Dantrolene, a Calcium-Induced Calcium Release Inhibitor, Prevents the Acquisition of Amygdaloid Kindling in Rats, a Model of Experimental Epilepsy

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1 Tohoku J. Exp. Med., 2006, Kindling 209, is Associated with Intracellular Ca 2+ Stores 303 Dantrolene, a Calcium-Induced Calcium Release Inhibitor, Prevents the Acquisition of Amygdaloid Kindling in Rats, a Model of Experimental Epilepsy MOTOAKI YOSHIDA and TOMOMI SAKAI 1 Department of Neurology, University of Health Science, Minamitsuru, Japan, and 1 Center for Integrative Medicine, Tsukuba University of Technology, Tsukuba, Japan YOSHIDA, M. and SAKAI, T. Dantrolene, a Calcium-Induced Calcium Release Inhibitor, Prevents the Acquisition of Amygdaloid Kindling in Rats, a Model of Experimental Epilepsy. Tohoku J. Exp. Med., 2006, 209 (4), Kindling phenomenon (an experimental model of acquired epilepsy) and long-term potentiation (LTP) have many features in common. LTP is associated with an increase in intracellular Ca 2+ concentration where intracellular Ca 2+ stores play an important role. Dantrolene has an inhibitory action against Ca 2+ release from intracellular Ca 2+ stores. This substance has been reported to inhibit the induction of LTP. We investigated whether or not the development of kindling would be inhibited by dantrolene. We first determined at the right amygdala afterdischarge threshold (ADT) that is the minimal electric current necessary to evoke afterdischarge. For investigating the effects of dantrolene on the development of kindling, rats were stimulated once daily at the right amygdala with a current, 10% larger than ADT, one hour after either dantrolene or vehicle had been injected intraperitoneally. We observed daily the progress of both intensities of behavioral seizures (stages of seizures) and the duration of afterdischarge. Dantrolene significantly delayed both the progression of kindling stages and the elongation of afterdischarge duration at a dose of 10 mg/kg or 20 mg/kg, as compared with control vehicle. To investigate the effects of dantrolene on seizures in already kindled rats, we determined generalized seizure threshold (GST) that is the minimum electric current necessary to evoke generalized seizures in fully kindled rats. Rats were stimulated at a current, 10% larger than GST, one hour after treated with the drug or vehicle. Dantrolene showed no anticonvulsant action against either the stages of seizures or afterdischarge durations in fully kindled rats. Therefore, the inhibitory effect of dantrolene on the acquisition of kindling was not attributed to an anticonvulsant action. These results suggest that the prevention of kindling acquisition is associated with an inhibitory action of dantrolene on calcium induced calcium release from intracellular Ca 2+ stores. Dantrolene; intracellular calcium stores; calcium-induced calcium release; LTP; kindling 2006 Tohoku University Medical Press Received March 7, 2006; revision accepted for publication May 20, Correspondence: Motoaki Yoshida, M.D., , Namiki, Tsukuba , Japan. motoaki-y@mwe.biglobe.ne.jp 303

2 304 M. Yoshida and T. Sakai Epilepsy is one of the most common neurological disorders. Recently, it has been postulated that environmental stresses to the nervous system in cases of symptomatic epilepsy would induce the alteration in the expression of specific genes that might produce the permanent hyperexcitability state in the brain (Delorenzo and Morris 1999). The kindling phenomenon is considered to be an experimental model for complex partial seizures (Pedley and Meldrum 1992). The kindling phenomenon is a process in which the repeated administration of an initially subconvulsive tetanic stimulus at regular intervals ultimately elicits stereotyped electrographic discharges and behavioral seizures (Goddard et al. 1969). It continues for an entire life when acquired. Therefore, it is suggested that some transcriptional alterations occur in the central nervous system. While it is debated whether or not long-term potentiation (LTP) constitutes the cellular basis of kindling, the two models of increased excitability (LTP and kindling) share many features. Studies on the elucidation of mechanisms underlying LTP have been applied to kindling and the results obtained have demonstrated many similarities (Pedley and Meldrum 1992). Studies on the mechanism underlying LTP have demonstrated that changes in gene transcription occurred with the induction of plasticity changes associated with the model (Hardingham and Bading 1998). LTP is associated with changes in the neuronal calcium level that was triggered by NMDA (N-methyl-D-aspartate) receptorregulated calcium entry (Bliss and Collingridge 1993). Since NMDA receptoractivated calcium entry is required for epileptogenesis, insights gained from studying LTP may relate to the mechanisms involved in regulating the gene expression in epilepsy (Delorenzo and Morris 1999). LTP is inhibited by NMDA receptor blockers (Gilbert and Mack 1990; Morimoto et al. 1991). Kindling phenomenon has also been reported to be inhibited by the blockade of NMDA receptors (McNamara et al. 1988). Stimulation of NMDA receptors produces entry of Ca 2+ into neuronal cells. It stimulates calcium-induced calcium release (CICR), which increases the cytoplasmic Ca 2+ concentration (Verkhratsky and Schmigol 1996). This alteration of cytoplasmic Ca 2+ concentration produces an alteration of Ca 2+ level in nuclei, which consequently induces gene transcriptions (Hardingham et al. 1997; Hu et al. 1999). Bading (2000) suggested that through the mediation of nuclear Ca 2+ alteration, physiological stimuli induced LTP and non-physiological stimuli produced long-term neuronal hyperexcitability such as kindling phenomenon. Dantrolene is a substance that acts on ryanodine receptors on endoplasmic reticulum in both neuronal cells and muscular cells. It blocks calcium-induced calcium release (CICR) from intracellular Ca 2+ stores and is consequently supposed to mediate an alteration of the nuclear Ca 2+ level. Clinically, it is used as muscle relaxants or for the treatment of malignant hyperthermia. We investigated whether or not dantrolene would inhibit the acquisition of amygdaloid kindling in rats. MATERIALS AND METHODS General procedure Male Wistar rats weighing from 300 g to 350 g at surgery were used. All rats were housed with 12 hrs light-dark cycle. Food and water were supplied ad libitum. A bipolar stainless steel enamel-coated electrode (0.2 mm in diameter) with tip separation of 0.5 mm was implanted into the right amygdaloid nucleus (coordinate A 6.0, L 5.0, H 8.5 ) using the stereotaxic atlas of Paxinos and Watson (1986). The electrode was used for both kindling stimulation and recording electroencephalography (EEG). A carvarial screw electrode was placed at the inion for reference. The implantation of electrodes was carried out under sodium pentobarbital anesthesia (50 mg/kg). After a recovery period of two weeks, tests were performed. The electric current used for stimulation was biphasic, 1 ms in width, 60 Hz, and 2 s in duration. EEG was recorded for 2 min before and for 5 min after the stimulation. Duration of afterdischarge (ADD) was measured. Behavioral seizure ranks were classified into the five-scale devised by Racine (1972). Dantrolene sodium (Sigma Co., Ltd., St. Louis, MO, USA) at a dose of 10 mg/kg, 20 mg/kg or 40 mg/kg was dissolved in a volume of 10 ml of 0.9% saline.

3 Kindling is Associated with Intracellular Ca 2+ Stores 305 These doses were chosen according to previous studies in rats (Niebauer and Gruenthal 1999). All substances were administered intraperitoneally one hour before the stimulation. The experimental procedures employed in this study were carried out in accordance with the guidelines set by the Animal Experimental Committee of Tsukuba College of Technology. Studies on effects of dantrolene on the development of kindling Afterdischarge thresholds were determined by an ascending manner. Beginning at 60 μa, a current was increased in intensity at 20 μa steps every 3 min until afterdischarge appeared. We regarded the spike of its amplitude over two-fold than the basic activity as afterdischarge. The second afterdischarge was counted as the first one when it appeared within 30 seconds after the first one disappeared. The current which was 10% higher than afterdischarge threshold was used to study the development of kindling. Kindling stimulation was given after the administration of control vehicle or dantrolene at a dose of either 10 mg/kg or 20 mg/kg (n = 6, respectively) once a day for ten days (drug session). Subsequently, rats were stimulated without the administration of any substances for four more days (drug-free session). Eighteen animals were totally included for this experiment. When rats showed class five seizures five times consecutively, they were regarded as fully kindled or completion of kindling epilepsy. Studies on effects of dantrolene on seizures in kindled rats Seven fully kindled rats that showed ten consecutive class five seizures were used (Yoshida et al. 1997). Generalized seizure thresholds (GST) were determined by administering a series of stimuli beginning at 60 μ A and increased in intensity at 20 μ A steps at an interval of 3 min until class five seizures appeared. It was defined as GST. We used a current 10% higher than GST as a current of stimulation. This current was delivered at the amygdala after the administration of control vehicle or dantrolene at a dose of either 20 mg/kg or 40 mg/kg. The same seven rats were used repeatedly to investigate effects of these different substances. A one-week period intervened between a test and the next test in each rat. Histology Rats were overdosed with sodium pentobarbital and perfused through the heart with 10% formalin followed by saline. The brain was removed and stored in 10% formalin. Coronal sections were cut at a thickness of 50 μm using a freezing microtome. They were mounted and stained with cresyl violet to verify the placement of electrodes. Only animals with electrodes lying within amygdaloid neclei were included in the analysis. Statistical analysis The number of stimuli required for the initial appearance of stage 5 seizures was analyzed using oneway ANOVA (analysis of variance). Effects of dantrolene on kindling development were analyzed using repeated measures ANOVA, followed by multiple comparisons using Scheffé s F test. Where significant, individual means at each day were analyzed using Mann- Whitney s U-test for seizure stages or one-way ANOVA for afterdischarge durations. Paired t-test was used for the analysis of ADD of seizures evoked in kindled rats. Wilcoxon signed-ranks test was used for the analysis of stages of seizures evoked in kindled rats. Statistical analysis was performed using Statcel 2 (Yanai 2003). RESULTS Effects of dantrolene on the development of kindling When we touched or pinched rats, we observed that rats given dantrolene (n = 12) were hypotonic and flaccid. Locomotor activity of rats treated with the drug was reduced. The intensity of limbic seizures was milder than that of control animals. For example, their backbones when rearing at the stage 4 or 5 seizures were slightly round, while the backbones of control rats were straight-forward. But no ataxic signs such as legsplaying were observed. While rats treated with vehicle (n = 6) all completed full kindling (consecutive five stage 5 seizures) within 10 days, no rats treated with either 10 mg/kg or 20 mg/kg of dantrolene (n = 6, respectively) completed full kindling within this period. While the number of stimuli which was necessary for the initial appearance of stage 5 seizures was 5.3 ± 0.6 (mean ± S.E.M) for vehicle-

4 306 M. Yoshida and T. Sakai treated rats, it was 9 ± 1.0 for rats treated with 10 mg/kg of dantrolene or 10.0 ± 1.1 for rats treated with 20 mg/kg of dantrolene. Thus, the number of stimuli required for the initial appearance of stage 5 seizures was significantly larger in rats treated with dantrolene at two different doses (p < 0.01 or p < 0.005, respectively, one-way ANOVA), as compared with that in control rats treated with vehicle. Since vehicle controls were all fully kindled within ten days, the kindling development was statistically analyzed using the data obtained in this period (Fig. 1). In the analysis of the development of seizure stages, the significant retardation of stage development was observed in the Fig. 1. Inhibitory effects of dantrolene on the development of kindling stages. Daily electrical stimulation at the amygdaloid nuclei produced the development of kindling whether pretreated with control vehicle or dantrolene. However, the development of kindling stages was significantly delayed by the pretreatment with dantrolene at a dose of either 10 mg/kg or 20 mg/kg (n = 6, p < 0.05 or p < 0.01 respectively, repeated measures ANOVA), as compared with control vehicle (n = 6). No significant dose-dependence was observed at the doses of dantrolene used in the present study (p > 0.05, Scheffé s F test)., saline vehicle;, 10 mg/kg of dantrolene;, 20 mg/kg of dantrolene;, the last day of drug session. * p < 0.05, ** p < 0.01 : statistical analysis of stages on each day by Mann-Whitney s U-test. groups treated with either 10 mg/kg or 20 mg/kg of dantrolene, as compared with the control group treated with vehicle (p < 0.05 or p < 0.01, respectively, repeated measures ANOVA). However, no significantly difference of inhibitory effects on the stage development was observed between the two groups treated with different doses of dantrolene (p > 0.05, Scheffé s F test). When seizure stages on each day were statistically analyzed among three groups, seizure stages of the group treated with 10 mg/kg or 20 mg/kg were significantly smaller than those of the vehicle control group on 7 days or 6 days respectively within the test period of initial 10 days ( * p < 0.05 or ** p < 0.01, Mann-Whitney s U-test). The development of ADD was significantly delayed in the groups treated with dantrolene at a dose of either 10 mg/kg or 20 mg/kg, as compared with that of the control group treated with vehicle (p < 0.05, respectively, repeated measures ANOVA) (Figs. 2 and 3). No significant difference of inhibitory effects on the development of ADDs was observed between the two groups treated with different doses of dantrolene (p > 0.05, Scheffé s F test). When ADDs on each day were statistically analyzed among three groups, ADDs of both groups treated with 10 mg/kg and with 20 mg/kg of dantrolene were significantly smaller than those of the vehicle control group on 6 days within the test period of initial 10 days ( * p < 0.05 or ** p < 0.01, one-way ANOVA). Effects of dantrolene on seizures in kindled rats This study was performed to investigate whether dantrolene had an action to suppress seizures, because an anticonvulsant action might retard the kindling development. When an electric current 10% higher than GST was delivered at the amygdala, all groups of fully kindled rats (n = 7, respectively) showed stage 5 seizures, whether they were pretreated with vehicle or with dantrolene with 20 mg/kg or 40 mg/kg. A dose of 40 mg/kg of dantrolene, which was higher than that used in the developmental study, showed no significant anticonvulsant

5 Kindling is Associated with Intracellular Ca 2+ Stores 307 Fig. 2. A specimen record of afterdischarges from amygdaloid nuclei in a test rat and a control rat on the 6th experimental day. The inhibition of afterdischarge by the continuous treatment of dantrolene was demonstrated. Mono P: monopolar lead, Bi P: bipolar lead. Vertical arrow: the beginning of afterdischarge. Horizontal line underlying EEG: the duration of afterdischarge. Horizontal arrow: continuation from the EEG record shown on upper row. Fig. 3. Inhibitory effects of dantrolene on the progression of afterdischarge duration in kindling. The development of afterdischarge duration was significantly delayed by dantrolene at a dose of either 10 mg/kg or 20 mg/kg, as compared with control vehicle (n = 6, p < 0.05, respectively, repeated measures ANOVA). No significant dose-dependence was observed at the doses used in the present study ( p > 0.05, Scheffé s F test)., saline vehicle;, 10 mg/kg of dantrolene;, 20 mg/kg of dantrolene;, the last day of drug session. * p < 0.05, ** p < 0.01: statistical analysis of ADDs on each day by one-way ANOVA.

6 308 M. Yoshida and T. Sakai effects on either seizure stages or ADDs (Fig. 4). DISCUSSION Effects of dantrolene on the development of kindling It was shown in the present study that dantrolene, a CICR blocker, significantly delayed the kindling development of either seizure stages or ADDs. No significantly different effects were observed between the two groups treated with either 10 mg/kg or 20 mg/kg of dantrolene. It could not be excluded that larger doses might produce significantly different effects. However, we did not try doses over 20 mg/kg in the kindling development study, because the chronic treatment with such larger doses elicited toxic effects. LTP and kindling, both of which are the models of enduring increased neuronal excitability, may constitute the common cellular basis in part (Pedley and Meldrum 1992). They require gene transcription and protein synthesis (Berridge 1998; Hardingham and Bading 1998). Nuclear calcium mediates enduring forms of plasticity-related events such as LTP or LTD. Nonphysiological stimuli may generate aberrant nuclear calcium signals that initiate inappropriate transcriptional responses. These could lead to long-term changes in neuronal excitability, such as those seen in kindling (Bading 2000). Nuclear calcium is to a large extent provided by intracellular calcium stores. Calcium influx through synaptic NMDA receptors triggers calcium release from intracellular calcium stores. This amplifies synaptic calcium signals and relays them in the form of regenerative calcium waves to the nucleus (Hardingham et al. 2001). Calcium from intracellular calcium stores is released via ryanodine receptor (RYR) channels or inositol triphosphate receptor (Ins P 3 R) channels; CICR or InsP 3 - induced calcium release (IICR), respectively (Berridge 1993; Kohda et al. 1995; Verkhratsky and Shmigol 1996). Ryanodine receptors are activated by the local accumulation of Ca 2+ flowing through NMDA receptors, thus allowing increased cytoplasmic Ca 2+ to elicit further increases in the cytoplasmic Ca 2+ by the process of CICR (Rutecki et al. 2002). When the trigger Ca 2+ entering Fig. 4. No anticonvulsant effects of dantrolene on the stage of seizures (upper panel) and the afterdischarge duration (lower panel) in kindled rats. Fully kindled rats (n = 7) were stimulated at the amygdala with the current 10% higher than the generalized seizure threshold. Rats reacted with stage 5 seizures whether they were pretreated with control vehicle or with dantrolene. Even when a dose of dantrolene was increased to 40 mg/kg, no significant alteration was observed. There were no statistically significant differences in effects of dantrolene on stages and afterdischarge durations as compared with control vehicle (p > 0.05, Wilcoxon signed-ranks test or paired t-test, respectively). across NMDA receptors can be amplified and relayed by the Ca 2+ release from intracellular Ca 2+ stores through the process of CICR, Ca 2+ may consequently gain access to the nucleus (Berridge 1998). Echevarria et al. (2003) recently identified a reticular network (nucleoplasmic reticulum) of nuclear calcium stores that is continuous with the

7 Kindling is Associated with Intracellular Ca 2+ Stores 309 endoplamic reticulum and the nuclear envelope. They showed it had the capacity to regulate calcium signals in localized subnuclear regions. It has been documented that dantrolene inhibited the binding of ryanodine on ryanodine receptors on endoplasmic reticulum and blocked CICR (Simpson et al. 1995). Recently it was reported that release of Ca 2+ from the nucleoplasmic reticulum evoked CICR within the nucleus, which could be suppressed by dantrolene (Marius 2006). Dantrolene has been reported to block the induction of LTP in a slice preparation of the hippocampal CA1 region (Obenaus et al. 1989), though there was a contradictory report (Xu and Krnjević 1990). Dantrolene might block the nuclear alteration of Ca 2+ levels by inhibiting tetanus-induced regenerative Ca 2+ waves toward the nucleus or directly inhibiting calcium release in the nucleus. The inhibition of nuclear increment in Ca 2+ concentration might block the gene transcription underlying kindling phenomena. Therefore, there was a possibility that inhibitory effects of dantrolene on the kindling acquisition in the present study were produced by the blockade of Ca 2+ release from ryanodine-receptors on intracellular stores. Effects of dantrolene on seizures in kindled rats It was demonstrated that dantrolene had no anticonvulsant action against kindled seizures, even at a dose larger than those used in the study on kindling development. It has been documented that dantrolene suppressed convulsive seizures in EL mice (Nagatomo et al. 2001). Dantrolene at very large doses (from 62.5 to 500 mg/kg, i.p.) has been described to inhibit limbic seizures induced by injections of 1S,3R-ACPD or 3,5-DHPG, mglur agonists, into the thalamus (Tizzano et al. 1993, 1995). Dantrolene has been also demonstrated to inhibit ictal discharges induced by pilocarpine or (RS)-3,5-dihydroxyphenylglycine (DHPG), a mglur agonist in hippocampal slice preparations of rats (Rudecki et al. 2002). On the other hand, it has been shown that dantrolene (5-20 mg/kg, i.p.) did not raise electroconvulsive thresholds in mice (Borowicz et al. 1997). EEG parameters have been shown to fail providing any evidence for an anticonvulsant effect of dantrolene (10 mg/kg, i.p.) in electrically-induced status epilepticus in rats (Niebaur and Gruenthal 1999). The results in the present study suggested that the inhibitory effect of dantrolene on the acquisition of kindling might not be related to an anticonvulsant action. On the other hand, an inhibitory action of dantrolene on CICR was suggested to delay the development of kindling phenomenon. Further studies are still necessary to clarify the relationship of intracellular Ca 2+ stores and a kindling development by using the chronic treatment with less toxic substances which modify intracellular Ca 2+ stores or directly measuring Ca 2+ content and transcriptional factors in nuclei. Less toxic substances which modify intracellular Ca 2+ stores would be beneficial to prevent acquired epilepsies. Acknowledgments I would like to express my great appreciation to Professor Pauly for improving the manuscript. References Bading, H. (2000) Transcriptional-dependent neuronal plasticity, The nuclear calcium hypothesis. Eur. J. Biochem., 267, Berridge, M.J. (1993) Inositol triphosphate and calcium signaling. Nature, 361, Berridge, M.J. (1998) Neuronal calcium signaling. Neuron, 21, Bliss, T.V.P. & Collingridge, G.L. (1993) A synaptic model of memory: long-term potentiation in the hippocampus. Nature, 361, Borowicz, K.K., Gasior, M., Kleinrok, Z. & Czuczwar, S.J. (1997) Influence of isradipine, niguldipine and dantrolene on the anticonvulsive action of conventional antiepileptics in mice. Eur. J. Pharmacol., 323, Delorenzo, R.J. & Morris, T.A. (1999) Long-term modulation of gene expression in epilepsy. Neuroscientist, 5, Echevarria, W., Leite, M.F., Guerra, M.T., Zipfel, W.R. & Nathanson, M.H. (2003) Regulation of calcium signals in the nucleus by a nucleoplasmic reticulum. Nat. Cell Biol., 5, Gilbert, M.E. & Mack, C.M. (1990) The NMDA antagonist, MK-801, suppresses long-term potentiation, kindling, and kindling-induced potentiation in the perforant path of the unanesthetized rat. Brain Res., 519, Goddard, G.V., McIntyre, D.C. & Leech, C.K. (1969) A permanent change in brain function resulting from daily electrical

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