Effects of ryanodine receptor activation on neurotransmitter release and neuronal cell death following kainic acid-induced status epilepticus

Epilepsy Research 65 (2005) 59–70

Research paper

Effects of ryanodine receptor activation on neurotransmitter release and neuronal cell death following kainic acid-induced status epilepticus Fumiaki Mori a,∗ , Motohiro Okada b , Masahiko Tomiyama c , Sunao Kaneko b , Koichi Wakabayashi a a

c

Department of Neuropathology, Institute of Brain Science, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan b Department of Neuropsychiatry, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan Department of Neurological Science, Institute of Brain Science, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan Received 21 December 2004; received in revised form 24 February 2005; accepted 23 April 2005 Available online 24 June 2005

Abstract Dynamic changes in intracellular free Ca2+ concentration play a crucial role in various neural functions. The inositol 1,4,5trisphosphate (IP3) receptor (IP3R) and the ryanodine (Ry) receptor (RyR) are involved in Ca2+ -induced Ca2+ -release (CICR). Recent studies have shown that type 3 IP3R is highly expressed in rat hippocampal neurons after kainic acid (KA)-induced seizures and that dantrolene, a RyR antagonist, reduces KA-induced neuronal cell death. We investigated the RyR-associated effects of CICR agents on basal and K+ -evoked releases of GABA and glutamate in rat hippocampus and the changes in expression of mRNA for RyRs in mouse brain after KA-induced seizures. The stimulatory effect of Ry on releases of GABA and glutamate was concentration-dependent in a biphasic manner. The inflection point in concentration–response curves for Ry on GABA release was lower than that for glutamate in both basal and K+ -evoked conditions, suggesting that hyperactivation of RyRassociated CICR produces the imbalance between GABAergic and glutamatergic transmission. Following KA-induced seizures, transient up-regulation of brain-type RyR mRNA was observed in the hippocampal CA3 region and striatum, and signals for c-Fos mRNA increased transiently in the hippocampus, dentate gyrus and deeper layers of the neocortex. Thereafter, some dead neurons with single-stranded DNA (ssDNA) immunoreactive fragmented nuclei appeared in these areas. These findings suggest that intracellular Ca2+ release via the RyR might be one of the mechanisms involved in KA-induced neuronal cell death. © 2005 Elsevier B.V. All rights reserved. Keywords: Ryanodine receptor; Inositol 1,4,5-trisphosphate receptor; Kainic acid; Neuronal cell death; In situ hybridization

1. Introduction ∗

Corresponding author. Tel.: +81 172 395131; fax: +81 172 395132. E-mail address: [email protected] (F. Mori). 0920-1211/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.eplepsyres.2005.04.006

A number of studies provide support for the “imbalance hypothesis” that epileptic seizures are preceded by

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a relative imbalance between excitatory and inhibitory neurotransmission (Hirose et al., 2000). Such imbalance consequently precipitates and propagates abnormal neuronal hyperexcitability in the CNS, i.e. epilepsy (Hirose et al., 2000). The “imbalance hypothesis” was also recently demonstrated in gene analysis of human epileptic patients (Baulac et al., 2001; Wallace et al., 2001). Indeed, mutations in genes encoding GABAA receptor subunits have been identified as a cause of several types of epilepsy (Baulac et al., 2001; Wallace et al., 2001). Kainic acid (KA), a cyclic analogue of glutamate, injected systemically or intracerebrally in rodents evokes seizures that are accompanied by nerve cell damage primarily in regions of the limbic system such as the amygdala, hippocampus, and entorhinal cortex (Lothman and Collins, 1981; Nadler et al., 1981; Schwob et al., 1980). The symptomatology of the KAinduced seizures and the subsequent neuronal damage resemble those seen in patients with severe long-lasting temporal lobe epilepsy. At present, the KA-seizure model is thus regarded as one of the best models for seizure-induced neuronal loss in patients with epilepsy (Ben-Ari, 1985). Dynamic changes in the intracellular free Ca2+ concentration ([Ca2+ ]i ) play a crucial role in various neural functions, including excitability, transmitter release, synaptic plasticity, gene expression and neurotoxicity (Simpson et al., 1995). Rises in [Ca2+ ]i are mediated by Ca2+ influx across the cell membrane via voltagedependent Ca2+ channels and ligand-gated ion channels, as well as output from intracellular Ca2+ store associated with endoplasmic reticulum, namely Ca2+ induced Ca2+ -release system (CICR) via ryanodine (Ry) receptors (RyRs) and inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs). Recently several studies have provided that the functional abnormalities of RyR, IP3R and Mg2+ /Ca2+ ATPase contribute to the elevation of [Ca2+ ]i associated with epileptic seizure (Pal et al., 1999, 2000, 2001; Raza et al., 2001, 2004; Parsons et al., 2000, 2001). Furthermore, a RyR antagonist, dantrolene, protects neurons against KA-induced apoptosis in vitro and in vivo (Popescu et al., 2002). Berg et al. (1995) suggested that the dantrolene-sensitive RyR might play a major role in seizure-triggered neuronal cell death. In the brain, neuronal RyR is thought to be responsible for CICR, which is analogous to muscular RyR

function, whereas IP3R is involved in IP3-induced Ca2+ release (McPherson and Campbell, 1993). At the molecular level, three isoforms of the RyR have so far been identified and have been named the skeletal muscle type (sRyR or RyR-1), cardiac type (cRyR or RyR-2) and brain type (bRyR or RyR-3) (Hakamata et al., 1992; Nakai et al., 1990; Takeshima et al., 1989). These three isoforms are structurally related, but are distinct in their functional properties and regulation (McPherson and Campbell, 1993). These three isoforms exist in the brain, although their distributions in the adult brain are clearly different from each other and from that of the IP3R (Furuichi et al., 1994; Giannini et al., 1995; McPherson and Campbell, 1993; Sharp et al., 1993). Changes in [Ca2+ ]i also play important roles during development, such as growth cone movement (Zheng, 2000), neuronal cell migration (Rakic and Komuro, 1995) and apoptosis (Sastry and Rao, 2000). However, it remains unknown how RyR expression is regulated and which type of RyR is involved in the process of CICR during the seizures induced by KA. Thus, on the basis of this previous knowledge, the present study was intended to clarify the mechanisms of epileptic seizure and neuronal damage induced by KA. We investigated the expression of mRNA for RyRs and c-Fos, c-Fos protein, and ssDNA and the histopathological changes in mouse brain after KA-induced seizures as well as the effects of RyR-associated CICR agents on exocytosis of GABA and glutamate in the rat hippocampus.

2. Methods 2.1. Extracellular levels of GABA and glutamate in the rat hippocampus All animal experiments in this study were performed in accordance with the Guidelines for Animal Experimentation, Hirosaki University, Japan. Each rat was placed in a stereotaxic frame and kept under halothane anesthesia (1.5% mixture of halothane and O2 with N2 O) (Okada et al., 2001). A concentric I-type dialysis probe (0.22 mm diameter; 3 mm exposed membrane; Eicom, Kyoto, Japan) was implanted in the rat hippocampus (anterior, 5.8 mm; lateral, 4.8 mm; ventral, 4.0 mm, relative to the bregma).

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Perfusion experiments commenced 18 h after recovery from anesthesia (Okada et al., 2001, 2004). The perfusion medium was initially modified Ringer’s solution (MRS) composed of: 145 mM Na+ , 2.7 mM K+ , 1.2 mM Ca2+ , 1.0 mM Mg2+ , 154.4 mM Cl− , and buffered with 2 mM phosphate buffer and 1.1 mM Tris buffer to adjust the pH to 7.4. At least 6 h after starting the perfusion, the levels of glutamate and GABA in the perfusion medium were measured. When the coefficients of variation of each neurotransmitter level reached less than 5% over 60 min (stabilization) (Okada et al., 2001, 2004), control data were obtained over a further 60 min; then the perfusion medium was switched from MRS to MRS containing target agent (Zhu et al., 2004a,b). In order to study the effects of target agent on K+ -evoked releases, the perfusion medium was initially MRS with or without (control) target agent (prestimulation period). After confirming stabilization, the perfusion medium was switched to MRS containing 50 mM K+ (HKMRS) with the same agent for 20 min (K+ -evoked stimulation). After the stimulations were evoked, the perfusion medium was switched to the pre-stimulation medium for 100 min again. The ionic composition was modified and isotonicity was maintained by an equimolar decrease of Na+ (Okada et al., 2001, 2004). Each hippocampal dialysate was injected into the high-performance liquid chromatography (HPLC) apparatus. The extracellular levels of glutamate and GABA were determined by HPLC with ophthalaldehyde-derived fluorescence detection. The analytical column (100 mm × 1.5 mm internal diameter) was packed with Mightysil RP-18 (particle size 3 ␮m; a gift from Kanto Chemicals, Tokyo, Japan) by Masis Inc. and was maintained at 30 ◦ C. The excitation and emission wavelengths of the fluorescence detector were set at 340 and 445 nm, respectively (Zhu et al., 2004a). A linear gradient elution program was performed over 30 min with mobile phases A (0.05 M phosphate buffer containing 25% methanol, pH 6.0) and B (0.05 M phosphate buffer containing 40% methanol, pH 3.5) at a flow rate of 400 ␮l/min (Okada et al., 2001). The chemical agents used in this study included the RyR agonist ryanodine (Ry; Calbiochem, San Diego, CA, USA) and the RyR antagonist ruthenium red (RR; Dainippon Pharmaceutical Co., Osaka, Japan).

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2.2. Experimental animals and tissue processing Adult male C57BL/6J mice (JCL; 19–20 g body weight) were administered intraperitoneally a 25 mg/kg dose of KA (0.2 ml of KA dissolved in saline/20 g of body weight) (420318; CalbiochemNovabiochem Co., San Diego, CA, USA), and the animals showing convulsions during the first hour after administration were selected. For in situ hybridization studies (n = 15) and histopathological examination (n = 15), mice showing convulsions were killed 2, 6, 12, 24, or 48 h after the initiation of convulsions. For histopathological studies, mice were anesthetized with sodium pentobarbital (30 mg/kg body weight) and perfused transcardially with 0.1 M phosphate buffer, pH 7.4, containing 0.25% (v/v) heparin, followed by 4% paraformaldehyde plus 0.1% glutaraldehyde in 0.1 M phosphate buffer. The brains were removed and post-fixed in the same fixative for 24 h. The right half of each brain was used for routine histological examination. The tissues were dehydrated in a graded ethanol series and embedded in paraffin. Serial 4-␮m-thick sections were cut from the block samples and stained with hematoxylin and eosin (H & E). An atlas of the mouse brain with an anatomical explanation of the terminology adopted by Paxinos and Franklin (2001) was used to identify brain regions. The left half of each brain was used for immunohistochemistry. Floating sections (50 ␮m thick) were cut with a microslicer (DTK-3000; Dosaka EM Co. Ltd., Kyoto, Japan) and the sections were stained using the avidin–biotin-peroxidase complex (ABC) method with a Vectastain kit (Vector, Burlingame, CA, USA). Rabbit polyclonal antibodies against c-Fos (SC52; Santa Cruz Biotechnology, Santa Cruz, CA, USA; 1:1000) and single-stranded DNA (ssDNA) (A4506; DakoCytomation, Kyoto, Japan; 1:100) were used as the primary antibodies. The anti-ssDNA antibody is known to detect drug-induced apoptosis and programmed cell death during embryogenesis (Naruse et al., 1994). For in situ hybridization studies, animals were killed by decapitation and the brain was quickly removed, frozen on powdered dry ice, and stored at −80 ◦ C. Sagittal brain sections (20 ␮m thick) were cut with a microtome cryostat at −15 ◦ C, thaw-mounted on glass slides precoated with 3-aminopropyltriethoxysilane, and kept at −80 ◦ C. RyR-1, RyR-2, and RyR-3 mRNAs were investigated by using 33 P-labeled oligonucleotide

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probes. The data presented here were obtained with sense and antisense oligonucleotide probes synthesized to correspond to the following cDNA regions: nucleotide residues 1541–1585 of mouse RyR-1 cDNA (accession no. X83932), 1195–1240 of mouse RyR2 cDNA (X83933) and 363–411 of mouse RyR-3 cDNA (D38218) (Mori et al., 2000). The procedures for probe labeling were the same as reported previously (Tomiyama et al., 1996). X-ray film autoradiograms were digitized with a flathead scanner (Epson, Tokyo, Japan) and evaluated semi-quantitatively in terms of gray levels with NIH Image (version 1.61). The data were normalized by subtracting the film background, and the mean value was calculated from three sections. The mean values of each case were analyzed using oneway ANOVA with post-hoc test. A double-labeling immunofluorescence study was performed on selected frozen sections with the monoclonal anti-RyR (MA3-925; Affinity Bioreagent, Inc., CO, USA; 1:100) and the polyclonal anti-ssDNA (1:50) antibodies. The second antibodies employed were fluorescein isothiocyanate-conjugated anti-mouse IgG (Vector; 1:50) and Texas Red-conjugated anti-rabbit IgG. Vector M.O.M.TM Immunodetection Kit (Vector) was used to prevent high background staining before applying the monoclonal anti-RyR antibody.

3. Results 3.1. Effects of RyR agents on releases of GABA and glutamate in the hippocampus The levels of basal and K+ -evoked releases were calculated by a previously published method (Okada et al., 2001, 2004). The RyR agonist, Ry, increased basal glutamate release concentration-dependently (P < 0.01) (Fig. 1a). Although neither basal releases of GABA nor glutamate in rat hippocampus were affected by RyR antagonist, RR (Fig. 1b), the pre-perfusion with 50 ␮M RR reduced the Ry-induced releases of GABA and glutamate (data not shown). The inflection point of the concentration–response curve for Ry on hippocampal basal GABA release occurred at 100 ␮M, since Ry increased basal GABA release concentrationdependently (P < 0.01) over the concentration range from 1 to 100 ␮M, whereas perfusion with 1000 ␮M Ry did not affect it (Fig. 1a).

Fig. 1. Concentration-dependent effects of RyR agents on basal releases of GABA and glutamate in rat hippocampus. Concentrationdependent effects of a RyR antagonist (RR) and a RyR agonist (Ry) on basal releases of GABA (open columns) and glutamate (closed columns) are indicated in Fig. 1a and b, respectively. Ordinates indicate the concentration of basal releases of GABA and glutamate (pmol/sample), and the abscissa shows the concentration of the RyR agents (␮M). The concentration-dependent effects of RyR agents on releases of GABA and glutamate were analyzed using one-way ANOVA with Tukey’s multiple comparison test (* P < 0.05; ** P < 0.01).

In contrast to basal release, releases of GABA and glutamate evoked by 50 mM K+ were reduced by RR in a concentration-dependent manner (P < 0.01) (Fig. 2a). Ry affected K+ -evoked releases of GABA and glutamate in a biphasic concentration-dependent manner (Fig. 2b). The inflection point of the concentration–response curve for Ry on hippocampal K+ -evoked GABA release was observed at 10 ␮M

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GABA release was shifted to the right by K+ -evoked stimulation (Figs. 1b and 2b). The inflection point of the concentration–response curve for Ry on hippocampal K+ -evoked glutamate release was observed at 100 ␮M (Fig. 2b). Ry increased K+ -evoked glutamate release concentration-dependently (P < 0.01) over the concentration range from 1 to 100 ␮M, whereas perfusion with 1000 ␮M Ry increased K+ -evoked glutamate release but the stimulatory effect was attenuated (Fig. 2b). 3.2. Histopathological examination of the mouse brain after KA-induced seizures 3.2.1. Conventional histological findings In KA-treated mice, pyknotic neurons were found in the hippocampus, dentate gyrus, amygdala, septum, piriform and entorhinal cortices, medial thalamus, and cerebral neocortex 2–12 h after treatment (Fig. 3a and b). At 24 and 48 h post-treatment, neuronal cell debris with fragmented nuclei was observed in the hippocampus, dentate gyrus, amygdala, septum, striatum, medial thalamus, piriform and entorhinal cortices, and cerebral neocortex. No degenerated neurons were observed in control mice.

Fig. 2. Concentration-dependent effects of RyR agents on K+ evoked releases of GABA and glutamate in rat hippocampus. Concentration-dependent effects of a RyR antagonist (RR) and a RyR agonist (Ry) on K+ -evoked releases of GABA (open columns) and glutamate (closed columns) are indicated in Fig. 2a and b, respectively. Ordinates indicate the concentration of K+ -evoked releases of GABA and glutamate (pmol/sample), and the abscissa shows the concentration of the RyR agents (␮M). The concentration-dependent effects of RyR agents on releases of GABA and glutamate were analyzed using one-way ANOVA with Tukey’s multiple comparison test (* P < 0.05; ** P < 0.01).

(Fig. 2b). Ry increased K+ -evoked GABA release concentration-dependently (P < 0.01) over the concentration range from 1 to 10 ␮M, whereas at concentrations higher than 10 ␮M the stimulatory effect of Ry on K+ -evoked GABA release was attenuated concentration-dependently (P < 0.05) (Fig. 2b). K+ evoked GABA release was reduced by pre-perfusion with 1000 ␮M Ry (P < 0.01) (Fig. 2b). The inflection point in the concentration–response curves for Ry on

3.2.2. c-Fos immunohistochemistry Epileptiform activity elicited neuronal expression of the Fos family of proto-oncogenes. Two hours after treatment, c-Fos immunoreactivity was seen in neurons in the pyramidal cell layers of the hippocampus and dentate gyrus (Fig. 3c and d). Neurons in the striatum, amygdala, septum, piriform and entorhinal cortices, and neocortices also demonstrated intense immunolabeling. Twenty-four hours after the drug injection, c-Fos immunoreactivity was absent from these areas. Control subjects did not exhibit significant immunoreactivity. 3.2.3. ssDNA immunohistochemistry A few ssDNA-positive cells were detected in the granule cell layer of the dentate gyrus at 2 h. Some ssDNA-positive cells were observed in the pyramidal cell layer of the CA1 and CA3 regions of the hippocampus at 6–24 h (Fig. 3e and f). At 48 h, ssDNA-positive cells were also distributed in the striatum, amygdala, septum, medial thalamus, piriform and entorhinal cortices, and cerebral neocortex. Control subjects did not exhibit significant immunoreactivity.

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Fig. 3. Histopathological findings in the mouse hippocampus stained with H & E (a, b), anti-c-Fos (c, d), and anti-ssDNA (e, f). (a) Moderate neuronal loss in the CA3 region (arrowheads) 2 h after kainic acid (KA)-induced seizure. (b) Higher magnification of the area indicated by arrowheads in (a), showing pyknotic neurons (arrows). (c) Strong c-Fos induction in the CA1–CA3 region and dentate gyrus 2 h after KAinduced seizure. (d) Higher magnification of the area indicated by arrowheads in (c), showing c-Fos immunoreactivity in the neuronal nuclei. (e) ssDNA immunoreactivity in the pyramidal layer of the CA3 (arrowheads) 6 h after KA-induced seizure. (f) Higher magnification of the area indicated by arrowheads in (e), showing ssDNA-immunoreactive fragmented nuclei (arrows). Bars = 200 ␮m for a, c, e; 50 ␮m for b, d; and 25 ␮m for f.

3.2.4. Expression of RyRs and c-Fos mRNA in the mouse brain after KA-induced seizures In control mice, the highest levels of RyR-1 mRNA were detected in the dentate gyrus of the hippocampus and the Purkinje cell layer of the cerebellum, with low to moderate levels in the olfactory mitral cell layer, olfactory tubercle, caudate–putamen and superficial regions of the cerebral cortex (Fig. 4a). Prominent signals for RyR-2 mRNA were observed in various brain regions, with the highest levels occurring in the Ammon’s horn and dentate gyrus of the hippocampus and in the granular layer of the cerebellum (Fig. 4c). Moderate levels of RyR-3 mRNA were detected in the hippocampus, striatum, and dorsal region of the thalamus (Fig. 4e). These spatial distributions were

consistent with previous results (Mori et al., 2000). Specificity was confirmed by blank autoradiograms after hybridization had been carried out in the presence of unlabeled oligonucleotides or with sense probes. Furthermore, similar expression patterns were obtained when another set of non-overlapping antisense probes was used (data not shown). The expression patterns of RyR-1 and RyR-2 mRNAs did not change after KA-induced seizures (Fig. 4b and d). On the other hand, RyR-3 mRNA levels increased in the hippocampal CA3 region and striatum 2 and 6 h after KA administration and decreased thereafter (Figs. 4f, 5, 6e and f). Strong signals for c-Fos mRNA appeared at 2 h after KA-induced seizures in the hippocampus, den-

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Fig. 4. Effects of KA-induced seizures on the expression of mRNA for RyR-1 (a, b), RyR-2 (c, d), RyR-3 (e, f), and c-Fos (g, h) in the brains of control mice (a, c, e, g) and injected mice 2 h after KA-induced seizure (b, d, f, h). There is no difference between the expression patterns of RyR-1 and RyR-2 mRNAs after KA-induced seizures (a–d). Higher RyR-3 mRNA levels are shown in the hippocampal CA3 region and striatum of KA-induced mice (f). Stronger signals for c-Fos mRNA are found at 2 h after KA-induced seizures in the hippocampus, dentate gyrus and deeper layers of the neocortex (h). Bar = 1 mm.

tate gyrus and deeper layers of the neocortex (Fig. 4h). Thereafter, the levels decreased gradually and disappeared by 12 h after KA-induced seizures (Fig. 6g and h). In control mice, weak to moderate signals for c-Fos mRNA were observed in the neocortex, hippocampus, dentate gyrus and granule cell layer of the cerebellum (Fig. 4g). 3.2.5. Co-expression of RyR and ss-DNA in pyramidal neurons in the hippocampus Double-labeling immunofluorescence revealed coexpression of RyR and ssDNA in the same pyramidal neurons in the hippocampal CA3 regions 6 h after KA administration (Fig. 7).

4. Discussion In the present study, transient up-regulation of mRNA expression of RyR-3 and c-Fos was observed in the hippocampus and dentate gyrus following KA-induced seizures. The expression of c-Fos mRNA, one of the immediately early genes, is activated by various agents. Up-regulation of c-Fos mRNA expression and its protein product indicates excessive excitability and increased [Ca2+ ]i of neuronal cells in the hippocampus, striatum and cerebral cortex (Gass et al., 1995; Ferrer et al., 2000). Moreover, type 3 IP3R is highly expressed in rat hippocampal neurons after KA administration (Blackshaw et al., 2000). These findings

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Fig. 5. Changes in expression of mRNA for RyR-3 in the mouse brain at 0 h (a), 2 h (b), 6 h (c), 12 h (d), 24 h (e), and 48 h (f) after KA-induced seizures. Increases of RyR-3 mRNA levels are found in the hippocampal CA3 region and striatum 2 h (b) and 6 h (c) after KA administration.

suggest that RyR-3 might be augmented in neuronal cells by excessive excitability following KA-induced seizures. Release of synaptic vesicles containing neurotransmitters is triggered by influx of Ca2+ through voltagesensitive Ca2+ channels. This influx, which increases intracellular Ca2+ from a basal level of 100 nM to more than 100 ␮M (Rettig and Neher, 2002; Sollner et

al., 1993), activates CICR (Berridge, 1998; Rettig and Neher, 2002). CICR regulates a wide variety of neuronal functions, including the Ca2+ -associated mechanism of neurotransmitter exocytosis (Berridge, 1998; Rettig and Neher, 2002). The present demonstration indicates that during resting stage, RyR system does not provide the neurotransmitter exocytosis; however, the potential of the RyR system in neurotransmitter exocytosis may be promoted by neuronal hyperexcitable stage. Furthermore, in the present study, Ry, a RyR agonist, enhanced releases of both glutamate and GABA, although the stimulatory effect of Ry on releases of GABA and glutamate was biphasic in a concentrationdependent manner. Especially, the inflection point in the concentration–response curves for Ry on releases of GABA and glutamate was shifted to the left by neuronal hyperactivation, from 100 ␮M during the resting stage to 10 ␮M during K+ -evoked stimulation and from more than 1000 ␮M during the resting stage to 100 ␮M during K+ -evoked stimulation, respectively. This shift is similar to that observed for striatal dopamine release (Zhu et al., 2004b). The noteworthy result is that the inflection point in the concentration–response curves for Ry on GABA release is lower than that on glutamate release in both resting and K+ -evoked conditions. Thus, these observations suggest that hyperactivation of RyR-associated CICR produces the imbalance between GABAergic and glutamatergic transmission, with a relative predominance of glutamatergic transmission rather than GABAergic transmission. In the present study, up-regulation of RyR-3 mRNA was observed in the hippocampal CA3 region and striatum following KA-induced seizures. Thereafter, RyR-positive pyramidal neurons died in these regions. Pelletier et al. (1999) reported that dantrolene, a RyR antagonist, prevents seizure-induced cell death in vitro. Those investigators suggested that an activation of RyR-associated CICR triggered by Ca2+ inflow to the intraneuronal space from the extraneuronal space plays an important role in seizure-associated neuronal cell death. Moreover, Popescu et al. (2002) reported that dantrolene protects neurons against KA-induced apoptosis in vitro and in vivo. In the brains of adult mice, the highest levels of RyR-1 mRNA are detected in the dentate gyrus of the hippocampus and the Purkinje cell layer of the cerebellum. Prominent signals for RyR-2 mRNA are ubiquitous in the brain regions, whereas RyR-3 mRNA is restricted to specific brain regions

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Fig. 6. Semi-quantification of RyR-1 (a, b), RyR-2 (c, d), RyR-3 (e, f), and c-Fos (g, h) mRNA in the CA1, CA3, dentate gyrus (DG), cerebral cortex (Cx), caudate–putamen complex (CP), and thalamus (Th) after KA-induced seizures. The mean values of each case were analyzed using one-way ANOVA with post-hoc test (* P < 0.05). Significant changes of signals for RyR-1 or RyR-2 mRNAs are not seen after KA-induced seizures (a–d). Significant up-regulation of RyR-3 mRNA levels (P < 0.05) is found in the hippocampal CA3 region (e) and striatum (f) 2 and 6 h after KA administration. Strong up-regulation of c-Fos mRNA is shown at 2 h after KA-induced seizures in the hippocampus and dentate gyrus (g, h).

Fig. 7. Double-labeling immunofluorescence demonstrating RyR-positive pyramidal neurons (a) and ssDNA-positive fragmented nuclei (b) in the hippocampal CA3 region. RyR appears green (a) and ss-DNA appears red (b). The overlap of RyR with ssDNA appears orange (c). Bar = 25 ␮m.

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such as CA1-3 stratum pyramidale, striatum, and dorsal thalamus (Furuichi et al., 1994; Mori et al., 2000). Zhao et al. (2001) indicated that both RyR-1 and RyR-3, but not RyR-2, might be targets for dantrolene inhibition in vitro. These findings suggest that up-regulation of RyR-3 mRNA expression in the hippocampal CA3 region and the striatum might be involved in neuronal cell death following KA-induced seizures. It has been well established that the multiple neuronal Ca2+ homeostatic system, including VSCC, ligand-gated ion channels, Mg2+ /Ca2+ ATPase and CICR, are affected by status epileptics (Pal et al., 1999, 2000, 2001; Raza et al., 2001, 2004; Parsons et al., 2000, 2001). The pilocarpine-induced status epileptics immediately reduced Mg2+ /Ca2+ ATPase (Parsons et al., 2000). The study using hippocampal neuronal culture model of epilepsy has demonstrated that activities of Mg2+ /Ca2+ ATPase and IP3R were chronically reduced and increased, respectively (Pal et al., 2001). Although RyR activity plays an important role in KAinduced apoptosis and seizure-triggered neuronal cell death (Berg et al., 1995; Popescu et al., 2002), RyR activity was not changed in hippocampal neuronal culture model of epilepsy (Pal et al., 2001). Taken together with these previous evidence, the present demonstrations suggest that inhibition of RyR activity contributes to the inhibition of neuronal events acutely after status epilepticus but does not affect chronically. Several mechanisms might be involved in KAinduced neurodegeneration (Malva et al., 1998; Zagulska-Szymczak et al., 2001; Popescu et al., 2002). First, KA activates excitatory pathways, leading to release of significant amounts of excitatory amino acids, which can cause cell death through excitotoxic mechanisms. Secondly, KA triggers epileptic seizures that are associated with brain edema and hypoxia, known to be responsible for neuronal injury. The third possibility is that KA causes apoptosis directly in cells when it binds kainate receptors through a mechanism similar to that encountered in neuronal cell cultures. It has been shown that KA can increase [Ca2+ ]i in cerebellar granule cells (Leski et al., 1999). In the present study, we demonstrated that excessive amounts of RyR agonist increase basal release of glutamate and decrease that of GABA in the hippocampus and that RyR-3 mRNA is up-regulated in the hippocampal CA3 region after administration of KA. These findings suggest that intracellular Ca2+ release via RyR might be

one of the mechanisms involved in KA-induced neuronal cell death.

Acknowledgments The authors thank Ms M. Nakata for technical assistance. This work was supported in part by Grantsin-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (15591208 and 16109006) and a grant from the Japan Epilepsy Research Foundation.

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