and present results indicate that neuroprotective activity of this compound is not restricted to an in vitro model, but may also occur in vivo, which seems to be particularly important to a possible future use in the clinic.

Neuroprotective effect of MTEP in rat hippocampus

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So far, there are few studies of other authors on the neuroprotective action of MTEP. Neuroprotective effect of MTEP in the animal model was previously shown in vitro by Lea et al. [35] whereas in in vivo model by Makarewicz et al. [36] and Szyd³owska et al. [64]. Moreover, many studies have demonstrated neuroprotective action of another selective mGluR5 antagonist, MPEP, both in vitro [7, 10, 35, 40, 46, 57, 64] and in vivo [6, 7, 10, 28, 31, 40, 57, 58, 70]. In our experiment we decided to use MTEP, as it seemed to be more specific and free from disadvantages described for MPEP [18]. It was also found that MTEP had better solubility and CNS penetrability than MPEP [18]. Moreover, some authors suggested the role of nonspecific blockade of NMDA receptors by MPEP in its neuroprotective effects [40, 46]. Both in vitro and in vivo characterization of MTEP indicates that the drug has no effect on other mGluR subtypes nor does it influence NMDA, AMPA or KA receptors [17, 18]. Summing up, all these results suggest that MTEP shows greater selectivity for mGluR5 than other known antagonists [2, 3, 17, 18]. A particularly important finding of our study is that MTEP may be neuroprotective even if it is applied 1–6 h after the onset of exposure to KA. The effectiveness of delayed treatment with MTEP against KA excitotoxicity is in line with our earlier findings on neuronal cultures, which revealed for the first time that MTEP may be neuroprotective given as late as 6 h after the onset of KA treatment [21]. Earlier in vitro studies by other authors [35] also showed that MTEP inhibited neurotoxic damage in primary cortical neuronal cultures, however, they did not investigate possibility of delayed treatment. The effectiveness of delayed treatment with MTEP was described by Makarewicz et al. [36] and Szyd³owska et al. [64], but the treatment was not as delayed as in our experiments. In our present in vivo study, the effectiveness of mGluR5 antagonist MTEP after delayed treatment was examined particularly after intrahippocampal microinjection. Our results showed for the first time the neuroprotective effect of MTEP injected as late as 6 h after KA, although that effect was considerably weaker than after less delayed injections (3 h, 1 h or 30 min after KA). A separate group of rats were treated with MTEP ip, since such a mode of treatment seemed to be more comparable to situation in the clinic. The delay of the first MTEP injection was set at 1 h after KA, as that time was very effective both in vitro [21] and in vivo

after intrahippocampal injection. It was found that ip administration of MTEP at the dose of 1 mg/kg (this dose blocks approximately 50% of mGluR5 receptors [11]) for 7 days, caused significant increase in the number of surviving neurons in CA pyramidal layer of hippocamus by ca. 74% in comparison to the KAlesioned hippocampi. On the basis of our results, we suppose that the effectiveness of delayed peripheral or intracerebral treatment may indicate a possibility of potential therapeutic use of similar compounds in patients to whom such neuroprotective treatment can be given a few hours after damage. As mentioned above, the neuroprotective effect of MTEP injected intraperitoneally was also described by some other authors, but they used different models of injury (usually ischemia and/or hypoxia), a different time schedule, higher doses of MTEP and a shorter delay [36, 64]. The aim of the present study was also to elucidate the mechanism responsible for the neuroprotective effect of MTEP. It is commonly known that KA excitotoxicity occurs mainly by excessive glutamate release [23, 24, 37]. Therefore, we tried to investigate possible influence of MTEP on KA-induced glutamate release in rat hippocampus using a microdialysis method. Our results have shown that, indeed MTEP strongly and significantly diminished the KA-induced increase in glutamate release almost to the basal level (see Fig. 4). It seems to play some role in mediating neuroprotective effect of MTEP. It is the first finding of this kind, because to date no other authors have studied the influence of MTEP on neuronal glutamate release. However, some earlier studies by other authors showed that another mGluR5 antagonist, MPEP, inhibited glutamate release by blocking presynaptically located mGlu5 receptors [57, 67, 68]. All these findings provide evidence for the role of inhibition of mGluR5-type receptors in the regulation of neuronal glutamate release in the central nervous system. In our study we investigated the neuroprotective effect of MTEP in the hippocampus. It has been reported that mGlu5 receptors are highly expressed in the hippocampus, especially in CA1 and CA3 regions, located both postsynaptically [33, 59–61] and presynaptically [39, 42]. Moreover, the hippocampal CA1 and CA3 regions in the rat show a high density of KA receptors, hence pyramidal neurons in hippocampal CA fields seem to be particularly sensitive to KA-induced neuronal excitation [19, 37, 43]. A lot of

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evidence indicates that excitotoxicity induced by KA involves activation of presynaptic KA receptors located on glutamatergic terminals in the hippocampus, thus causing the excessive release of glutamate [14, 23, 24] and dysregulation of Ca2+ homeostasis [4]. A critical signal of glutamate release from presynaptic terminals is the influx of Ca2+ through voltagedependent and ligand-gated channels in the plasma membrane [38]. Because activation of mGlu5 receptors evokes an increase in intracellular Ca2+ as a result of inositol trisphosphate formation and protein kinase C activation [16], our experiment suggests that the inhibition of presynaptic mGlu5 receptors by MTEP may prevent the influx of Ca2+ into presynaptic glutamatergic terminals and may thus inhibit the calcium ion concentration dependent release of glutamate [63]. While discussing the potential neuroprotective mechanism of the mGluR5 antagonist MTEP, it is noteworthy that mGluR5s are also located postsynaptically, and that their activation leads to enhance NMDA-dependent effects [5, 56, 69] and contributes to the development of excitotoxic neuronal death [8–10, 16]. Therefore, it is likely that application of the mGluR5 antagonist may indirectly lead to attenuation of postsynaptic NMDA function, thereby causing an effect similar to that obtained by using an antagonist of this receptor [62]. Therefore, on the basis of the above findings we suppose that the blockade of mGlu5 receptors by MTEP in our experiment may lead to reduction of glutamatergic transmission, which contributes to its neuroprotective effect in rat hippocampus.

Conclusions In conclusion, the present results demonstrate a strong significant neuroprotective potential of mGluR5 antagonist MTEP against KA-induced excitotoxicity in an in vivo model. In addition, our study shows for the first time that MTEP significantly reduces the KAinduced glutamate release, which may be one of the main mechanisms of neuroprotective action of MTEP. Our results suggest that mGluR5 antagonists could be suitable neuroprotective drugs, since they appear to attenuate excitotoxicity without completely blocking fast excitatory transmission. Moreover, it seems of crucial importance that MTEP may be effective even 1058

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when it is applied 1–6 h after the toxin. The effectiveness of such a late treatment may raise hopes for a potential therapeutic use of similar compounds in patients to whom neuroprotective treatment can be administered a few hours after damage.

Acknowledgments: The study was supported by the KBN Grant no. 2P05A 114 28, and also by funds for the statutory activity of the Institute of Pharmacology, Polish Academy of Sciences, Kraków, Poland, and MS&HE scientific network 28/E-32/SN-0053/2007.

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Received: September 14, 2010; in the revised form: November 19, 2010.

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