Hideo Tsuda

Regional excitatory and inhibitory amino acid concentrations in pentylenetetrazol kindling and kindled rat brain

We determined regional concentrations of excitatory and inhibitory amino acids in pentylenetetrazol (PTZ) kindling and kindled rat brains in order to investigate the mechanisms responsible for the PTZ kindling process and kindled state. Compared with control rats, PTZ kindling rats had significantly higher concentrations of aspartate in the striatum and the temporal cortex 24 h after the 14th injection of PTZ. Glutamate and GABA concentrations were also significantly higher in the brainstem of PTZ kindling rats 24 h after the 14th injection of PTZ. These findings suggest that the striatum and temporal cortex may participate in a pathway for propagation of the PTZ kindling process, and that the brainstem may be a primary site of PTZ kindling epileptogenesis or a part of the pathways for propagation. On the other hand, the concentrations of amino acids in PTZ kindled rats and controls did not differ 4 weeks after the 14th injection. This suggests that the alterations we detected in amino acid metabolism are not related to retention of the PTZ kindled state, and that some other mechanism for it must exist.

Biochemical Study on the Critical Period for Treatment of the Mottled Brindled Mouse

Abstract: Hemizygous mottled brindled mice (Mobr/y mice) were treated by subcutaneous injection of copper and were decapitated on postnatal day 14. Cytochrome c oxidase (COX) activity of the brain mitochondria in the mice given 10 μg of copper/g on day 4 or 7 showed significant increases compared with that of untreated Mobr/y animals, and these mice had no neurological symptoms. Mice given 10 μg of copper/g on day 12 showed neither increases in COX activity nor clinical improvement. The brain levels of copper, nor‐adrenaline, and dopamine in the mice treated on day 12 were the same as those in animals treated on day 4 or 7. The in vitro activities of dopamine‐β‐hydroxylase of the brain were also the same among the treated mice, irrespective of the date of treatment. The results indicate that delays in copper treatment produce irreversible changes in COX activity of the brain and lead to clinical unresponsiveness to treatment.

Involvement of the noradrenergic system in the seizures of epileptic El mice

We studied the role of the noradrenergic system in the seizures of epileptic El mice. To this end, the anticonvulsant activity of adrenergic drugs was tested with a scoring method, and the binding of [3H]dihydroalprenolol, [3H]prazosin and [3H]yohimbine was evaluated in whole brains and various brain regions from stimulated and unstimulated El mice, and their maternal ddy mice. The seizures of El mice were inhibited by noradrenaline, phenylephrine, oxymetazoline, clonidine and yohimbine in a dose-dependent manner. These preventive effects of alpha-adrenoceptor agonists were antagonized by pretreatment with alpha-adrenoceptor antagonists. The preventive effect of yohimbine was reversed by pretreatment with clonidine or alpha-methyl-p-tyrosine, although the latter drug did not affect the anticonvulsant effect of clonidine. The binding of [3H]dihydroalprenolol was the same in the three groups of mice. More [3H]prazosin was bound in the cerebellum and striatum, and there were more [3H]yohimbine binding sites in the whole brain, cerebral cortex, hippocampus and brainstem of stimulated and unstimulated El mice than in the same areas of ddy mice. These findings suggest that up-regulated alpha 1- and alpha 2-adrenoceptors are involved in the inhibition of the seizures of El mice.

Age- and seizure-related changes in noradrenaline and dopamine in several brain regions of epileptic El mice

Noradrenaline (NA) and dopamine (DA) levels in six brain regions of stimulated and nonstimulated El (El[s] and El[ns]) mice and their maternal ddY mice were determined at various ages and various times after a convulsion. The NA levels in the striatum and hippocampus of 12-week-old El[s] and El[ns] mice were lower than in ddY mice, and remained lower in 23-week-old El[s] mice, but not in El[ns] mice. DA levels were lower in the striatum of El[s] mice than in El[ns] and ddY mice at 16 and 23 weeks of age. NA levels decreased during seizure in the striatum and hippocampus of El[s] mice, and returned to preconvulsive levels 1 hr after convulsion in the striatum and 30 min in the hippocampus. DA levels in the striatum of El[s] mice decreased during convulsion and increased from 1 to 10 min after convulsion. These changes suggest that the NAergic systems in the striatum and hippocampus and the DAergic system in the striatum have important roles in relation to seizure susceptibility in El mice.

Depth EEG in mutant epileptic El mice: demonstration of secondary generalization of the seizure from the hippocampus

Mutant epileptic E1 mice are thought to have focal epilepsy of hippocampal origin because glucose utilization is increased in the hippocampus (HPC) during seizures in these mice. However, direct electrographic evidence is still lacking for the notion. We recorded electroencephalograms (EEGs) using depth electrodes in E1 and non-epileptic ddY mice. All the mice were subjected to a conventional seizure-provoking maneuver during EEG recording; each mouse was placed on a mesh floor and observed for 3 min, and then tossed up in the air. When the E1 mice showed signs of abortive seizures or prodromal symptoms including squeaking, running and myoclonus, sporadic spikes or sharp waves were generated exclusively in the HPC. When generalized convulsions followed these prodromes, the sporadic discharges evolved into a burst of generalized spikes which again predominated in the HPC. We also observed the cerebral cortex, amygdaloid, caudate, centro-median thalamic and ventral postero-lateral thalamic nuclei, all of which were found to be only secondarily involved. These findings provide the first electrical evidence that E1 mice have a secondarily generalized seizure that has its initiating focus in the HPC.

Effect of penicillin on GABA-gated chloride ion influx

To investigate the mechanism of penicillin-induced convulsions, we have studied the effects of penicillin G (PC-G) on GABA-gated chloride ion influx in brain ‘microsac’ preparations of mice. In the presence of 10−4 M GABA, PC-G inhibited GABA-gated chloride ion influx in a dose-dependent manner. The dose-response curve for GABA in the presence of 10−3 M PC-G was shifted rightward and there was a decrease in maximum response. The inhibitory effects of PC-G were not reversed by RO 15-1788, an antagonist of benzodiazepine (BZ) receptors, but were reversed by washing the ‘microsac’ membranes. Therefore, PC-G probably exerts its proconvulsant effect by inhibiting GABA-gated chloride ion influx. However, it appears not to act through the BZ receptor of the GABA/BZ receptor complex.

GABA-gated chloride ion influx in brains of epileptic El mice

GABA-gated chloride ion influx was measured in brain “microsac” preparations of epileptic El mice. There was significantly greater sensitivity to GABA in stimulated El mice (which had 14–18 convulsions induced at weekly intervals) than in unstimulated El mice (which had not experienced convulsions) or ddY mice. GABA-gated chloride ion influx was significantly decreased 20 min after a single convulsion, and returned to the preconvulsion level 60 min after a convulsion. These findings suggest that the functional state of GABA-gated chloride channel in El mice is changed secondarily by single or repeated convulsions.

Decreased benzodiazepine receptor binding in epileptic El mice: A quantitative autoradiographic study

Benzodiazepine receptors and subtypes were examined in El mice and normal ddY mice with a quantitative autoradiographic technique. Specific [3H]flunitrazepam binding in stimulated El mice, which had experienced repeated convulsions, was significantly lower in the cortex and hippocampus than in ddY mice and unstimulated El mice. In the amygdala, specific [3H]flunitrazepam binding in stimulated El mice was lower than in ddY mice. There was a tendency for the [3H]flunitrazepam binding in these regions in unstimulated El mice to be intermediate between that in stimulated El mice and that in ddY mice, but there was no significant difference between unstimulated El mice and ddY mice. [3H]Flunitrazepam binding displaced by CL218,872 was significantly lower in the cortex of stimulated El mice than in that of the other two groups, and in the hippocampus of stimulated than of unstimulated El mice. These data suggest that the decrease in [3H]flunitrazepam binding in stimulated El mice may be due mainly to that of type 1 receptor and may be the result of repeated convulsions.

Association of NMDA receptor sites and seizures of El mice

To investigate the possible role of N-methyl-D-aspartate (NMDA) receptors in the seizures of El mice, a genetic animal model of epilepsy, we measured [3H]3-[(+)-2-(carboxypiperazin-4-yl)][1,2-3H]- propyl-1-phosphonic acid (CPP) binding in several brain regions of El and ddY mice. At 22-24 weeks of age, the maximum number of binding sites (Bmax) of [3H]CPP was lower only in the cerebral cortex of both stimulated and unstimulated El mice (El(+) and El(-), respectively) than in that of ddY mice. A reduction in Bmax values of cortical [3H]CPP binding of El mice was detected after the age of 12 weeks. Cortical [3H]CPP binding in El(+) mice decreased further transiently after evoked seizures. No significant change was observed in El(-) mice after postural stimulation. These results suggest that El(+) and El(-) mice share seizure propensity and that activation of NMDA receptors is involved in the seizures of El mice.

NMDA-sensitive l-[3H]glutamate binding in cerebral cortex of El mice

NMDA-sensitive L-[3H]glutamate binding was examined in the brains of El mice, a genetic animal model of epilepsy, and in ddY mice. In whole brain, Scatchard analysis showed that both stimulated and unstimulated El mice had significantly lower Bmax values for binding than did ddY mice. In regional studies, the binding of NMDA-sensitive L-[3H]glutamate was significantly less in the cerebral cortex of both stimulated and unstimulated El mice than in that of ddY mice. These data suggest that NMDA receptors may be involved in the genetic susceptibility of El mice to seizures.