Brain Advance Access originally published online on November 25, 2005
Brain 2006 129(1):96-107; doi:10.1093/brain/awh673
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Up-regulation of hippocampal metabotropic glutamate receptor 5 in temporal lobe epilepsy patients
Rudolf Magnus Institute of Neuroscience, Departments of 1 Pharmacology and Anatomy, 2 Neurosurgery and 3 Neurology, 4 Department of Pathology, University Medical Center Utrecht, Utrecht, The Netherlands and 5 Department of Pharmaceutical Sciences, University of Toronto, Toronto, Ontario, Canada 6 Present address: Department of Pathology, The Ottawa Hospital, Ottawa, Ontario K1Y 4E9, Canada
Correspondence to: Dr R. G. E. Notenboom, Rudolf Magnus Institute of Neuroscience, Department of Pharmacology and Anatomy, University Medical Center Utrecht, P.O. Box 85060, 3508 AB Utrecht, The Netherlands E-mail: r.g.e.notenboom{at}med.uu.nl
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Summary |
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Metabotropic glutamate receptors (mGluRs) are G protein-coupled receptors involved in the regulation of glutamatergic transmission. Recent studies indicate that excitatory group I mGluRs (mGluR1 and mGluR5) contribute to neurotoxicity and hyperexcitability during epileptogenesis. In this study, we examined the distribution of mGluR1
and mGluR5 immunoreactivity (IR) in hippocampal resection tissue from pharmaco-resistant temporal lobe epilepsy (TLE) patients. IR was detected with panels of receptor subtype specific antisera in hippocampi from TLE patients without (non-HS group) and with hippocampal sclerosis (HS group) and was compared with that of non-epileptic autopsy controls (control group). By immunohistochemistry and immunoblot analysis, we found a marked increase of mGluR5 IR in hippocampi from the non-HS compared with the control group. High mGluR5 IR was most prominent in the cell bodies and apical dendrites of hippocampal principal neurons and in the dentate gyrus molecular layer. In the HS group, this increase in neuronal mGluR5 IR was even more pronounced, but owing to neuronal loss the number of mGluR5-immunoreactive neurons was reduced compared with the non-HS group. IR for mGluR1 was found in the cell bodies of principal neurons in all hippocampal subfields and in stratum oriens and hilar interneurons. No difference in mGluR1 IR was observed between neurons in both TLE groups and the control group. However, owing to neuronal loss, the number of mGluR1-positive neurons was markedly reduced in the HS group. The up-regulation of mGluR5 in surviving neurons is probably a consequence rather than a cause of the epileptic seizures and may contribute to the hyperexcitability of the hippocampus in pharmaco-resistant TLE patients. Thus, our data point to a prominent role of mGluR5 in human TLE and indicate mGluR5 signalling as potential target for new anti-epileptic drugs.
Key Words: hippocampus; human; immunocytochemistry; metabotropic glutamate receptors; temporal lobe epilepsy
Abbreviations: HS = hippocampal sclerosis; IR = immunoreactivity; mGluR = metabotropic glutamate receptor; TLE = temporal lobe epilepsy
Received February 7, 2005. Revised August 30, 2005. Accepted September 30, 2005.
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Introduction |
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Mesial temporal lobe epilepsy (TLE) is the most common form of epilepsy in adult humans and usually associated with hippocampal sclerosis (HS), a characteristic pattern of hippocampal cell loss (Margerison and Corsellis, 1966
; Houser, 1999), but can also be lesional or cryptogenic (Engel, 1996). Approximately 30% of the patients suffering from TLE are pharmaco-resistant, which means that seizures cannot be controlled by tolerable doses of anti-epileptic drugs. Surgical resection of the epileptogenic tissue, in particular the hippocampus, most often results in seizure control, implicating the hippocampus in the generation and/or propagation of seizures in these patients (Mathern et al., 1995b; Zentner et al., 1995).
The molecular mechanisms underlying human TLE are still largely unknown (reviewed, Dalby and Mody, 2001). Excessive synaptic excitation mediated by glutamate interacting with ionotropic and metabotropic glutamate receptors (mGluRs) has been recognized as an important process in the pathophysiology underlying TLE. In hippocampal resection tissue from TLE patients, alterations in AMPA (-amino-3-hydroxy-5-methyl-4-isoxazole propionate), kainate and NMDA (N-methyl-D-aspartate) receptor subunit number or distribution have been reported (reviewed, Meldrum et al., 1999; see also Mathern et al., 1998, 1999). Only limited information is available on the expression of mGluRs in human TLE (Blümcke et al., 2000; Lie et al., 2000; Tang and Lee, 2001; Tang et al., 2001).
The mGluRs are G protein-coupled receptors, which modulate neuronal excitability and synaptic transmission by regulating the activity of various membrane ion channels and intracellular second-messenger systems (for a review see Cartmell and Schoepp, 2000). Eight mammalian mGluR subtypes (mGluR1–mGluR8) have been cloned and assigned to three groups on the basis of their sequence homology, second messenger coupling and pharmacology. Group I consists of mGluR1 and mGluR5, which couple primarily to the activation of phospholipase C/phosphoinositide hydrolysis in heterologous expression systems. Group II and III include all other subtypes and couple negatively to adenylyl cyclase/cyclic AMP formation but differ in their agonist selectivity. In general, group I mGluRs are considered to be excitatory, whereas group II and III receptors have predominantly inhibitory properties (Cartmell and Schoepp, 2000).
In vitro and in vivo studies have shown a pivotal role of hippocampal group I mGluRs in epileptogenesis (reviewed, Bordi and Ugolini, 1999; see also Merlin, 1999, 2002). Further diversity in group I mGluRs is generated by alternative splicing at the C-terminal cytoplasmic domain. Three splice variants have been described for the human mGluR1 receptor ( or a, ß or b, and d) (Desai et al., 1995; Laurie et al., 1996; Stephan et al., 1996; Lin et al., 1997) and three for human mGluR5 (a, b and d) (Minakami et al., 1994; Daggett et al., 1995; Malherbe et al., 2002).
In situ hybridization studies have indicated a widespread, but discrete, distribution of group I mGluR subtypes in the human brain (Daggett et al., 1995; Lin et al., 1997; Malherbe et al., 2002). In the human hippocampus, mGluR1 mRNAs are expressed with the highest density in neurons of the CA3 region and dentate gyrus, whereas mGluR5 mRNAs are most predominant in the CA1 region. A similar distribution pattern has been reported for both receptor subtypes by immunohistochemistry (Blümcke et al., 1996).
To date only two studies have been published on the expression of group I mGluRs in human TLE. The first one studied mGluR1 and mGluR5 in the hippocampus of non-HS and HS TLE patients (Blümcke et al., 2000). These authors report up-regulation of mGluR1 immunoreactivity (IR) and similar or slightly reduced mGluR5 expression in the dentate gyrus molecular layer of TLE patients as compared with a limited number of peritumoral control specimens. Yet the second immunocytochemical study, using only biopsies of HS patients, describes high mGluR1 and mGluR5 IR in the dentate gyrus molecular layer and of mGluR5 in hippocampal pyramidal neurons (Tang et al., 2001).
In view of the importance of group I mGluRs as potential targets for pharmacological treatment of medically intractable chronic epilepsies (see Spooren et al., 2001; Gasparini et al., 2002) a detailed description of their expression in human TLE is essential. We, therefore, analysed mGluR1 and mGluR5 IR in the hippocampus of pharmaco-resistant TLE patients with and without HS and compared it with non-epileptic autopsy controls.
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Materials and methods |
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Patient evaluation and tissue collection
Hippocampal tissue of pharmaco-resistant TLE patients with complex partial seizures was obtained at surgery. Patients were selected for epilepsy surgery according to the criteria of the Dutch Epilepsy Surgery Program (Debets et al., 1991
). Surgical removal of the hippocampus was necessary in all patients to achieve seizure control and was performed under general or local anaesthesia. The excision was based on clinical evaluations, interictal and ictal EEG studies (video EEG monitoring), MRI, and intraoperative electrocorticography. Informed and written consent was obtained from the patients for all procedures. After en bloc resection, the hippocampus was cut into three slices perpendicular to its long (rostrocaudal) axis and the middle portion was used for analysis. Tissue samples for immunohistochemistry were immediately immersion-fixed in 4% formaldehyde (pH 7.4) overnight at room temperature and embedded in paraffin. Samples for immunoblotting were freshly frozen on powdered dry ice and stored at –80°C until further use. Hippocampal control tissue was obtained at autopsy and processed as described for surgical resection samples.
To determine post-mortem stability of mGluR1 and mGluR5 epitopes human neocortical tissue was subjected to autopsy-like conditions. In brief, neocortical tissue resected from HS TLE patients during selective amygdalohippocampectomy was cut into 10 mm thick slices and was immersion-fixed in 4% formaldehyde immediately or after 12 or 24 h storage at ambient temperature in PIPES (1,4-piperazinediethanesulphonic acid) buffer (in mM: 120 NaCl, 5 KCl, 1 CaCl2, 1 MgCl2, 20 Pipes, 25 D-glucose; pH 7.0) until further processing as described for hippocampal tissue. mGluR5 IR was found to be stable for at least 24 h post-mortem and mGluR1 IR for at least 12 h. At 24 h post-mortem overall mGluR1 IR and the number of mGluR1-immunoreactive neurons was slightly decreased. Therefore, in this study we have used autopsy controls with post-mortem delays <17 h. In this autopsy control group no difference could be detected in hippocampal mGluR1 or mGluR5 between patients with various post-mortem delays (ranging from 5 to 17 h).
For neuropathological evaluation representative paraffin sections (7 µm) were stained with cresyl violet (Nissl stain). HS was diagnosed and classified according to Wyler (Wyler et al., 1992). Only tissue samples from non-HS (Wyler grade 0; n = 12) and severe HS (Wyler grade 4; n = 12) cases were used. In the non-HS group, pathological diagnoses included oligodendroglioma (n = 1), astrocytoma and related low-grade astrocytic tumours (n = 2) or glioneuronal neoplasms (n = 4), cavernous haemangioma (n = 1), neocortical gliosis (n = 2) or dysplasia (n = 1). None of these focal lesions extended into the hippocampus proper. In one patient, neither hippocampal nor extrahippocampal pathology could be demonstrated by MRI and histopathological examination. The HS group included patients without focal lesions, who generally had a clinical history of an initial precipitating injury and in whom the hippocampus was characterized by extensive neuronal loss and gliosis in all CA segments (Fig. 2C and F; compare with Fig. 2A and B, and 2D and E, respectively; see also Proper et al., 2000). Initial precipitating injuries (Mathern et al., 1995a) included febrile convulsions (n = 4), infantile meningitis with febrile seizures (n = 2) or significant head trauma (n = 1). Three patients had no known initial insult and for two patients these data had not been recorded. One HS patient had dual pathology with a low-grade ganglioglioma in the neocortex. At the time of surgery, all TLE patients received anti-epileptic medication either as monotherapy or polytherapy. Two non-HS and one HS TLE patient were treated with carbamazepine monotherapy. Two non-HS patients were on monotherapy with oxcarbazepine and one patient with clobazam. Seven non-HS and eleven HS TLE patients received various combinations of the following anti-epileptic drugs: carbamazepine, clobazam, clonazepam, lamotrigine, oxcarbazepine, phenytoin, topiramate and valproic acid. In the autopsy control group (n = 12), cardiovascular failure (n = 3), cerebral (n = 2) or cerebellar (n = 1) haemorrhage, cerebral metastases (n = 1), cerebral trauma (n = 1), herniation due to acute vascular accident (n = 1), ileus (n = 1) or septic shock (n = 2) was recorded as cause of death. None of the autopsy controls had a history of neurological or psychiatric disorders and all hippocampal specimens were normal as confirmed by neuropathological examination.
Relevant clinical data for autopsy controls and TLE patients included in this study are summarized in Table 1. Multiple group comparison or 
2 tests between the autopsy control, non-HS and HS group revealed only one significant difference in all the clinical parameters. The mean age at tissue collection in the control group was higher than in the non-HS group (P = 0.02, post hoc Bonferroni test) but did not differ between the control and HS group (post hoc P = 0.14) or the non-HS and HS group (post hoc P = 1.00).
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Antibodies
Panels of affinity purified rabbit antisera against mGluR1
or mGluR5 were employed. The mGluR1 antiserum (#AB1595; Chemicon, Temecula, CA) used in this study is directed against amino acids 1116–1130 of the rat mGluR1 splice variant (Houamed et al., 1991; Masu et al., 1991). This amino acid sequence differs only at one residue from the human mGluR1 1108–1124 (Desai et al., 1995; Stephan et al., 1996) and exhibits no similarity to other known mGluRs based on alignment of reported sequences. The mGluR5 antiserum (#06–451; Upstate, Lake Placid, NY) was raised against a synthetic peptide containing the C-terminal 20 amino acids of the receptor. This amino acid sequence is common to rat mGluR5a (Abe et al., 1992) and mGluR5b (Minakami et al., 1993), and to the human a, b and d splice variants (Minakami et al., 1994; Daggett et al., 1995; Malherbe et al., 2002). Additional group I mGluR antisera (listed in Table 2) included three mGluR1 and two mGluR5 affinity purified rabbit anti-peptide antibodies. Specificity characteristics of these antisera have been described elsewhere (Hampson et al., 1994; Ferraguti et al., 1998; Alvarez et al., 2000). Glial fibrillary acidic protein (GFAP) was detected with a mouse monoclonal antibody (clone G-A-5; Boehringer, Mannheim, Germany; diluted 1 : 50).
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Immunoblotting
Homogenates from human hippocampus were prepared as reported earlier (van der Hel et al., 2005
). The subiculum was removed from all hippocampal specimens before homogenization. Synaptosomal plasma membranes were isolated from hippocampal homogenates as described by Kristjansson et al. (1982). Rat brain microsomal proteins were from Upstate. Proteins were heated for 30 min at 37°C, separated on 7% SDS–PAGE (sodium dodecyl sulphate–polyacrylamide gel electrophoresis) gels, and transferred to nitrocellulose membranes by semi-dry blotting. Equal protein loading was checked by Ponceau-S staining. Blots were stained with antibodies according to the manufacturer's instructions (Upstate). IR was detected with peroxidase-conjugated goat anti-rabbit immunoglobulins (diluted 1 : 15 000–30 000; Sigma Chemical Co., St Louis, MO). The blots were developed with enhanced chemiluminescent substrate (SuperSignal® West Dura Extended Duration Substrate; Pierce, Rockford, IL) and apposed to high performance chemiluminescence film (HyperfilmTM ECL; Amersham, Buckinghamshire, UK). Prestained Precision ProteinTM standards were from Bio-Rad (Hercules, CA). In negative controls, the primary antiserum was omitted. On quantitative immunoblots, chemiluminescent signals were captured with a Fluor-S® Multi-Imager (Bio-Rad) and analysed using Bio-Rad's Quantity One® software. Band intensities were measured in duplicate for each homogenate under conditions of signal linearity. Relative optical densities corrected for background were averaged per patient and the mean was used for statistical analysis.
Immunohistochemistry
Immunostaining was performed on dewaxed, 7 µm thick serial sections by the indirect unlabelled antibody peroxidase–antiperoxidase method (Sternberger et al., 1970). Per primary antiserum, tissue sections from all patients were stained simultaneously to minimize variation in the immunohistochemical reactions. Endogenous peroxidase activity was blocked with 3% H2O2 in phosphate-buffered saline (PBS). Tissue sections to be stained for mGluR1 or with the mGluR5 US Biological (Swampscott, MA; #M3884–79) antiserum were immersed in 10 mM sodium citrate (pH 6.0) for 20 min and microwaved in the same solution for 7 min at 750 W to expose immunoreactive sites (Shi et al., 1991). Non-specific binding of immunoglobulins was reduced with 0.5% non-fat dry milk powder in 10 mM Tris–HCl, 5 mM EDTA (ethylenediaminetetraacetate), 150 mM NaCl, 0.25% gelatin and 0.05% Tween-20 (pH 8.0). Primary antisera (diluted in PBS) were applied overnight at room temperature. Optimal working concentrations (Table 2) were determined by serial dilutions on rat brain and human autopsy tissue. Goat anti-rabbit immunoglobulin G (H + L) serum adsorbed with human, mouse and rat serum proteins (Jackson Immunoresearch, West Grove, PA) or rabbit anti-mouse immunoglobulin G (H + L) serum (diluted 1 : 250) and the appropriate peroxidase–antiperoxidase immunocomplexes (diluted 1 : 750) (Nordic, Tilburg, The Netherlands) were applied in PBS for 1.5 h each. For pre-adsorption controls, the primary antisera were incubated overnight at 4°C with 50 (mGluR1) or 100 (mGluR5) µg/ml of blocking peptide and then applied to the sections. Antiserum samples without blocking peptide were run in parallel. After each incubation, the sections were thoroughly rinsed in PBS. IR was visualized with 0.05% 3,3'-diaminobenzidine tetrahydrochloride (Sigma Chemical Co.) in 25 mM imidazole, 1 mM EDTA (pH 7.0) containing 0.01% H2O2. Colour was developed for 15 min. Controls without the primary antisera did not reveal staining (data not shown).
Specificity of the mGluR antisera
Adult male Wistar (U:WU; GDL, Utrecht, The Netherlands; 250–300 g; n = 3) or Sprague–Dawley (Hsd:SD; Harlan Winkelmann GmbH, Borchen, Germany; 250–300 g; n = 3) rats were used in testing the specificity of the mGluR antisera. Rat experiments conformed to institutional guidelines. Rats were either killed by decapitation or by transcardial perfusion (5–10 min) with 4% formaldehyde under deep pentobarbital (200 mg/kg body weight, i.p.) anaesthesia. Their brains were rapidly removed from the skull and then processed and immunostained as described for human tissue. No differences in the staining pattern of the different mGluR antibodies were observed between the two rat strains or fixation methods.
The specificity of the mGluR antisera was evaluated in several ways. First, on immunoblots loaded with rat brain microsomal proteins, the mGluR1 and mGluR5 antisera labelled a single protein band with an estimated molecular weight of 150 kDa and 145 kDa, respectively (Fig. IA in the Supplementary material), consistent with previous reports (cf. Baude et al., 1993; Shigemoto et al., 1993; Hampson et al., 1994; Romano et al., 1995). Immunoreactive bands on immunoblots of synaptosomal plasma membrane (Fig. 1A) or homogenate (e.g. Fig. 6A) proteins from human hippocampus showed similar molecular weights as in rat. Secondly, in immunohistochemistry on whole rat brain sections, both antisera produced distinct and highly reproducible IR patterns in various brain regions (Supplementary Fig. IB, C, D, H, J and N). The specificity of the mGluR5 antiserum was further confirmed in regions known to express high levels of mGluR1 IR (cerebellar cortex, thalamus) but virtually no or only low mGluR5 (cf. Martin et al., 1992; Baude et al., 1993; Shigemoto et al., 1993; Hampson et al., 1994; Romano et al., 1995). In addition, the labelling pattern observed with the mGluR1 and mGluR5 antisera (in rat hippocampus and cerebellum) was identical to the immunostaining obtained, respectively, with the mGluR1 #m1a (Supplementary Fig. IE and K) or Diasorin (Stillwater, MN; #24426) (Supplementary Fig. IF and L) and the mGluR5 US Biological antiserum (data not shown). These antisera have been characterized in detail previously (Hampson et al., 1994; Ferraguti et al., 1998; Alvarez et al., 2000). Finally, in immunohistochemistry on human and rat brain tissues, pre-adsorption of the antisera with appropriate peptide sequences from rat mGluR1 (amino acids 1116–1130; Diasorin) or mGluR5 (amino acids 1159–1171; US Biological) resulted in a loss of signal (e.g. Fig. 1B; see also Supplementary Fig. IG, I, M and O).
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The other mGluR antisera tested on rat and human hippocampal sections produced no reliable (Chemicon's mGluR5, #AB5232) or an overlapping IR pattern (Chemicon's mGluR1
, #AB1551) with the mGluR5 antiserum used in this study (data not shown). This latter antiserum (#AB1551; directed against the C-terminal 20 amino acids) should, therefore, probably be considered a group I rather than an mGluR1 specific antiserum (cf. Ferraguti et al., 1998; Alvarez et al., 2000) and, therefore, was not further used in this study.
Statistical analysis
Densitometric measurements were analysed for significant group differences using one-way ANOVA, combined with post hoc Bonferroni test as a multiple comparison method. P < 0.05 was considered significant.
Nomenclature
The hippocampal nomenclature used in this study is essentially as described by Amaral and Insausti (1990), except that the proximal part of the CA3 pyramidal layer (also termed ‘end blade’ or ‘endfolium;’ Margerison and Corsellis, 1966) that inserts into the dentate gyrus is referred to as CA4 as in our previous studies (Proper et al., 2000).
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mGluR1
immunoreactivityThe mGluR1
antiserum labelled the perikarya and proximal apical dendrites of principal neurons and interneurons in the control hippocampus (Figs 2G, 3A, 4A and 5A). The strongest IR was observed within the hippocampus proper in pyramidal neurons of CA2 and CA3 (Fig. 2G). Labelling of CA1 and CA4 pyramidal neurons was less intense. Immunoreactive neuronal cell bodies were also observed in CA1–3 stratum oriens, consistent with a distribution of a subpopulation of somatostatin-containing interneurons (cf. Chan-Palay, 1987). In the dentate gyrus, granule cells and polymorphic hilar neurons were consistently immunoreactive (Figs 2G and 5A). Glial cells as identified by GFAP IR in adjacent sections were negative for mGluR1 IR. A similar pattern of mGluR1 IR was obtained with the mGluR1 #m1a and Diasorin antisera (data not shown).
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Non-HS and HS TLE hippocampi showed a similar mGluR1
IR pattern as the controls (Fig. 2H and I; compare with Fig. 2G). However, owing to severe neuronal loss as revealed by Nissl staining, fewer mGluR1-immunopositive neurons were present in the HS hippocampus (Fig. 2C and I; compare with Fig. 2A and B, and 2G and H, respectively). No consistent difference in the staining intensity for mGluR1 IR was found between neurons in the control and non-HS or HS group (Figs 3A–C, 4A–C and 5A–C). In both patient groups, glial cells were negative for mGluR1 IR.
mGluR5 immunoreactivity
The mGluR5 antiserum yielded an immunoreactive staining pattern in control tissue similar to that described previously for the human hippocampus (Blümcke et al., 1996). A dense and homogeneously distributed neuropil staining decorated the pyramidal cell layer and stratum lacunosum-moleculare of CA1–3 (Fig. 2J). Staining of these layers was strongest in CA1 and less intense in CA3. IR for mGluR5 in CA4 was extremely weak (Figs 2J and 4D). In the dentate gyrus (Figs 2J and 5D), mGluR5 IR consistently labelled the outer molecular layer. In contrast, IR in the inner molecular layer was variable. The polymorphic layer of the dentate hilus exhibited virtually no detectable staining. Neuronal cell bodies in the control hippocampus were almost devoid of IR, except in CA1 where pyramidal neurons showed a dense, cytoplasmic staining of the perikarya (Fig. 3D; compare with e.g. Fig. 4D). The cytoplasm within dendritic shafts of CA1 pyramidal cells exhibited no or only weak IR. In the control tissue, no staining for mGluR5 was seen in individual glial cells.
The most striking finding in both TLE patient groups was an increased intracellular staining of principal neurons. In the HS group, the perikarya and apical dendrites of virtually all surviving pyramidal neurons displayed this strong, cytoplasmic staining (Figs 3F and 4F). In the dentate gyrus, granule cell bodies and basal dendrites often were also intensely stained (Fig. 5F). This staining pattern was also apparent in the non-HS group, but fewer strongly mGluR5-immunoreactive neurons were found and in these neurons strong staining was more confined to pyramidal shafts (Figs 1B and 3E) or dendritic processes (Fig. 4E). In the non-HS group, mGluR5 IR of the granule cells (cell bodies and basal dendrites) varied between patients. In some patients granule cells were almost devoid of staining, whereas in others about one-third of the cell population was moderately stained (Fig. 5E).
A further interesting finding was the strong neuropil staining in the non-HS and HS group compared with control tissue. However, in the HS group in regions with marked sclerosis, such as CA1 and CA4, neuropil IR was less dense (Fig. 2L; compare with Fig. 2C, asterisks). In both the non-HS and HS group, prominent mGluR5 IR was apparent in the neuropil of the dentate gyrus outer molecular layer (Fig. 2K and L). The staining for mGluR5 IR in this sublayer was consistently stronger in the HS group (Fig. 5F) than in the non-HS group (Fig. 5E). Except for some strongly immunoreactive dendritic profiles in the supragranular zone, IR for mGluR5 in the inner molecular layer of the HS hippocampus did not differ much from that in the control hippocampus (Fig. 5F; compare with Fig. 5D). In contrast, in the non-HS hippocampus the inner molecular layer showed a stronger neuropil staining compared with control and sclerotic tissue (Fig. 5E; compare with Fig. 5D and F). Moreover, in the non-HS group, a discrete, punctate staining decorated the granule cell apical dendrites in the supragranular zone.
In the HS group, mGluR5 IR was also observed in a subset of strongly GFAP-immunoreactive glial cells (data not shown); most notably in those hippocampal segments with substantial neuronal loss and gliosis. Although the nature of these cells was not further investigated, they resembled reactive astrocytes by their morphological appearance. In the non-HS hippocampus, mGluR5-immunoreactive glial cells were only observed in regions of poor neuropil staining such as the alveus or close to the hippocampal fissure.
All mGluR5 IR was specific, because immunostaining (i.e. including that in non-HS or HS neuronal cell bodies and apical dendrites) could be abolished by pre-adsorption with the mGluR5 blocking peptide (Fig. 1B). Moreover, similar IR patterns were obtained with the US Biological antibody (data not shown).
Immunoblot analysis of mGluR5 in control, non-HS and HS hippocampi revealed a single immunoreactive band of 
145 kDa (Fig. 6A) in all samples. Consistent with our immunohistochemical data, mGluR5 IR was significantly increased in hippocampal homogenates from the non-HS compared with the control and HS group (15-fold and 7.5-fold, respectively) (Fig. 6B; P < 0.001), but mGluR5 IR did not differ significantly between the control and the HS group (Fig. 6B; post hoc P = 1.00).
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The main conclusion that emerges from this study is that mGluR5 IR is significantly up-regulated in the epileptogenic hippocampus from TLE patients as compared with non-epileptic controls. The most drastic increase in mGluR5 IR was observed in hippocampal principal neurons (i.e. pyramidal and granule cells) and in the dentate gyrus molecular layer. This increase in mGluR5 IR was found in the hippocampus of both TLE patient groups. No differences in mGluR1
IR were observed between hippocampi from TLE patients with or without HS, and non-epileptic controls.
IR for mGluR1 in the hippocampus from autopsy controls and in the rat was localized to the somata and proximal dendrites of interneurons, particularly in the stratum oriens and the hilus, and was also detectable in principal neurons. This distribution pattern was observed with all three mGluR1 antibodies (against a unique proline/glutamine-rich region of the mGluR1 molecule; Houamed et al., 1991; Masu et al., 1991) and could be completely abolished by immuno-adsorption with a corresponding synthetic mGluR1 peptide. The pattern correlates well with that found by in situ hybridization for mRNAs coding for all mGluR1 (Masu et al., 1991; Shigemoto et al., 1992; Lin et al., 1997) and by immunohistochemistry for the human ß splice variant (Blümcke et al., 1996). In situ hybridization studies aimed to identify the expression of individual mGluR1 splice variants, however, have failed to detect mGluR1 transcripts in hippocampal principal neurons (Berthele et al., 1998). In view of the relatively low abundance of mGluR1 protein in principal neurons it is not surprising that others had difficulties in detecting the mGluR1 transcript. Previous immunohistochemical studies on rat brain also do not consistently report mGluR1 IR in hippocampal principal neurons (Martin et al., 1992; Baude et al., 1993; Hampson et al., 1994; Ferraguti et al., 1998, 2004; Alvarez et al., 2000; but see Defagot et al., 2002). Most likely, the inconsistency in detecting mGluR1 IR in hippocampal principal neurons is due to limits in sensitivity of methodologies used. Here we used 7 µm paraffin sections and antigen unmasking techniques based on microwave irradiation (Shi et al., 1991). In fact, using serial dilutions of the mGluR1 antibodies staining was first lost in principal neurons in CA1, then in CA3, and finally in interneurons. Thus, we found staining in all principal neurons varying from strong IR in CA3 to light IR in CA1. The presence of mGluR1 in hippocampal principal neurons is consistent with electrophysiological data showing a role for mGluR1 in regulating pyramidal cell function (Mannaioni et al., 2001).
The mGluR1 IR patterns in the hippocampus of the non-HS and the HS group and autopsy controls were similar. As expected, sclerosis-related alterations in hippocampal morphology were visible in the neuronal mGluR1-staining pattern in the HS group. HS is characterized by severe neuronal cell loss and dispersion of the granule cell layer (Margerison and Corsellis, 1966; Houser, 1990, 1999; Proper et al., 2000). Associated with these pathological alterations, we detected a decrease in the number of mGluR1-immunoreactive neurons (most pronounced in CA1 and CA4) and a broader band of mGluR1-immunoreactive granule cells in the HS hippocampus. We did not observe an increased mGluR1 IR in the dentate gyrus molecular layer in TLE patients, as reported by Tang et al. (2001) and Blümcke et al. (2000), but found such an increase for mGluR5 IR. This apparent discrepancy may not be so surprising, because in control experiments on rat brain tissue (see Material and methods) we found that the mGluR1 antiserum used in the study of Blümcke shows cross-reactivity with mGluR5. Other studies using this antibody, including studies in mice lacking mGluR1, confirm cross-reactivity with mGluR5 (Ferraguti et al., 1998; Alvarez et al., 2000). It is, therefore, likely that the increased labelling in the dentate gyrus molecular layer in the aforementioned human studies represents mGluR5 rather than mGluR1 IR. The mGluR1 antiserum that we used in this study was raised against unique epitopes of the rat mGluR1 splice variant and showed a consistent neuronal somatodendritic distribution in rat and human hippocampus.
In the control group, mGluR5 IR was most pronounced in the dendritic compartments (cf. Blümcke et al., 1996), but in TLE patients mGluR5 IR was also detected in hippocampal principal neurons and glial cells. The most drastic increase in mGluR5 IR in TLE patients (compared with autopsy controls) was observed in the cell bodies and main dendritic processes of pyramidal neurons and granule cells. IR for mGluR5 in neuronal cell bodies has also been described in other CNS regions (Romano et al., 1995; Alvarez et al., 2000). Thus, whereas neuronal mGluR5 IR was weak in autopsy controls, it was markedly increased in the non-HS group and very prominent in the HS group. Immunoblot experiments confirmed the increase in mGluR5 IR in the non-HS hippocampus, but the increase in the HS hippocampus did not reach significance. Conceivably, the severe neuron loss in the HS hippocampus masks the large increase of mGluR5 in individual neurons.
The increase in mGluR5 IR in hippocampal neurons in TLE patients is not due to a reduced IR in the autopsy control group (e.g. as a result of post-mortem protein degradation and/or modification) as control experiments with freshly resected neocortical biopsies kept under post-mortem conditions showed mGluR5 IR to be stable for at least 24 h. Moreover, the distribution of mGluR5 IR in our autopsy tissue closely resembled the pattern described for surgically removed (i.e. freshly excised) peritumoral hippocampal tissue from non-epileptic patients (Blümcke et al., 1996). Of course, the observed increase in neuronal mGluR5 IR in the HS compared with the non-HS group cannot be attributed to post-mortem effects. Thus, our results show that mGluR5 IR is up-regulated in hippocampal neurons of patients with pharmaco-resistant TLE.
A further interesting finding was the increase in mGluR5 IR in the outer molecular layer of the dentate gyrus in the HS and the non-HS group. In the dentate inner molecular layer mGluR5 IR is decreased in the HS compared with the non-HS group. Because patients in both groups have a long history of seizures, this decrease in mGluR5 IR is likely to be pathology-related rather than seizure-related. In HS, extensive synaptic reorganization occurs in the inner molecular layer concomitant with mossy fibre sprouting (reviewed, Houser, 1999; see also Proper et al., 2000). Possibly decreased expression of mGluR5 in this hippocampal sublayer is part of a compensatory mechanism in response to increased, recurrent excitation caused by mossy fibre sprouting.
The increase in mGluR5 IR in hippocampal neurons and the dentate molecular layer is in agreement with a study by Tang et al. (2001). These authors studied mGluR5 localization in TLE patients by light and electron microscopy and found high mGluR5 IR in both post-synaptic and pre-synaptic elements in the molecular layer of the dentate gyrus and hippocampal CA1 region, but this study did not include a non-HS nor an autopsy control group. Blümcke et al. (2000) have described a slight decrease in mGluR5 IR in the dentate gyrus molecular layer of TLE patients with HS, as compared with peritumoral hippocampal specimens from non-epileptic controls, but did not illustrate this.
In the HS group, mGluR5 IR was also found in glia-like cells. Since in these patients massive gliosis has been described (Margerison and Corsellis, 1966; Wyler et al., 1992; Proper et al., 2000), these cells may represent reactive astrocytes. Up-regulation of hippocampal mGluR5 expression in reactive astrocytes has been documented in kainic acid-lesioned rats (Ulas et al., 2000) and mice (Ferraguti et al., 2001), and in rats after electrically-induced status epilepticus (Aronica et al., 2000). Expression of mGluR5 in glia is consistently found in gliotic human brain tissue from epileptic (Aronica et al., 2001; Tang et al., 2001) as well as non-epileptic (Blümcke et al., 1996; Condorelli et al., 1997; Geurts et al., 2003) patients. Whether the mGluR5-immunoreactive cells in the HS hippocampus are reactive astrocytes needs to be investigated.
It is important to realize that studies on human epileptic tissue suffer from a number of limitations. For instance, only a selected group of TLE patients is treated for their epilepsy by surgical removal of the epileptogenic tissue. These pharmaco-resistant TLE patients suffer from a highly progressed state of epilepsy and have had seizures for many years. Thus, it is not easy to determine whether the up-regulation of mGluR5 IR in hippocampal neurons is an underlying cause or merely a consequence of the repeated seizures (for a discussion see Meldrum, 1997). Conceivably, the up-regulation of mGluR5 receptors is a consequence rather than a cause of the epileptic seizures. It is unlikely that the increase in mGluR5 expression is drug-induced, because anti-epileptic drug treatment did not differ between TLE groups. Also the broad spectrum of anti-epileptic drugs prescribed makes a general effect on mGluR5 expression unlikely. This up-regulation of mGluR5 in surviving neurons may contribute to the hyperexcitability of the hippocampus. Alternatively, it may be part of a mechanism to protect against overexcitation and neurotoxicity (Nicoletti et al., 1999). Although activation of mGluR5 receptors normally seems to enhance glutamatergic transmission, their function may switch to inhibiting glutamatergic transmission when mGluR5 receptors become desensitized upon exposure to glutamate (Herrero et al., 1998; Rodríguez-Moreno et al., 1998; reviewed, De Blasi et al., 2001). Further physiological and pharmacological studies must clarify whether high mGluR5 expression in TLE corresponds to increased functional receptor activity. If high mGluR5 expression indeed contributes to hyperexcitability of the hippocampal network, mGluR5 signalling might be a potential target for new anti-epileptic drugs.
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TopSummaryIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
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Supplementary data are available at Brain Online.
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Acknowledgements |
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We wish to thank Drs Cas Kruitwagen and Wiebe Pestman (Centre for Biostatistics, Utrecht University) for statistical analyses, and Marina de Wit and Marije Rensen for assistance with the immunoblotting experiments. This work was supported by The Epilepsy Fund of The Netherlands (grant 98–16) and the Foundation Focal Epilepsies of Childhood.
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|---|
Abe T, Sugihara H, Nawa H, Shigemoto R, Mizuno N, Nakanishi S. Molecular characterization of a novel metabotropic glutamate receptor mGluR5 coupled to inositol phosphate/Ca2+ signal transduction. J Biol Chem 1992; 267: 13361–8.
Alvarez FJ, Villalba RM, Carr PA, Grandes P, Somohano PM. Differential distribution of metabotropic glutamate receptors 1a, 1b, and 5 in the rat spinal cord. J Comp Neurol 2000; 422: 464–87.[CrossRef][Web of Science][Medline]
Amaral DG, Insausti R. Hippocampal formation. In: Paxinos G, editor. The human nervous system. San Diego: Academic Press; 1990. p. 711–55.
Aronica E, van Vliet EA, Mayboroda OA, Troost D, Lopes da Silva FH, Gorter JA. Upregulation of metabotropic glutamate receptor subtype mGluR3 and mGluR5 in reactive astrocytes in a rat model of mesial temporal lobe epilepsy. Eur J Neurosci 2000; 12: 2333–44.[CrossRef][Web of Science][Medline]
Aronica E, Yankaya B, Jansen GH, Leenstra S, van Veelen CWM, Gorter JA, et al. Ionotropic and metabotropic glutamate receptor protein expression in glioneuronal tumours from patients with intractable epilepsy. Neuropathol Appl Neurobiol 2001; 27: 223–37.[CrossRef][Web of Science][Medline]
Baude A, Nusser Z, Roberts JDB, Mulvihill E, McIlhinney RAJ, Somogyi P. The metabotropic glutamate receptor (mGluR1) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction. Neuron 1993; 11: 771–87.[CrossRef][Web of Science][Medline]
Berthele A, Laurie DJ, Platzer S, Zieglgänsberger W, Tölle TR, Sommer B. Differential expression of rat and human type 1 metabotropic glutamate receptor splice variant messenger RNAs. Neuroscience 1998; 85: 733–49.[CrossRef][Web of Science][Medline]
Blümcke I, Behle K, Malitschek B, Kuhn R, Knöpfel T, Wolf HK, et al. Immunohistochemical distribution of metabotropic glutamate receptor subtypes mGluR1b, mGluR2/3, mGluR4a and mGluR5 in human hippocampus. Brain Res 1996; 736: 217–26.[CrossRef][Web of Science][Medline]
Blümcke I, Becker AJ, Klein C, Scheiwe C, Lie AA, Beck H, et al. Temporal lobe epilepsy associated up-regulation of metabotropic glutamate receptors: correlated changes in mGluR1 mRNA and protein expression in experimental animals and human patients. J Neuropathol Exp Neurol 2000; 59: 1–10.[Web of Science][Medline]
Bordi F, Ugolini A. Group I metabotropic glutamate receptors: implications for brain diseases. [Review]. Prog Neurobiol 1999; 59: 55–79.[CrossRef][Web of Science][Medline]
Cartmell J, Schoepp DD. Regulation of neurotransmitter release by metabotropic glutamate receptors. [Review]. J Neurochem 2000; 75: 889–907.[CrossRef][Web of Science][Medline]
Chan-Palay V. Somatostatin immunoreactive neurons in the human hippocampus and cortex shown by immunogold/silver intensification on vibratome sections: coexistence with neuropeptide Y neurons, and effects in Alzheimer-type dementia. J Comp Neurol 1987; 260: 201–23.[CrossRef][Web of Science][Medline]
Condorelli DF, Dell'Albani P, Corsaro M, Giuffrida R, Caruso A, Trovato Salinaro A, et al. Metabotropic glutamate receptor expression in cultured rat astrocytes and human gliomas. Neurochem Res 1997; 22: 1127–33.[CrossRef][Web of Science][Medline]
Daggett LP, Sacaan AI, Akong M, Rao SP, Hess SD, Liaw C, et al. Molecular and functional characterization of recombinant human metabotropic glutamate receptor subtype 5. Neuropharmacology 1995; 34: 871–86.[CrossRef][Web of Science][Medline]
Dalby NO, Mody I. The process of epileptogenesis: a pathophysiological approach. [Review]. Curr Opin Neurol 2001; 14: 187–92.[CrossRef][Web of Science][Medline]
Debets RM, van Veelen CW, van Huffelen AV, van Emde Boas W. Presurgical evaluation of patients with intractable partial epilepsy: the Dutch epilepsy surgery program. [Review]. Acta Neurol Belg 1991; 91: 125–40.[Web of Science][Medline]
De Blasi A, Conn PJ, Pin J-P, Nicoletti F. Molecular determinants of metabotropic glutamate receptor signaling. [Review]. Trends Pharmacol Sci 2001; 22: 114–20.[CrossRef][Medline]
Defagot MC, Villar MJ, Antonelli MC. Differential localization of metabotropic glutamate receptors during postnatal development. Dev Neurosci 2002; 24: 272–82.[CrossRef][Web of Science][Medline]
Desai MA, Burnett JP, Mayne NG, Schoepp DD. Cloning and expression of a human metabotropic glutamate receptor 1: enhanced coupling on co-transfection with a glutamate transporter. Mol Pharmacol 1995; 48: 648–57.[Abstract]
Engel J Jr. Introduction to temporal lobe epilepsy. [Review]. Epilepsy Res 1996; 26: 141–50.[CrossRef][Web of Science][Medline]
Ferraguti F, Conquet F, Corti C, Grandes P, Kuhn R, Knopfel T. Immunohistochemical localization of the mGluR1ß metabotropic glutamate receptor in the adult rodent forebrain: evidence for a differential distribution of mGluR1 splice variants. J Comp Neurol 1998; 400: 391–407.[CrossRef][Web of Science][Medline]
Ferraguti F, Corti C, Valerio E, Mion S, Xuereb J. Activated astrocytes in areas of kainate-induced neuronal injury upregulate the expression of the metabotropic glutamate receptors 2/3 and 5. Exp Brain Res 2001; 137: 1–11.[CrossRef][Web of Science][Medline]
Ferraguti F, Cobden P, Pollard M, Cope D, Shigemoto R, Watanabe M, et al. Immunolocalization of metabotropic glutamate receptor 1 (mGluR1) in distinct classes of interneuron in the CA1 region of the rat hippocampus. Hippocampus 2004; 14: 193–215.[CrossRef][Web of Science][Medline]
Gasparini F, Kuhn R, Pin J-P. Allosteric modulators of group I metabotropic glutamate receptors: novel subtype-selective ligands and therapeutic perspectives. [Review]. Curr Opin Pharmacol 2002; 2: 43–9.[CrossRef][Web of Science][Medline]
Geurts JJG, Wolswijk G, Bö L, van der Valk P, Polman CH, Troost D, et al. Altered expression patterns of group I and II metabotropic glutamate receptors in multiple sclerosis. Brain 2003; 126: 1755–66.
Hampson DR, Theriault E, Huang X-P, Kristensen P, Pickering DS, Franck JE, et al. Characterization of two alternatively spliced forms of a metabotropic glutamate receptor in the central nervous system of the rat. Neuroscience 1994; 60: 325–36.[CrossRef][Web of Science][Medline]
Herrero I, Miras-Portugal MT, Sánchez-Prieto J. Functional switch from facilitation to inhibition in the control of glutamate release by metabotropic glutamate receptors. J Biol Chem 1998; 273: 1951–8.
Houamed KM, Kuijper JL, Gilbert TL, Haldeman BA, O'Hara PJ, Mulvihill ER, et al. Cloning, expression, and gene structure of a G protein-coupled glutamate receptor from rat brain. Science 1991; 252: 1318–21.
Houser CR. Granule cell dispersion in the dentate gyrus of humans with temporal lobe epilepsy. Brain Res 1990; 535: 195–204.[CrossRef][Web of Science][Medline]
Houser CR. Neuronal loss and synaptic reorganization in temporal lobe epilepsy. [Review]. Adv Neurol 1999; 79: 743–61.[Medline]
Kristjansson GI, Zwiers H, Oestreicher AB, Gispen WH. Evidence that the synaptic phosphoprotein B-50 is localized exclusively in nerve tissue. J Neurochem 1982; 39: 371–8.[CrossRef][Web of Science][Medline]
Laurie DJ, Boddeke HWGM, Hiltscher R, Sommer B. HmGlu1d, a novel splice variant of the human type I metabotropic glutamate receptor. Eur J Pharmacol 1996; 296: R1–3.[CrossRef][Web of Science][Medline]
Lie AA, Becker A, Behle K, Beck H, Malitschek B, Conn PJ, et al. Up-regulation of the metabotropic glutamate receptor mGluR4 in hippocampal neurons with reduced seizure vulnerability. Ann Neurol 2000; 47: 26–35.[CrossRef][Web of Science][Medline]
Lin FF, Varney M, Sacaan AI, Jachec C, Daggett LP, Rao S, et al. Cloning and stable expression of the mGluR1b subtype of human metabotropic receptors and pharmacological comparison with the mGluR5a subtype. Neuropharmacology 1997; 36: 917–31.[CrossRef][Web of Science][Medline]
Malherbe P, Kew JNC, Richards JG, Knoflach F, Kratzeisen C, Zenner M-T, et al. Identification and characterization of a novel splice variant of the metabotropic glutamate receptor 5 gene in human hippocampus and cerebellum. Brain Res Mol Brain Res 2002; 109: 168–78.[Medline]
Mannaioni G, Marino MJ, Valenti O, Traynelis SF, Conn PJ. Metabotropic glutamate receptors 1 and 5 differentially regulate CA1 pyramidal cell function. J Neurosci 2001; 21: 5925–34.
Margerison JH, Corsellis JAN. Epilepsy and the temporal lobes: a clinical, electroencephalographic and neuropathological study of the brain in epilepsy, with particular reference to the temporal lobes. Brain 1966; 89: 499–530.
Martin LJ, Blackstone CD, Huganir RL, Price DL. Cellular localization of a metabotropic glutamate receptor in rat brain. Neuron 1992; 9: 259–70.[CrossRef][Web of Science][Medline]
Masu M, Tanabe Y, Tsuchida K, Shigemoto R, Nakanishi S. Sequence and expression of a metabotropic glutamate receptor. Nature 1991; 349: 760–5.[CrossRef][Medline]
Mathern GW, Pretorius JK, Babb TL. Influence of the type of initial precipitating injury and at what age it occurs on course and outcome in patients with temporal lobe seizures. J Neurosurg 1995a; 82: 220–7.[Web of Science][Medline]
Mathern GW, Babb TL, Pretorius JK, Melendez M, Lévesque MF. The pathophysiologic relationships between lesion pathology, intracranial ictal EEG onsets, and hippocampal neuron losses in temporal lobe epilepsy. Epilepsy Res 1995b; 21: 133–47.[CrossRef][Web of Science][Medline]
Mathern GW, Pretorius JK, Kornblum HI, Mendoza D, Lozada A, Leite JP, et al. Altered hippocampal kainate-receptor mRNA levels in temporal lobe epilepsy patients. Neurobiol Dis 1998; 5: 151–76.[CrossRef][Web of Science][Medline]
Mathern GW, Pretorius JK, Mendoza D, Leite JP, Chimelli L, Born DE, et al. Hippocampal N-methyl-D-aspartate receptor subunit mRNA levels in temporal lobe epilepsy patients. Ann Neurol 1999; 46: 343–58.[CrossRef][Web of Science][Medline]
Meldrum BS. First Alfred Meyer Memorial Lecture. Epileptic brain damage: a consequence and a cause of seizures. [Review]. Neuropathol Appl Neurobiol 1997; 23: 185–202.[CrossRef][Web of Science][Medline]
Meldrum BS, Akbar MT, Chapman AG. Glutamate receptors and transporters in genetic and acquired models of epilepsy. [Review]. Epilepsy Res 1999; 36: 189–204.[CrossRef][Web of Science][Medline]
Merlin LR. Group I mGluR-mediated silent induction of long-lasting epileptiform discharges. J Neurophysiol 1999; 82: 1078–81.
Merlin LR. Differential roles for mGluR1 and mGluR5 in the persistent prolongation of epileptiform bursts. J Neurophysiol 2002; 87: 621–5.
Minakami R, Katsuki F, Sugiyama H. A variant of metabotropic glutamate receptor subtype 5: an evolutionally conserved insertion with no termination codon. Biochem Biophys Res Commun 1993; 194: 622–7.[CrossRef][Web of Science][Medline]
Minakami R, Katsuki F, Yamamoto T, Nakamura K, Sugiyama H. Molecular cloning and the functional expression of two isoforms of human metabotropic glutamate receptor subtype 5. Biochem Biophys Res Commun 1994; 199: 1136–43.[CrossRef][Web of Science][Medline]
Nicoletti F, Bruno V, Catania MV, Battaglia G, Copani A, Barbagallo G, et al. Group-I metabotropic glutamate receptors: hypotheses to explain their dual role in neurotoxicity and neuroprotection. [Review]. Neuropharmacology 1999; 38: 1477–84.[CrossRef][Web of Science][Medline]
Proper EA, Oestreicher AB, Jansen GH, Veelen CW, van Rijen PC, Gispen WH, et al. Immunohistochemical characterization of mossy fibre sprouting in the hippocampus of patients with pharmaco-resistant temporal lobe epilepsy. Brain 2000; 123: 19–30.
Rodríguez-Moreno A, Sistiaga A, Lerma J, Sánchez-Prieto J. Switch from facilitation to inhibition of excitatory synaptic transmission by group I mGluR desensitization. Neuron 1998; 21: 1477–86.[CrossRef][Web of Science][Medline]
Romano C, Sesma MA, McDonald CT, O'Malley K, van den Pol AN, Olney JW. Distribution of metabotropic glutamate receptor mGluR5 immunoreactivity in rat brain. J Comp Neurol 1995; 355: 455–69.[CrossRef][Web of Science][Medline]
Shi S-R, Key ME, Kalra KL. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 1991; 39: 741–8.[Abstract]
Shigemoto R, Nakanishi S, Mizuno N. Distribution of the mRNA for a metabotropic glutamate receptor (mGluR1) in the central nervous system: an in situ hybridization study in adult and developing rat. J Comp Neurol 1992; 322: 121–35.[CrossRef][Web of Science][Medline]
Shigemoto R, Nomura S, Ohishi H, Sugihara H, Nakanishi S, Mizuno N. Immunohistochemical localization of a metabotropic glutamate receptor, mGluR5, in the rat brain. Neurosci Lett 1993; 163: 53–7.[CrossRef][Web of Science][Medline]
Spooren WPJM, Gasparini F, Salt TE, Kuhn R. Novel allosteric antagonists shed light on mglu5 receptors and CNS disorders. [Review]. Trends Pharmacol Sci 2001; 22: 331–7.[CrossRef][Medline]
Stephan D, Bon C, Holzwarth JA, Galvan M, Pruss RM. Human metabotropic glutamate receptor 1: mRNA distribution, chromosome localization and functional expression of two splice variants. Neuropharmacology 1996; 35: 1649–60.[CrossRef][Web of Science][Medline]
Sternberger LA, Hardy PH Jr, Cuculis JJ, Meyer HG. The unlabeled antibody enzyme method of immunohistochemistry preparation and properties of soluble antigen-antibody complex (horseradish peroxidase-antihorseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem 1970; 18: 315–33.[Abstract]
Tang F-R, Lee W-L. Expression of the group II and III metabotropic glutamate receptors in the hippocampus of patients with mesial temporal lobe epilepsy. J Neurocytol 2001; 30: 137–43.[CrossRef][Web of Science][Medline]
Tang F-R, Lee W-L, Yeo TT. Expression of the group I metabotropic glutamate receptor in the hippocampus of patients with mesial temporal lobe epilepsy. J Neurocytol 2001; 30: 403–11.[CrossRef][Web of Science][Medline]
Ulas J, Satou T, Ivins KJ, Kesslak JP, Cotman CW, Balázs R. Expression of metabotropic glutamate receptor 5 is increased in astrocytes after kainate-induced epileptic seizures. Glia 2000; 30: 352–61.[CrossRef][Web of Science][Medline]
van der Hel WS, Notenboom RGE, Bos IWM, van Rijen PC, van Veelen CWM, de Graan PNE. Reduced glutamine synthetase in hippocampal areas with neuron loss in temporal lobe epilepsy. Neurology 2005; 64: 326–33.
Wyler AR, Dohan FC Jr, Schweitzer JB, Berry III AD. A grading system for mesial temporal pathology (hippocampal sclerosis) from anterior temporal lobectomy. J Epilepsy 1992; 5: 220–5.[CrossRef][Web of Science]
Zentner J, Hufnagel A, Wolf HK, Ostertun B, Behrens E, Campos MG, et al. Surgical treatment of temporal lobe epilepsy: clinical, radiological, and histopathological findings in 178 patients. J Neurol Neurosurg Psychiatry 1995; 58: 666–73.
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