Research paperEffects of ryanodine receptor activation on neurotransmitter release and neuronal cell death following kainic acid-induced status epilepticus
Introduction
A number of studies provide support for the “imbalance hypothesis” that epileptic seizures are preceded by 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 KA-induced 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 voltage-dependent 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, Pal et al., 2000, Pal et al., 2001, Raza et al., 2001, Raza et al., 2004, Parsons et al., 2000, Parsons et al., 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.
Section snippets
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 N2O) (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).
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, Okada et al., 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 μM RR reduced the Ry-induced releases of GABA and glutamate (data not shown). The inflection point of the
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,
Acknowledgments
The authors thank Ms M. Nakata for technical assistance. This work was supported in part by Grants-in-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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2010, Neuroscience ResearchCitation Excerpt :Two-way ANOVA indicated a significant interaction between acute administration of VPA (perfusion with VPA: 500 and 1000 μM) and ryanodine (1–1000 μM) on extracellular levels of GABA [two-way ANOVA: Fryanodine(4, 105) = 49.1, P < 0.01; FVPA(2, 105) = 27.4, P < 0.01; Fryanodine × VPA(8, 105) = 9.1, P < 0.01] and glutamate [two-way ANOVA: Fryanodine(4, 105) = 110.7, P < 0.01; FVPA(2, 105) = 37.9, P < 0.01; Fryanodine × VPA(8, 105) = 4.5, P < 0.01] (Fig. 3). Perfusion with ryanodine (1–1000 μM) increased the hippocampal extracellular glutamate level in a concentration-dependent manner (Fig. 3B), similar to previous demonstration (Yoshida et al., 2005, 2007; Mori et al., 2005). The hippocampal extracellular GABA level also increased following perfusion with 1–100 μM ryanodine; however, perfusion with 1000 μM ryanodine did not affect that of GABA (Fig. 3A), similar to previous demonstration (Yoshida et al., 2005, 2007; Mori et al., 2005).