Noriko Tsuru

Simultaneous monitoring of the seizure-related changes in extracellular glutamate and γ-aminobutyric acid concentration in bilateral hippocampi following development of amygdaloid kindling

We simultaneously monitored the seizure-related changes in extracellular hippocampal glutamate (Glu) and gamma-aminobutyric acid (GABA) concentration in brain dialysates in order to clarify the role of Glu and GABA in the development of kindling. Brain dialysates were collected every 5 min from 10 min prior to 80 min after stimulus in the three developing conditions consisting of pre-kindling state, stage 3 (C-3), and five consecutive stage 5 (5*C-5) following kindling in the same rat. Extracellular Glu level increased rapidly, lasting for only 5 min after stimulus. The post-stimulus ratio of Glu increase in partially kindled rats (C-3) was 2.5-3.5 times of the baseline, and in fully kindled rats it was about 5 times of the baseline. Extracellular GABA concentration enhanced gradually, reaching a plateau level at 15-20 min and lasting for several hours after stimulus at each stage. The enhancement of GABA level was about 1.5 times of the baseline in partially kindled stage, and was about 2.5 times of the baseline in fully kindled stage. There was no significant difference between the two hemispheres with respect to either the time-course or the magnitude of Glu and GABA increase respectively. These data show that progressive, transient and stimulus-induced enhancement of extracellular Glu levels combined with long-lasting elevation of extracellular GABA levels in the bilateral ventral hippocampi results in imbalance between the excitatory and inhibitory neuronal systems, causing excessive propagation of seizure activity, culminating in the secondary generalized seizure of amygdaloid kindling.

Bilateral seizure-related changes of extracellular glutamate concentration in hippocampi during development of amygdaloid kindling

To monitor the seizure-related changes of extracellular hippocampal glutamate (Glu) concentration during the development of amygdaloid kindling, we used brain dialysates and an enzymatic cycling technique for Glu determination with a highly sensitive assay and high time resolution (1 min). The extracellular Glu level was transiently (for 3 min) enhanced after stimulus and returned rapidly to baseline. In partially kindled rats (stage 3), the extracellular Glu level during the first minute post stimulus was 2.5-3.5-fold that of baseline, while fully kindled rats exhibited about a 5-fold increase in Glu level. Amygdaloid kindling is accompanied by a progressive, transient, stimulus-induced enhancement of extracellular Glu levels during the first minute post stimulus in both hippocampi.

Expression of glutamate transporters and ionotropic glutamate receptors in GLAST knockout mice

In order to investigate the molecular mechanism underlying high seizure susceptibility of GLAST knockout mice, we carried out Western blotting for the expression of GLT-1, EAAC-1, and several kinds of glutamate receptors in the hippocampus and the cortex. Although no significant difference was observed between GLAST (+/+) and (-/-) mice in terms of expression of GLT-1 and EAAC-1 in the hippocampus, these proteins were over-expressed in the frontal cortex in GLAST (-/-) mice (GLT-1, about 210% increase; EAAC-1, about 180% increase). Expression of hippocampal Glu-R1 and Glu-R2 in GLAST (-/-) mice was remarkably increased (Glu-R1, about 140% increase; Glu-R2, about 160% increase), while Glu-R3 and NMDA receptors levels (NMDA-R1, 2A and 2B) were equal to those in control. Cortical levels of Glu-R1, -R2 and -R3 receptors in GLAST (-/-) mice were remarkably decreased (Glu-R1, about 60% decrease; Glu-R2, about 60% decrease; Glu-R3, about 70% decrease), while NMDA receptors were remarkably increased in comparison to those in GLAST (+/+) mice (N-R1, about 150% increase; N-R2A, about 150% increase; N-R2B, about 140% increase). These data suggest that the increased susceptibility to seizures in GLAST (-/-) mice might be derived from increased expression of Glu-R1 in the hippocampus coupled with decreased cortical expression of Glu-R2 and increased NMDA-R1 and -2A, -2B expression.

Effect of Unilateral Dentate Nucleus Lesions on Amygdaloid Kindling in Rats

Summary: Amygdala kindling is used to investigate cerebellar dentate nucleus participation in the neuronal plasticity of the cerebrum. We studied the behavioral change, the occurrence of epileptiform events, and the duration of afterdischarge during amygdaloid kindling under control conditions and after a dentate lesion of the contralateral cerebellum. Our results indicate that the dentate lesion induced the facilitation of the behavioral development of the amygdaloid kindling, whereas there was only a slight increase in the duration of afterdischarge and the spontaneous epileptiform potential of the amygdala. There was a discrepancy between the behavioral kindling and the electroencephalographic epileptic activity. Therefore the dentate lesion must play a role in controlling the generalization mechanism of epilepsy. We also found that on a right amygdala that had been influenced by a left dentate lesion there were few spikes, while on the left amygdala there were frequent spikes. These results suggest that the dentate nucleus plays an important role in amygdaloid kindling as well as in the neuronal plasticity of the amygdala.

Effects of acute treatment and long-term treatment with MK-801 against amygdaloid kindled seizures in rats

Effects of both acute and long-term treatments with a non-competitive NMDA-antagonist, MK-801, were studied in rats in order to investigate whether NMDA receptors would be involved in the maintenance of kindling. Intraperitoneally administered MK-801 at doses of 0.5, 1.0 and 2.0 mg/kg inhibited fully amygdaloid kindled seizures (stage 5) behaviorally and significantly reduced the duration of after discharge and postictal spikes in a dose-dependent manner. Long-term (28 days) administration of MK-801 at three dosages neither abolished behavioral kindled seizures nor significantly altered generalized seizure thresholds. However the after discharge duration and postictal spikes were significantly reduced in rats treated with MK-801 at doses of 0.5 and 1.0 mg/kg, while no significant alterations were observed in rats treated with saline or MK-801 at a dose of 2.0 mg/kg. Acute effects of MK-801 suggested that NMDA receptors were involved in the maintenance of kindling. On the other hand, long-term treatments with MK-801 remained to show such an unequivocal effect.

Amygdaloid kindling in glutamate transporter (GLAST) knockout mice

Summary:  Purpose: Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system. We previously reported abnormal glutamate release during seizures after kindling. GLAST and GLT‐1 are astrocytic glutamate transporters, highly concentrated in the cerebellum and the telencephalon, respectively. We have investigated whether stages of amygdala kindling in knockout (KO) mice deficient in GLAST are the same as those of wild mice.

Neuronal Firing Pattern Following Amygdaloid Kindling in Unrestrained Rats

Summary: Chronic recordings of amygdaloid neurons were performed on freely moving rats following kindling. Satisfactory recordings were obtained from 22 amygdaloid neurons of the contralateral amygdala before, during, and after unilateral kindling. Kindling stimulations were given once per hour. Seven cells disappeared during kindling. Seven cells were recorded during the full course of kindling. These units showed (a) an increase in spontaneous firing, (b) a development of high‐frequency bursts (the peak interval of the interval histogram decreased from 18 to 2 ms), and (c) high‐frequency firings during spontaneous activity that were similar to the firings recording during afterdischarge.

Functional role of GABA transporters for kindling development in GLAST KO mice.

Kindling-induced after discharge in electroencephalograms depends on the protein associated with glutamatergic and/or GABAergic neuronal transmission. In glutamate transporter knockout (GLAST KO) mice, the kindling phenomena in GLAST KO developed more slowly while the after discharge duration (ADD) was briefer than that of the control C57BL-6J mice. These findings indicate that either the excitatory function was suppressed or the inhibitory function was enhanced in GLAST KO kindling. To explain these phenomena, we used Western blotting to evaluate the alterations in the expression of hippocampal GABA transporter proteins, and the estimation of the effect on the process of epileptogenesis. Although no alterations were observed in the GAT-3 expression, the hippocampal GAT-1 expression was significantly suppressed in comparison to that of C57BL-6J mice. A decreased GAT-1 level in the hippocampus, which might be associated with the increased extracellular GABA level, may therefore inhibit both ADD and seizure propagation as shown by the amygdaloid kindling phenomenon observed in GLAST KO mice.

Effect of riluzole on the acquisition and expression of amygdala kindling

PURPOSE Riluzole possesses various synaptic effects including an inhibitory action on glutamate release. The drug has been shown to inhibit kindled seizures, while its effect on the acquisition of kindling has not been reported. We investigated effects of riluzole on the kindling development in addition to effects on kindled seizures. METHODS A bipolar electrode was implanted in the right amygdala of rats. Riluzole was injected intraperitoneally 30 min before kindling stimulation. To investigate effects of riluzole on the kindling development, rats were stimulated once daily for the drug session of 14 days at a current of 200 microA, 60 Hz, 1 ms for 2 s and thereafter stimulated without drugs (drug-free session) until completion of kindling. Seizure ranks and after discharge duration were observed every day. To investigate effects of riluzole on kindled seizures, fully-kindled rats were stimulated at the current of generalized seizure threshold (GST) before and after the administration of riluzole. Seizure ranks and after discharge duration were measured. GST after the treatment was also determined. RESULTS The number of stimuli required for the first appearance of stage five seizure was significantly larger in rats treated with 8 mg/kg of riluzole than in vehicle controls. Riluzole at a dose of 8 mg/kg significantly retarded the development of seizure stages in the drug session. By comparison, effects on the duration of after discharge was relatively mild, though significantly different from the vehicle control. Riluzole at a dose of either 4 or 8 mg/kg markedly inhibited behavioral seizures and reduced the duration of after discharge in kindled seizures provoked by GST. The drug also significantly increased GST at both doses, suggesting that the anticonvulsant effects were attributed to the increase in GST. CONCLUSION It was demonstrated that inhibitory effects of riluzole on both kindled seizures and the development of behavioral seizures in kindling acquisition with relatively mild correlation to afterdischarge duration. These effects might be attributed to inhibitory actions of riluzole on glutamate release and NMDA-receptor mediated events.

Lack of substantial effect of the H3-antagonist thioperamide and of the non-selective mixed H3-antagonist/H1-agonist betahistine on amygdaloid kindled seizures

We investigated whether some histamine H(3)-antagonists would attenuate amygdaloid kindled seizures in rats. Thioperamide, a standard H(3)-antagonist, did not significantly reduce either seizure ranks or afterdischarge duration (ADD). Betahistine which has both H(3)-antagonistic activity and H(1)-agonistic activity significantly reduced ADD, albeit mild at a toxic dose, though seizure ranks were not affected. In addition, L-histidine, the precursor of histamine, affected neither seizure ranks, nor ADD. It was shown that H(3)-antagonists have no significant inhibitory action against amygdaloid kindled seizures, probably because released histamine was unable to inhibit those seizures.