Group II metabotropic glutamate receptor activation by agonist LY379268 treatment increases the expression of brain derived neurotrophic factor in the mouse brain

Neuroscience 165 (2010) 863– 873

GROUP II METABOTROPIC GLUTAMATE RECEPTOR ACTIVATION BY AGONIST LY379268 TREATMENT INCREASES THE EXPRESSION OF BRAIN DERIVED NEUROTROPHIC FACTOR IN THE MOUSE BRAIN V. DI LIBERTO,1 A. BONOMO,1 M. FRINCHI, N. BELLUARDO AND G. MUDÒ*

tribution. Many studies have demonstrated that activation of these receptors can result in neuroprotection (Flor et al., 2002). Particular attention has been focused on group-II mGlu receptors, which include mGlu2 and mGlu3 (mGlu2/3) receptors (Emile et al., 1996; Flor et al., 1995), for their established role on neuroprotection (Bruno et al., 2001) and for the recent availability of potent and selective ligands (Schoepp et al., 1999). A number of in vitro studies indicated a neuroprotective role for mGlu2/3 receptors in various models of neuronal injury, such as against glutamate-mediated neurodegeneration (Nicoletti et al., 1996; O’Neill, 2001) and against beta-amyloid–induced toxicity (Copani et al., 1995). mGlu2/3 agonists are also reported to be neuroprotective in several in vivo models of brain insults (Bond et al., 2000; Chiamulera et al., 1992; Henrich-Noack et al., 1998; Miyamoto et al., 1997; Poli et al., 2003). For example treatment with mGlu2/3 receptor agonist LY379268, which display a high specificity for mGlu2/3 receptors (Schoepp et al., 1999), was found to be protective against hypoxic brain damage in a goldfish anoxia model, whereas application of mGlu2/3 receptor antagonist LY341495 significantly exacerbated hypoxia-induced injury in the same system (Poli et al., 2003). Systemic treatment with LY379268 has been found to reduce neurodegeneration in corpus striatum and substantia nigra in a mouse model of Parkinson’s disease or in brain trauma (Corti et al., 2007; Movsesyan and Faden, 2006) and to induce neuroprotection in a model of global ischemia (Bond et al., 2000). In contrast to a growing data on mGlur2/3 neuroprotective role no clear information are available on the mechanism involved. In this context mGlu2/3 receptors, negatively coupled to adenylyl cyclase, are thought to act as presynaptic autoreceptors regulating glutamate transmission (Cartmell and Schoepp, 2000; Shigemoto et al., 1997) and therefore to limit the neuronal excitotoxicity by inhibiting glutamate release (Battaglia et al., 1997; Cozzi et al., 1997). Although a reduction of glutamate release is an attractive hypothesis, there is a body of evidence, especially in vitro, suggesting that a regulation of glutamate release may not always account for the observed neuroprotective activity of mGlu2/3 receptor agonists. An interplay among neurotrophic factors and mGlu2/3 receptors signalling system has been suggested in view of neurotrophic factors key role on neuroprotection. According to this, activation of mGlu2/3 receptors present in neurons or astrocytes or oligodendroglia might stimulate the production of neurotrophic factors, such as transforming-growth factor-␤1 (TGF-␤1) (Bruno et al., 1998; D’Onofrio et al.,

Department of Experimental Medicine, Division of Human Physiology, Laboratory of Molecular Neurobiology, University of Palermo, Corso Tukory 129, 90134 Palermo, Italy

Abstract—A number of in vitro and in vivo studies using selective agonists have indicated a neuroprotective role for group-II metabotropic glutamate (mGlu2/3) receptors in various models of neuronal injury. Although an interplay among neurotrophic factors and mGlu2/3 receptors signalling system has been suggested as possible mechanism involved on neuroprotection, at present poor information are available concerning the in vivo regulation by mGlu2/3 receptors activation of specific neurotrophic factors. By using in situ hybridization and western blotting methods the aim of present study was to analyse the potential regulatory role of selective mGluR2/3 agonist LY379268 treatment on brain derived neurotrophic factor (BDNF) expression in the mouse brain. The treatment with LY379268 evidenced a significant upregulation of BDNF mRNA levels in the cerebral cortex and in the hippocampal formation with a peak at 3 h from treatment and its disappearance already at 6 h from treatment. An analysis of dose-effect curve revealed that LY379268 may significantly enhance BDNF mRNA expression already at dose of 0.250 mg/kg b.w. The upregulation of BDNF mRNA expression was followed by a significant increase of BDNF protein levels at 24 h from LY379268 treatment. These effects of LY379268 treatment on BDNF expression were restricted to neuronal cells and were blocked by the new selective mGlu2/3 receptor antagonist LY341495, suggesting a receptor specificity. Taken together these findings suggest that several previous observed neuroprotective and trophic actions of mGluR2/3 agonists treatment may be mediated, at least in the cerebral cortex and hippocampal formation, by upregulation of BDNF expression. © 2010 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: LY379268, mGlu2/3 receptors, BDNF, neurotrophic factors, hippocampus, cerebral cortex.

Metabotropic glutamate (mGlu) receptors are a large family of receptor subtypes with diverse properties in terms of transduction coupling, pharmacology, and anatomical dis1 V. Di Liberto and A. Bonomo contributed equally to this work. *Corresponding author. Tel: ⫹39-091-6555849; fax: ⫹39-091-6555854. E-mail address: [email protected] (G. Mudò). Abbreviations: BDNF, brain-derived neurotrophic factor; GDNF, glial cell line-derived neurotrophic factor; LY379268, (⫺)-2-Oxa-4aminobicyclo[3.1.0]hexane-4,6-dicarboxylic acid; mGlu, metabotropic glutamate; NeuN, neuronal marker neuron-specific DNA-binding protein; NGF, nerve growth factor; OD, optical density; PBS, phosphate-buffered saline; PLSD, Protected Last Significant Difference; TGF-␤1, transforming-growth factor-␤1.

0306-4522/10 $ - see front matter © 2010 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2009.11.012

863

864

V. Di Liberto et al. / Neuroscience 165 (2010) 863– 873

2001), nerve growth factor (NGF) (Ciccarelli et al., 1999), brain derived neurotrophic factor (BDNF) (Matarredona et al., 2001), and very recently glial cell line-derived neurotrophic factor (GDNF) (Battaglia et al., 2009). However, among these findings only the induction of TGF-␤1 and GDNF have been demonstrated in the brain by systemic injection of mGlu2/3 receptors agonist. In fact, the NGF was found to be regulated in cultured astrocytes and BDNF was upregulated after in vivo local injection of mGlu2/3 receptors agonist. In addition all these neurotrophic factors, with the exclusion of GDNF, were found upregulated in glial cells and not in neuronal cells, which express mGlu2/3 receptors in several brain regions, such as cerebral cortex and hippocampus (Mudo et al., 2007; Ohishi et al., 1993, 1998). Although it has been reported that systemic injection of LY379268, using a single large dose (10 mg/kg), is ineffective in inducing BDNF in the rat brain (Bond et al., 2000), the role of mGlu2/3 receptors activation by systemic injection of LY379268 on in vivo regulation of BDNF expression remains to be elucidated in view of fact that a complete dose-effect and time-course study is not available. Therefore this study was designed in order to explore the potential regulatory role of selective mGluR2/3 agonist LY379268 treatment on BDNF expression in the mouse brain, by performing a dose-effect and time-course study and by using in situ hybridization and western blotting procedures.

EXPERIMENTAL PROCEDURES Animals Adult male C57/BL6 mice (25–28 g b.w.) from local stock have been used for the present study. The mice were kept under environmentally controlled conditions, ambient temperature 24 °C, humidity 40% and 12-h light/dark cycle with food and water ad libitum. Procedures involving animals and their care were conducted in conformity with the institutional guidelines that are in compliance with national (D.L. n. 116,G.U., suppl. 40, 18 Febbraio 1992) and international laws and policies (EEC Council Directive 86/609, OJ L 358,1, Dec. 12, 1987; NIH Guide for the Care and Use of Laboratory Animals, NIH Publication no. 80-23, 1985 and Guidelines for the Use of Animals in Biomedical Research, Thromb. Haemost. 58, 1078 –1084,1987). All efforts were made to minimize the number of animals used and their suffering and all experiments were approved by the local ethical committee.

Treatments Groups of at least four mice for each experiment performed were treated i.p. with saline, or with different doses of (⫺)-2-oxa-4aminocyclo[3.1.0]hexane-4,6-dicarboxylic acid LY379268, kindly provided by Eli Lilly & Company (Indianapolis, IN, USA), ranging from 0.100 mg/kg b.w. to 3 mg/kg b.w., dissolved in saline. A time-course study was also performed using LY379268 at dose of 0.250 mg/kg. The preferential mGluR2/3 antagonist (2S)-2-Amino-2[(1S,2S)-2-carboxycycloprop-1-yl]-3-(xanth-9-yl) propanoic acid LY341495 (Tocris Cookson Ltd., Bristol, UK), was injected i.p. at dose of 1 mg/kg b.w. 30 min before of LY379268. Mice were killed under deep anesthesia and brain was rapidly frozen in cooled isopentane and stored at ⫺70 °C until use.

BDNF probe labelling The cDNA fragments used for generation of BDNF riboprobes specific for exon V corresponds to the coding sequence at bp 43–771 on BDNF cDNA (gene accession number X67108) subcloned into the Bluescript/SK⫹plasmid (Hansson et al., 2000) (Stratagene, San Diego, CA, USA). The plasmid was linearized with HindIII and used as a template for T7 RNA polymerase to generate the antisense probe. The radiolabelling of the riboprobe was performed as follows. The template was incubated for 60 min at 37 °C in the presence of a transcription buffer made of 40 mm Tris–HCl pH 7.5, 6 mM MgCl2 and 2 mM spermidine and supplemented with 12.5 nmol each of ATP, CTP and GTP, 500 pmol UTP, 125 pmol 35S alpha-UTP (PerkinElmer, Boston, USA; 18047– 019), 1 IU/␮l RNAse inhibitor and 1 IU/␮l of T7 or T3 polymerase. The cDNA template was digested by adding 20 ng/ml Deoxyribonuclease I (DNAse I; Invitrogen 18047-019) at 37 °C for 15 min. Transcripts were purified using ProbeQuant G-50 micro columns (GE Healthcare, formerly Amersham, Europe GmbH – Filiale Italiana, Milan, Italy).

In situ hybridization Serial sagittal (lateral 1.70 –1.00 mm) cryostat sections (14 ␮m) of mouse brain were prepared according to mouse atlas (Lehmann, 1974). Tissue sections were processed for in situ hybridization as previously described by (Belluardo et al., 2005). Following fixation in 4% paraformaldehyde for 15 min, slides were rinsed twice in phosphate-buffered saline (PBS) and once in distilled water. Tissue was deproteinated in 0.2 M HCl for 10 min, acetylated with 0.25% acetic anhydride in 0.1 M ethanolamine for 20 min, and dehydrated with increasing concentrations of ethanol. Slides were incubated for 16 h in a humidified chamber at 52 °C with 8⫻105 cpm probe in 70 ␮l hybridization cocktail (50% formamide, 20 mM Tris–HCl pH 7.6, 1 mM EDTA pH 8.0, 0.3 M NaCl, 0.1 M dithiothreitol, 0.5 ␮g/ml yeast tRNA, 0.1 ␮g/ml poly-A-RNA, 1⫻ Denhardt’s solution, and 10% dextran sulfate), washed twice in 1⫻SSC (1⫻SSC⫽150 mM NaCl, 15 mM sodium citrate, pH 7.0) at 62 °C for 15 min, and then in formamide: SSC (1:1) at 62 °C for 30 min. After an additional wash in 1⫻SSC at 62 °C, singlestranded RNA was digested by RNAse treatment (10 ␮g/ml) for 15 min at 37 °C in 0.5 M NaCl, 20 mM Tris–HCl pH 7.5, 2 mM EDTA. Slides were washed twice with 1⫻SSC at 62 °C for 30 min before dehydration in ethanol and air-drying. For cell localization of mRNA, hybridized sections were coated with NTB Autoradiography Emulsion diluted in water (1:1) (Catalog number 8895666, Eastman-Kodak, Rochester, NY, USA), and stored in desiccated light-tight boxes at 4 °C for 4 weeks. Slides were developed with D19 (Eastman-Kodak, Rochester, NY, USA), fixed with Al-4 (Agfa Gevaert, Kista, Sweden), and counterstained with Cresyl Violet. Semiquantitative data on mRNA levels were obtained by measuring the optical density (OD) values of the labelling in the film autoradiograms on a personal computer using NIH ImageJ software (Rasband, W.S., ImageJ, USA. National Institutes of Health, Bethesda, MD, USA, http:// rsb.info.nih.gov/ij/, 1997–2007). The values for each region measured were defined as those obtained by subtracting the non-specific background values. Values represent the average of readings from at least three sagittal sections sampled in the corresponding lateral 1.70 –1.00 mm region level of mouse brain (Lehmann, 1974). The densitometric values for cerebral cortex have been obtained by measuring all the cortex present in the representative sagittal brain sections examined, and therefore including all layers of frontal, parietal and occipital cortex. Evaluation of silver grains over the individual cells from emulsion dipped slides was performed using image analysis system (IAS-Counter, Delta-Sistemi, Roma, Italy).

V. Di Liberto et al. / Neuroscience 165 (2010) 863– 873

In situ hybridization combined with immunolabeling We have used a combination of in situ hybridization for BDNF mRNA expression and immunohistochemical techniques for the neuronal marker neuron-specific DNA-binding protein (NeuN) in order to identify the cells showing increased expression of the BDNF mRNA. Brain sagittal cryostat sections of 14 ␮m thick were processed for immunohistochemistry immediately after the last washing of the in situ hybridization procedure, which was carried out exactly as detailed above. To this end, sections were washed with PBS, and incubated for 15 min in blocking buffer consisting of 2.5% normal goat serum and 0.3% Triton X-100 in PBS. Subsequently, sections were incubated overnight at 4 °C in the presence of the primary antibody mouse anti-NeuN diluted 1:500 (Chemicon Int. Temecula, CA, USA; MAB377) in PBS supplemented with 1.5% blocking serum. Sections were then washed three times for 5 min in PBS, and incubated at room temperature for 1 h with a biotinylated anti-mouse antiserum (GE Healthcare, formerly Amersham, Europe GmbH – Filiale Italiana, Milan, Italy), diluted

865

1:200. After three washing with PBS for 5 min, the sections were incubated for 1 h with a horseradish peroxidase-streptavidin complex (Vector, Burlingame, CA, USA), diluted 1:100 in PBS. After on washing in PBS and one in Tris–HCl buffer (0.1 M pH 7.4), the peroxidase reaction was developed in the same buffer containing 0.05% 3,3-diaminobenzidine-4 HCl and 0.003% hydrogen peroxide. The reaction was stopped in Tris–HCl buffer and after a short washing with H2O, the sections were fixed again as described in the in situ hybridization procedure, dehydrated in an ascending alcohol series, coated in NTB emulsion and processed as described above for autoradiographic development.

Western blotting analysis Orbitofrontal cortex (AP: 6.2–7.3 mm) according to mouse atlas (Lehmann, 1974) and hippocampus of both sides of different experimental groups were rapidly dissected under stereomicroscopy, frozen and processed for western blotting. The dissected tissue was homogenized in cold buffer containing 50 mM Tris–HCl

Fig. 1. Effects of LY379268 on BDNF mRNA expression in the cerebral cortex and hippocampal formation of adult mouse. (A) Representative photomicrograph of film autoradiograms of sagittal brain sections showing the expression of BDNF mRNA levels in control mice (saline) or treated with 0.250 mg/kg b.w. or 1 mg/kg b.w. of LY379268 and sacrificed at 3 h from treatment. (B) dose-effect of LY379268 treatment on BDNF mRNA levels in three different brain regions of mice sacrificed 3 h following LY379268 treatment: cerebral cortex (CTX), dentate gyrus (DG), and pyramidal layers of the hippocampal formation (CA1–CA3). (C) Time-course of LY379268 (0.250 mg/kg) effects on BDNF mRNA levels in the hippocampal formation and cerebral cortex. Data in (B) and (C) are reported as means⫾SEM of optical density (OD) values obtained from film autoradiograms. The data were evaluated by one-way ANOVA with intergroup differences analyzed by the Fisher’s PLSD test. PLSD test, asterisks indicate significant differences from controls: * P⬍0.05, ** P⬍0.01, *** P⬍0.001. Dose-effect ANOVA, CTX: F(6,24)⫽2.39, P⫽0.054; CA1–CA3: F(6,24)⫽9.39, P⫽0.008; DG: F(6,24)⫽9.93, P⬍0.0001. Time-course ANOVA, CTX: F(3,34)⫽2.19, P⫽0.11; CA1–CA3: F(3,34)⫽10.57, P⬍0.0001; DG: F(3,34)⫽5.74, P⫽0.0031. Scale bar: 2.5 mm.

866

V. Di Liberto et al. / Neuroscience 165 (2010) 863– 873

pH 7.4, 150 mM NaCl, 1% triton, 0.1% SDS, H2O and protease inhibitor cocktail (P8340, Sigma-Aldrich S.r.l., Milan, Italy). The homogenate was left on ice for 30 min and then centrifuged at 13,000 rpm for 30 min at 4 °C. The supernatants were stored at ⫺20 °C and aliquots were taken for protein determination by the method of Lowry et al. (1951). The samples with 60 ␮g of protein and mol.wt. markers (161-0375, Bio-Rad Laboratories S.r.l., Segrate (MI), Italy), were run on 10% polyacrylamide gel at 100 V and electrophoretically transferred onto nitrocellulose membrane (Hybond-C-extra, GE Healthcare, formerly Amersham, Europe GmbH – Filiale Italiana, Milan, Italy). Following 1 h of incubation with 5% nonfat milk, the membrane was incubated overnight at ⫹4 °C with anti-BDNF polyclonal antibody (1:1000) raised in rabbit (N-20 Sc-546; Santa Cruz Biotechnology, CA, USA). After washing the membrane was incubated for 1 h at room temperature with anti-rabbit IgG horseradish peroxidase-conjugated diluted 1:5000 (Sc 200 4, Santa Cruz Biotechnology) and BDNF band was visualized with chemiluminescence reagent (ECL, GE Healthcare, formerly Amersham, Europe GmbH – Filiale Italiana, Milan, Italy) according to the manufacturer’s instructions. The ECL-films were developed in Kodak D19 developer and fixer (Eastman-Kodak, Rochester, NY, USA), and the densitometric evaluation of bands was performed by measuring the optical density (O.D.) using NIH ImageJ software and results expressed as arbitrary units.

Quantitative evaluation and statistical analysis All data were analyzed in Prism 5 software (Graph-Pad, San Diego, CA, USA). Data are presented as mean⫾SEM and are representative of three independent experiments. One-way analysis was used to evaluate from film autoradiograms the dose effect and the time course of LY379268 treatment on BDNF mRNA expression. Significant effects were further evaluated by Fisher’s Protected Last Significant Difference (PLSD) test, corrected by Bonferoni’s procedure for dependent samples. t-test analysis was used to evaluate the LY379268 effects on BDNF mRNA, both as number of labeled cells and silver grains density over the individual cells from emulsion dipped slides, and on BDNF protein expression. The number of BDNF mRNA labeled cells in the cerebral cortex and in the CA3 and DG regions of hippocampal formation was estimated by counts made by systematic sampling of brain sections (total of 10 sections), every third section, corresponding to region level (lateral 1.70 –1.00 mm) of mouse brain (Lehmann, 1974). All the counts were made in four mice for each group, and were carried out in a double blind manner, by mean of both optical and computerized image analysis software counts (IAS-Counter, Delta-Sistemi, Roma, Italy). Optical counts were performed using a Leica microscope and 100⫻ magnification. The percentage of labeled cells was determined by counts of labeled and total cells with a micrometric quadriculated reticulum from three to five random fields per section (250 ␮m2 squares) and was expressed as means⫾SEM value of labeled cells found in 10 brain sections.

enhance the BDNF mRNA expression in the cerebral cortex and the hippocampal formation, including dentate gyrus and pyramidal layers (CA1–CA3). The dose of 2 mg/kg significantly increased BDNF mRNA in the hippocampal formation, whereas the highest dose used 3 mg/kg produced a statistically non-significant increase of BDNF mRNA expression (Fig. 1B). Time-course study. Using in situ hybridization we performed a time-course study of BDNF mRNA expression following LY379268 treatment. For this investigation we tested the dose of 0.250 mg/kg in the following time points: 2 h, 3 h, and 6 h. The analysis showed a significant increase of BDNF mRNA expression in the cerebral cortex and the hippocampal formation reaching the maximum levels 3 h following LY379268 treatment (Fig. 1C). This change of BDNF mRNA expression disappeared 6 h after LY379268 (Fig. 1C). Pre-treatment with mGluR2/3 antagonist LY341495. In order to define the receptors specificity of LY379268 effects we performed a pre-treatment with mGluR2/3 antagonist LY341495 (1 mg/kg) which was injected 30 min before 0.250 mg/kg of LY379268 and mice were sacrificed 3 h following LY379268 treatment. The results shown in Fig. 2 evidenced that pre-treatment with mGluR2/3 antagonist LY341495 blocks completely LY379268 effects. The treatment with the antagonist LY341495 alone did not influence BDNF mRNA expression (Fig. 2). Autoradiographic analysis. To further corroborate the data obtained by measuring the optical density of the labelling in the film autoradiograms we analysed the ex-

RESULTS Effects of LY379268 treatment on BDNF mRNA expression Dose-effect study. Using in situ hybridization we performed a dose-effect study of LY379268 treatment on BDNF mRNA expression. For this investigation we tested the following doses: 0.100 mg/kg, 0.250 mg/kg, 1 mg/kg, 2 mg/kg and 3 mg/kg. The different groups of mice were sacrificed 3 h after LY379268 treatment. The results, shown in Fig. 1A, B, evidenced that LY379268 treatment was not effective at dose of 0.100 mg/kg, whereas at dose of 0.250 mg/kg and 1 mg/kg was able to significantly

Fig. 2. Effect of pre-treatment with mGluR2/3 antagonist LY341495 on LY379268 treatment inducing BDNF mRNA expression. LY341495 (1 mg/kg) was injected 30 min before 0.250 mg/kg b.w. of LY379268 and mice were sacrificed 3 h following LY379268 treatment. Brain regions examined: cerebral cortex (CTX), dentate gyrus (DG) and CA1–CA3 pyramidal layers of the hippocampal formation. Data are Means⫾SEM of optical density (OD) values obtained from film autoradiograms. The data were evaluated by one-way ANOVA with intergroup differences analyzed by the Fisher’s PLSD test. PLSD test, asterisks indicate significant differences from controls: *** P⬍0.0001. ANOVA, CTX: F(3,20)⫽27.03, P⬍0.0001; CA1–CA3: F(3,20)⫽42.77, P⬍0.0001; DG: F(3,20)⫽93.47, P⬍0.0001.

V. Di Liberto et al. / Neuroscience 165 (2010) 863– 873

pression levels of BDNF mRNA in brain sections processed autoradiographically and stained with Cresyl-Violet. The BDNF mRNA levels were evaluated in the cerebral cortex and hippocampal sub-regions, CA3 pyramidal cell layer and dentate gyrus, by analysis of autoradiographic silver grain density over the individual cells, performed using image analysis system (see material and methods), and by counting the cells expressing BDNF.

867

In the cerebral cortex BDNF mRNA levels following LY379268 (0.250 mg/kg) treatment were increased mainly in the external layers and particularly in the layer II as compared to control (Fig. 3). This upregulation involved mainly the levels of BDNF mRNA expressed per cell and in less extent the increased number of cells expressing BDNF (Table 1). Similarly, in the CA3 pyramidal cell layer of hippocampal formation BDNF mRNA levels increase

Fig. 3. Bright-field microautoradiographs from Cresyl Violet stained brain sections showing BDNF mRNA labeled cells (black grains) in the layers II of cerebral cortex (CTX), in the CA3 pyramidal cell layer (CA3) and in the dentate gyrus (DG) of the hippocampal formation. By combining in situ hybridization for BDNF mRNA and immunolabeling for neuronal marker NeuN, in the bright-field microtoautoradiographs of lower panels are shown BDNF-labeled cells (black grains cluster) co-localizing with NeuN-positive cells (brown staining) in the layers II of cerebral cortex (BDNF/NeuN) in control mouse (saline) and mouse treated with 0.250 mg/kg b.w. of LY379268 and sacrificed after 3 h from treatment. Scale bar 30 ␮m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

868

V. Di Liberto et al. / Neuroscience 165 (2010) 863– 873

Table 1. Relative BDNF mRNA levels per cell and percentage of cells expressing BDNF mRNA in the cerebral cortex and in the CA3 pyramidal layer or in the dentate gyrus of hippocampal formation of adult mouse following LY379268 treatment (0.250 mg/kg b.w.) % of BDNF mRNA labelled cells

Grains density per cell

Brain regions

Control

LY379268

Control

LY379268

Cerebral cortex layer II CA3 pyramidal layer Dentate gyrus

22.7⫾2.4

24.2⫾4.7 n.s. 85.7⫾10.6 n.s. 22.0⫾1.1 P⬍0.0003

28.1⫾2.9

37⫾4.6 P⬍0.05 88.6⫾11.8 P⬍0.05 23.7⫾0.8 n.s.

72.8⫾16.8 9.4⫾1.2

59.0⫾11.6 18.3⫾4.3

The relative BDNF mRNA levels per cell and the percentage of cells expressing BDNF mRNA have been evaluated as described in material and methods. Statistical analysis of means⫾SD. The data were evaluated by t-test.

involved mainly the levels of BDNF mRNA expressed per cell (Fig. 3, Table 1). In contrast, this result was opposite in the dentate gyrus where it was observed a significant increase in the number of cells expressing BDNF instead of BDNF mRNA expressed per cell (Fig. 3, Table 1). Identification of cells expressing BDNF mRNA. We have used a combination of in situ hybridization for BDNF mRNA expression and immunohistochemistry for neuronal marker NeuN in order to identify both in saline and LY379268 treated mice the cells expressing BDNF. This study revealed that both in cerebral cortex and the hippocampal formation of saline or LY379268 (0.250 mg/kg b.w.) treated mice all the cells expressing BDNF mRNA were NeuN-positive, as shown in representative microphotoautoradiographs in the Fig. 3. This result indicated that the main effect of mGluR2/3 receptor activation involves neuronal cells, although mGluR3 receptor is also expressed in non neuronal cells, such as astrocytes and oligodendrocytes (Mudo et al., 2007; Ohishi et al., 1993). Effects of LY379268 treatment on BDNF protein levels. The BDNF protein levels were determined in the hippocampus and among the cerebral cortex regions in the orbitofrontal cortex, which was chosen in order to define the BDNF change in a specific cortical region implicate in depression, in view of recent observation that mGluR2/3 agonists might produce antidepressant effects (Matrisciano et al., 2007). To investigate the effect of mGluR2/3 activation on BDNF protein levels we chose to examine the dose of 0.250 and 2 mg/kg b.w. The anti-BDNF antibody used recognize both the mature and pro-BDNF respectively detected as single band at 14 and 28 kDa. The 28 kDa band may correspond to pro-BDNF form with molecular weight of 31–35 kDa, since the lower size detected is probably due to deglycosylation of pro-BDNF (Mowla et al., 2001). Western blot analysis 24 h after LY379268 treatment with the dose of 0.250 mg/kg showed a significant increase of mature and pro-BDNF protein expression in the hippocampal formation and of pro-BDNF in the orbitofrontal cortex (Fig. 4). The dose of 2 mg/kg showed a significant increase

of mature and pro-BDNF in both the orbitofrontal cortex and the hippocampal formation at the time point examined of 24 h (Fig. 4). In order to explore the effects of repeated injections of LY379268 on BDNF protein levels we designed two experiments with different length of LY379268 (0.250 mg/kg) treatment time: in one LY379268 was injected once daily for 3 days and 24 h after the last injection mice were killed; in the other one LY379268 was injected once daily for 7 days and 24 h after the last injection mice were killed. In both experiments after LY379268 treatment in the orbitofrontal cortex the BDNF level was found unchanged or even not significantly decreased in the mature form (Fig. 5). In the hippocampus, BDNF level was still found increased but it was not statistically significant, with the exclusion of mature form in mice treated for 3 days with LY379268 (Fig. 5). All together the results revealed that repeated injections of LY379268 are less effective than acute single injection.

DISCUSSION In the present work we could show that mGlu2/3 receptor agonist LY379268 treatment induces BDNF expression in the cerebral cortex and the hippocampal formation. The analysis of dose-effect curve revealed that LY379268 may already induce the maximum levels of BDNF mRNA upregulation at dose of 0.250 mg/kg b.w. and higher doses of LY379268 (1 and 2 mg/kg) did not produce a further increase of BDNF mRNA expression. The observed effects were non significant at dose of 3 mg/kg b.w. of LY379268, suggesting a differentially involvement of mGluR2/3 or recruitment of other subtype receptors at relatively high concentrations of LY379268 (Schoepp et al., 1999). This BDNF upregulation by treatment with LY379268 was localized only in neuronal cells and involved mainly the levels of BDNF mRNA expressed per cell and in less extent the increased number of labelled cells. In addition, BDNF upregulation was transient and blocked by the new selective mGlu2/3 receptor antagonist LY341495 (Kingston et al., 1998), suggesting a receptor specificity of the observed effect. Finally, we could show in both brain regions examined, orbitofrontal cortex and hippocampal region, that the upregulation of BDNF mRNA levels was followed by a significant BDNF protein increase involving both the mature and pro-BDNF forms with the exclusion of mature BDNF in the orbitofrontal cortex. Using repeated injections of LY379268 we could show that a chronic treatment did not produce cumulative effects on BDNF levels and it can be even less effective than acute single injection. This less promising effect of repeated daily injections of LY379268 could potentially depend on desensitization and/or internalization of target receptor (Dhami and Ferguson, 2006; Iacovelli et al., 2009). From the present data, because LY379268 does not distinguish between mGluR2 and mGluR3, we cannot define which subtype receptor is involved on BDNF upregulation. However recently, using knockout mice for mGluR2 or for mGluR3, Corti et al. (2007) could show that LY379268 neuroprotection is dependent on mGluR3 acti-

V. Di Liberto et al. / Neuroscience 165 (2010) 863– 873

869

Fig. 4. Western blot analysis of BDNF protein levels following LY379268 treatment. Representative image displaying mature and pro-BDNF protein levels respectively detected at 14 and 28 kDa in the hippocampus and the orbitofrontal cortex of control mice (saline) and mice treated with LY379268 (0.250 or 2 mg/kg) and sacrificed 24 h following treatment. Histograms showing the arbitrary levels of mature and pro-BDNF calculated by densitometry of the immunoreactve bands. Data are means⫾SEM of optical density (OD) values obtained from films and expressed as arbitrary units. t-test: * P⬍0.05; ** P⬍0.001; *** P⬍0.0001.

vation, whereas the mGluR2 might be even harmful to neurons exposed to toxic insult. More recently we showed

(Battaglia et al., 2009) a regulation of GDNF in the striatum as target of mGluR3 receptors activation by LY379268.

870

V. Di Liberto et al. / Neuroscience 165 (2010) 863– 873

Fig. 5. Western blot analysis of BDNF protein levels following repeated injections of LY379268. Representative image displaying mature and pro-BDNF protein levels respectively detected at 14 and 28 kDa in the hippocampus and the orbitofrontal cortex of control mice (saline) and mice treated with LY379268 (0.250 mg/kg) for 3 or 7 days (one injection per day) and sacrificed 24 h after the last injection. Histograms showing the arbitrary levels of mature and pro-BDNF calculated by densitometry of the immunoreactve bands. Data are means⫾SEM of optical density (OD) values obtained from films and expressed as arbitrary units. t-test: ** P⬍0.03.

Therefore, because the upregulation of BDNF in the hippocampal formation and cerebral cortex may be linked to

neuroprotective role of LY379268, indirectly we argue that mGluR3 could also be responsible for the present results.

V. Di Liberto et al. / Neuroscience 165 (2010) 863– 873

Previously Bond et al. (2000) have shown that high dose of LY379268 (10 mg/kg), although neuroprotective in a model of global ischemia, did not induce expression of specific neurotrophic factors, such as BDNF and NGF. More recently, it has be reported that activation of Group-II mGlu receptors, carried out using the agonist (2S,2=R,3=R)2-(2=,3=-dicarboxycyclopropyl)glycine (DCG-IV) injected in the rat striatum, can protect against MPTP neurotoxicity by increasing BDNF in reactive microglia (Matarredona et al., 2001; Venero et al., 2002). Both of these available investigations did not evidence an effect of mGluR2/3 agonists on neuronal BDNF upregulation as instead we found in the present study. These different results may be attributed to the following reasons: the local against the systemic injection of the agonist used; the different doses of drugs used; the different time-points examined; and the procedure used such as immunohistochemistry analysis which may be less sensitive for quantitative analysis when compared to in situ hybridization or western blotting. Concerning the dose examined, it appears clear from the present results of dose-effect curve that there is a restricted window of effective doses of LY379268 on BDNF upregulation. Therefore the previous negative data reported by Bond et al. (2000) on BDNF changes could be attribute to high dose of LY379268 (10 mg/kg) used. Several data are cumulating on neuroprotective effects of mGlu2/3 receptor agonists treatment including LY379268 (see review D’Onofrio et al., 2001). Concerning the possible mechanisms involved on the neuroprotective role of mGluR2/3 activation it is through that receptors located on glutamatergic terminals might limit the excitotoxicity by inhibiting glutamate release (Battaglia et al., 1997; Buisson and Choi, 1995; Cozzi et al., 1997; Rouse et al., 2000). Alternatively, it has been also examined the possibility that activation of mGlu2/3 receptors present in neurons or astrocytes or oligodendrocytes (Mudo et al., 2007; Petralia et al., 1996) might stimulate the production of neurotrophic factors, such as, TGF-␤1 (Bruno et al., 1998) or NGF (Ciccarelli et al., 1999) in cultured astrocytes and BDNF in reactive astrocytes (Matarredona et al., 2001). In the present investigation we could reveal an involvement of mGluR2/3 receptors activation on BDNF expression both as mRNA and protein with specific time course, dose-effect pattern and cell type involved. Therefore, potentially this effect on BDNF expression may give account for such neuroprotective response observed in the brain following mGluR2/3 receptors activation. In fact, BDNF is known to promote the survival, differentiation and plasticity of neuronal populations in many regions of the adult brain, and this role fits with the widespread distribution of BDNF in the brain and the co-localization of BDNF and its receptor TrkB at glutamatergic synapes (Binder and Scharfman, 2004; Bramham and Messaoudi, 2005; Lessmann, 1998; Lessmann et al., 2003; McAllister et al., 1999; Poo, 2001). According to this, the regulation of BDNF by LY379268 treatment occurs in two brain regions, cerebral cortex and hippocampal formation, largely studied in in vivo models to examine the neuroprotective effects of group-II receptor

871

agonists treatment. In this contest a complete protection has been observed in the hippocampal formation after global ischemia in gerbils (Bond et al., 2000) or in the rat retrosplenial cortex after administration of NMDA receptor antagonists (Okamura et al., 2003) or in brain injury produced by systemic MK-801 injection (Carter et al., 2004) and by kainate-induced seizures (Miyamoto et al., 1997). In the present work, LY379268 treatment was not able to induce regulation of BDNF in the striatum and it was in contrast to neuroprotective effects of mGluR2/3 agonists, including LY379268, found by several groups against nigro-striatal degeneration in models of Parkinson’s Disease (Battaglia et al., 2003; Corti et al., 2007; Matarredona et al., 2001; Murer et al., 2001). However, very recently we showed that GDNF increases in the striatum following LY379268 treatment, giving account for neuroprotection against nigro-striatal degeneration in a model of Parkinson’s Disease (Battaglia et al., 2009). Additionally, it is not possible exclude that the enhancement of BDNF levels observed in the cerebral cortex following LY379268 treatment may also be an afferent supply to the target neurons in the striatum since this neurotrophin can be anterogradely transported (Altar et al., 1997). It has been largely showed that BDNF has an important role in the regulation of hippocampal learning and memory processes, and that it has a key role in the structural and functional plasticity of this brain region (Cirulli et al., 2004; McAllister et al., 1999; Poo, 2001; Schuman, 1999; Thoenen, 1995, 2000). According to this, the upregulation of BDNF in the hippocampal formation by LY379268 treatment suggests a potential role of mGluR2/3 receptor activation in the learning and memory processes as reported by a restricted number of studies (Aultman and Moghaddam, 2001; Higgins et al., 2004). Additionally, because BDNF plays a pivotal role in the action of antidepressants drugs (Martinowich et al., 2007), the enhanced effect of LY379268 treatment on hippocampal or cerebral cortex, particularly the orbitofrontal cortex, BDNF expression levels could also give account of recent observation that mGluR2/3 agonists might produce antidepressant effects particularly when combined with classical antidepressant (Matrisciano et al., 2007). Taken together the findings of present work suggest that several previous observed neuroprotective and trophic actions of mGluR2/3 agonist treatment, at least in the cerebral cortex and hippocampal formation, may be mediated by upregulation of BDNF expression. Acknowledgments—We thank Eli Lilly (Indianapolis, IN, USA) for generously supplying of LY379268. We are grateful to Dr. A. Hansson for providing BDNF riboprobe. This work was supported by grants of MIUR (Prin 2004, 2004052809_003); Progetti di Ateneo (Università di Palermo). DLV, BA and FM are supported by Ateneo of Palermo.

REFERENCES Altar CA, Cai N, Bliven T, Juhasz M, Conner JM, Acheson AL, Lindsay RM, Wiegand SJ (1997) Anterograde transport of brain-derived neurotrophic factor and its role in the brain. Nature 389(6653):856 – 860.

872

V. Di Liberto et al. / Neuroscience 165 (2010) 863– 873

Aultman JM, Moghaddam B (2001) Distinct contributions of glutamate and dopamine receptors to temporal aspects of rodent working memory using a clinically relevant task. Psychopharmacology (Berl) 153(3):353–364. Battaglia G, Busceti CL, Pontarelli F, Biagioni F, Fornai F, Paparelli A, Bruno V, Ruggieri S, Nicoletti F (2003) Protective role of group-II metabotropic glutamate receptors against nigro-striatal degeneration induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in mice. Neuropharmacology 45(2):155–166. Battaglia G, Molinaro G, Riozzi B, Storto M, Busceti CL, Spinsanti P, Bucci D, Di Liberto V, Mudò G, Corti C, Corsi M, Nicoletti F, Belluardo N, Bruno V (2009) Activation of mGlu3 receptors stimulates the production of GDNF in striatal neurons. PLoS One 4(8):e6591. Battaglia G, Monn JA, Schoepp DD (1997) In vivo inhibition of veratridine-evoked release of striatal excitatory amino acids by the group II metabotropic glutamate receptor agonist LY354740 in rats. Neurosci Lett 229(3):161–164. Belluardo N, Olsson PA, Mudo’ G, Sommer WH, Amato G, Fuxe K (2005) Transcription factor gene expression profiling after acute intermittent nicotine treatment in the rat cerebral cortex. Neuroscience 133(3):787–796. Binder DK, Scharfman HE (2004) Brain-derived neurotrophic factor. Growth Factors 22(3):123–131. Bond A, Jones NM, Hicks CA, Whiffin GM, Ward MA, O’Neill MF, Kingston AE, Monn JA, Ornstein PL, Schoepp DD, Lodge D, O’Neill MJ (2000) Neuroprotective effects of LY379268, a selective mGlu2/3 receptor agonist: investigations into possible mechanism of action in vivo. J Pharmacol Exp Ther 294(3):800 – 809. Bramham CR, Messaoudi E (2005) BDNF function in adult synaptic plasticity: the synaptic consolidation hypothesis. Prog Neurobiol 76(2):99 –125. Bruno V, Battaglia G, Casabona G, Copani A, Caciagli F, Nicoletti F (1998) Neuroprotection by glial metabotropic glutamate receptors is mediated by transforming growth factor-beta. J Neurosci 18(23):9594 –9600. Bruno V, Battaglia G, Copani A, D’Onofrio M, Di IP, De BA, Melchiorri D, Flor PJ, Nicoletti F (2001) Metabotropic glutamate receptor subtypes as targets for neuroprotective drugs. J Cereb Blood Flow Metab 21(9):1013–1033. Buisson A, Choi DW (1995) The inhibitory mGluR agonist, S-4-carboxy-3-hydroxy-phenylglycine selectively attenuates NMDA neurotoxicity and oxygen-glucose deprivation-induced neuronal death. Neuropharmacology 34(8):1081–1087. Carter K, Dickerson J, Schoepp DD, Reilly M, Herring N, Williams J, Sallee FR, Sharp JW, Sharp FR (2004) The mGlu2/3 receptor agonist LY379268 injected into cortex or thalamus decreases neuronal injury in retrosplenial cortex produced by NMDA receptor antagonist MK-801: possible implications for psychosis. Neuropharmacology 47(8):1135–1145. Cartmell J, Schoepp DD (2000) Regulation of neurotransmitter release by metabotropic glutamate receptors. J Neurochem 75(3):889 – 907. Chiamulera C, Albertini P, Valerio E, Reggiani A (1992) Activation of metabotropic receptors has a neuroprotective effect in a rodent model of focal ischaemia. Eur J Pharmacol 216(2):335–336. Ciccarelli R, Di IP, Giuliani P, D’Alimonte I, Ballerini P, Caciagli F, Rathbone MP (1999) Rat cultured astrocytes release guaninebased purines in basal conditions and after hypoxia/hypoglycemia. Glia 25(1):93–98. Cirulli F, Berry A, Chiarotti F, Alleva E (2004) Intrahippocampal administration of BDNF in adult rats affects short-term behavioral plasticity in the Morris water maze and performance in the elevated plus-maze. Hippocampus 14(7):802– 807. Copani A, Bruno V, Battaglia G, Leanza G, Pellitteri R, Russo A, Stanzani S, Nicoletti F (1995) Activation of metabotropic glutamate receptors protects cultured neurons against apoptosis induced by beta-amyloid peptide. Mol Pharmacol 47(5):890 – 897.

Corti C, Battaglia G, Molinaro G, Riozzi B, Pittaluga A, Corsi M, Mugnaini M, Nicoletti F, Bruno V (2007) The use of knock-out mice unravels distinct roles for mGlu2 and mGlu3 metabotropic glutamate receptors in mechanisms of neurodegeneration/neuroprotection. J Neurosci 27(31):8297– 8308. Cozzi A, Attucci S, Peruginelli F, Marinozzi M, Luneia R, Pellicciari R, Moroni F (1997) Type 2 metabotropic glutamate (mGlu) receptors tonically inhibit transmitter release in rat caudate nucleus: in vivo studies with (2S,1=S,2=S,3=R)-2-(2=-carboxy-3=-phenylcyclopropyl)glycine, a new potent and selective antagonist. Eur J Neurosci 9(7):1350 –1355. D’Onofrio M, Cuomo L, Battaglia G, Ngomba RT, Storto M, Kingston AE, Orzi F, De BA, Di IP, Nicoletti F, Bruno V (2001) Neuroprotection mediated by glial group-II metabotropic glutamate receptors requires the activation of the MAP kinase and the phosphatidylinositol-3-kinase pathways. J Neurochem 78(3):435– 445. Dhami GK, Ferguson SS (2006) Regulation of metabotropic glutamate receptor signaling, desensitization and endocytosis. Pharmacol Ther 111(1):260 –271. Emile L, Mercken L, Apiou F, Pradier L, Bock MD, Menager J, Clot J, Doble A, Blanchard JC (1996) Molecular cloning, functional expression, pharmacological characterization and chromosomal localization of the human metabotropic glutamate receptor type 3. Neuropharmacology 35(5):523–530. Flor PJ, Battaglia G, Nicoletti F, Gasparini F, Bruno V (2002) Neuroprotective activity of metabotropic glutamate receptor ligands. Adv Exp Med Biol 513:197–223. Flor PJ, Lindauer K, Puttner I, Ruegg D, Lukic S, Knopfel T, Kuhn R (1995) Molecular cloning, functional expression and pharmacological characterization of the human metabotropic glutamate receptor type 2. Eur J Neurosci 7(4):622– 629. Hansson AC, Cintra A, Belluardo N, Sommer W, Bhatnagar M, Bader M, Ganten D, Fuxe K (2000) Gluco- and mineralocorticoid receptor-mediated regulation of neurotrophic factor gene expression in the dorsal hippocampus and the neocortex of the rat. Eur J Neurosci 12(8):2918 –2934. Henrich-Noack P, Hatton CD, Reymann KG (1998) The mGlu receptor ligand (S)-4C3HPG protects neurons after global ischaemia in gerbils. Neuroreport 9(6):985–988. Higgins GA, Ballard TM, Kew JN, Richards JG, Kemp JA, Adam G, Woltering T, Nakanishi S, Mutel V (2004) Pharmacological manipulation of mGlu2 receptors influences cognitive performance in the rodent. Neuropharmacology 46(7):907–917. Iacovelli L, Molinaro G, Battaglia G, Motolese M, Di ML, Alfiero M, Blahos J, Matrisciano F, Corsi M, Corti C, Bruno V, De BA, Nicoletti F (2009) Regulation of group II metabotropic glutamate receptors by G protein-coupled receptor kinases: mGlu2 receptors are resistant to homologous desensitization. Mol Pharmacol 75(4):991– 1003. Kingston AE, Ornstein PL, Wright RA, Johnson BG, Mayne NG, Burnett JP, Belagaje R, Wu S, Schoepp DD (1998) LY341495 is a nanomolar potent and selective antagonist of group II metabotropic glutamate receptors. Neuropharmacology 37(1):1–12. Lehmann A (1974) Atlas stereotaxique du cerveau de la souris. Paris: Editions du Centre national de la recherche scientifique. Lessmann V (1998) Neurotrophin-dependent modulation of glutamatergic synaptic transmission in the mammalian CNS. Gen Pharmacol 31(5):667– 674. Lessmann V, Gottmann K, Malcangio M (2003) Neurotrophin secretion: current facts and future prospects. Prog Neurobiol 69(5): 341–374. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265– 275. Martinowich K, Manji H, Lu B (2007) New insights into BDNF function in depression and anxiety. Nat Neurosci 10(9):1089 –1093. Matarredona ER, Santiago M, Venero JL, Cano J, Machado A (2001) Group II metabotropic glutamate receptor activation protects striatal

V. Di Liberto et al. / Neuroscience 165 (2010) 863– 873 dopaminergic nerve terminals against MPP⫹-induced neurotoxicity along with brain-derived neurotrophic factor induction. J Neurochem 76(2):351–360. Matrisciano F, Panaccione I, Zusso M, Giusti P, Tatarelli R, Iacovelli L, Mathe AA, Gruber SH, Nicoletti F, Girardi P (2007) Group-II metabotropic glutamate receptor ligands as adjunctive drugs in the treatment of depression: a new strategy to shorten the latency of antidepressant medication? Mol Psychiatry 12(8):704 –706. McAllister AK, Katz LC, Lo DC (1999) Neurotrophins and synaptic plasticity. Annu Rev Neurosci 22:295–318. Miyamoto M, Ishida M, Shinozaki H (1997) Anticonvulsive and neuroprotective actions of a potent agonist (DCG-IV) for group II metabotropic glutamate receptors against intraventricular kainate in the rat. Neuroscience 77(1):131–140. Movsesyan VA, Faden AI (2006) Neuroprotective effects of selective group II mGluR activation in brain trauma and traumatic neuronal injury. J Neurotrauma 23(2):117–127. Mowla SJ, Farhadi HF, Pareek S, Atwal JK, Morris SJ, Seidah NG, Murphy RA (2001) Biosynthesis and post-translational processing of the precursor to brain-derived neurotrophic factor. J Biol Chem 276(16):12660 –12666. Mudo G, Trovato-Salinaro A, Caniglia G, Cheng Q, Condorelli DF (2007) Cellular localization of mGluR3 and mGluR5 mRNAs in normal and injured rat brain. Brain Res 1149:1–13. Murer MG, Yan Q, Raisman-Vozari R (2001) Brain-derived neurotrophic factor in the control human brain, and in Alzheimer’s disease and Parkinson’s disease. Prog Neurobiol 63(1):71–124. Nicoletti F, Bruno V, Copani A, Casabona G, Knopfel T (1996) Metabotropic glutamate receptors: a new target for the therapy of neurodegenerative disorders? Trends Neurosci 19(7):267–271. O’Neill MJ (2001) Pharmacology and neuroprotective actions of mGlu receptor ligands. Dev Med Child Neurol Suppl 86:13–15. Ohishi H, Neki A, Mizuno N (1998) Distribution of a metabotropic glutamate receptor, mGluR2, in the central nervous system of the rat and mouse: an immunohistochemical study with a monoclonal antibody. Neurosci Res 30(1):65– 82. Ohishi H, Shigemoto R, Nakanishi S, Mizuno N (1993) Distribution of the messenger RNA for a metabotropic glutamate receptor, mGluR2, in the central nervous system of the rat. Neuroscience 53(4):1009 –1018. Okamura N, Hashimoto K, Shimizu E, Koike K, Ohgake S, Koizumi H, Kumakiri C, Komatsu N, Iyo M (2003) Protective effect of LY379268, a selective group II metabotropic glutamate receptor

873

agonist, on dizocilpine-induced neuropathological changes in rat retrosplenial cortex. Brain Res 992(1):114 –119. Petralia RS, Wang YX, Niedzielski AS, Wenthold RJ (1996) The metabotropic glutamate receptors, mGluR2 and mGluR3, show unique postsynaptic, presynaptic and glial localizations. Neuroscience 71(4):949 –976. Poli A, Beraudi A, Villani L, Storto M, Battaglia G, Di Giorgi Gerevini V, Cappuccio I, Caricasole A, D’Onofrio M, Nicoletti F (2003) Group II metabotropic glutamate receptors regulate the vulnerability to hypoxic brain damage. J Neurosci 23(14):6023– 6029. Poo MM (2001) Neurotrophins as synaptic modulators. Nat Rev Neurosci 2(1):24 –32. Rouse ST, Marino MJ, Bradley SR, Awad H, Wittmann M, Conn PJ (2000) Distribution and roles of metabotropic glutamate receptors in the basal ganglia motor circuit: implications for treatment of Parkinson’s disease and related disorders. Pharmacol Ther 88(3):427– 435. Schoepp DD, Jane DE, Monn JA (1999) Pharmacological agents acting at subtypes of metabotropic glutamate receptors. Neuropharmacology 38(10):1431–1476. Schuman EM (1999) Neurotrophin regulation of synaptic transmission. Curr Opin Neurobiol 9(1):105–109. Shigemoto R, Kinoshita A, Wada E, Nomura S, Ohishi H, Takada M, Flor PJ, Neki A, Abe T, Nakanishi S, Mizuno N (1997) Differential presynaptic localization of metabotropic glutamate receptor subtypes in the rat hippocampus. J Neurosci 17(19):7503–7522. Thoenen H (1995) Neurotrophins and neuronal plasticity. Science 270(5236):593–598. Thoenen H (2000) Neurotrophins and activity-dependent plasticity. Prog Brain Res 128:183–191. Venero JL, Santiago M, Tomas-Camardiel M, Matarredona ER, Cano J, Machado A (2002) DCG-IV but not other group-II metabotropic receptor agonists induces microglial BDNF mRNA expression in the rat striatum. Correlation with neuronal injury. Neuroscience 113(4):857– 869.

APPENDIX Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.neuroscience.2009.11.012.

(Accepted 4 November 2009) (Available online 10 November 2009)