Eiichi Sugaya

Behavior of intracellular cyclic nucleotide and calcium in pentylenetetrazole-induced bursting activity in snail neurons

To elucidate the intracellular mechanism of the bursting activity which is characteristic of seizure discharge, the behavior of the intracellular cyclic nucleotide and the intracellular calcium during pentylenetetrazole (PTZ)-induced bursting activity in snail neurons was investigated. Cyclic AMP was increased about 3-fold by the incubation of ganglia with PTZ. The effect of PTZ on phosphodiesterase activity measured using either cyclic AMP or cyclic GMP as substrate showed a slight increase in cyclic AMP phosphodiesterase activity. The release of calcium from the lysosome fraction was increased by the incubation of ganglia with dibutyryl cyclic AMP. Protein kinase activity was stimulated by the incubation of ganglia with PTZ. Adenylate cyclase activity was stimulated by the incubation of ganglia with PTZ. These findings suggest that PTZ-induced bursting activity in snail neurons is initiated by an intracellular increase of cyclic AMP, which promotes calcium release from lysosomes and induces protein kinase activation.

Pentylenetetrazole-induced bursting activity and cellular protein phosphorylation in snail neurons

To elucidate the intracellular mechanism of seizure discharge, phosphorylation of cellular protein during pentylenetetrazole (PTZ)-induced bursting activity in snail neurons was investigated. PTZ markedly enhanced the phosphorylation of proteins of 34,000 and 50,000 molecular weight. Similar effects were observed by application of a calcium ionophore, A23187. Calmodulin antagonist, N-(6-aminohexyl)-5-chloronaphthalenesulfonamide hydrochloride (W-7), inhibited the PTZ-induced increased phosphorylation of these two proteins. Dibutyryl cyclic AMP and iosobutylmethylxanthine (IBMX) showed no significant effect on the phosphorylation pattern. Bath application of the calcium ionophore produced bursting activity followed by long-lasting hyperpolarization. Bath application of W-7 completely inhibited the PTZ-induced bursting activity. These results suggest that the phosphorylation of proteins of 34,000 and 50,000 in molecular weight is related to the generation of bursting activity by PTZ.

Cloning and expression of SEZ-6, a brain-specific and seizure-related cDNA

To clarify the molecular mechanism of neuronal bursting activity of seizures, we have constructed a cDNA library from mouse cerebrum cortex-derived cells treated with pentylentetrazole (PTZ), one of the convulsant drugs. Using a differential screening technique, several cDNA clones whose expressions change with PTZ-treatment were obtained. Among these clones, SEZ-6 was characterized by increased expression with PTZ. Detailed northern analysis showed that expression of SEZ-6 was limited to the brain and increased by the administration of PTZ not only in in vitro cultured cells but also in vivo. Analysis of SEZ-6 cDNA revealed multiple motifs, including typical signal sequence, threonine-rich domain, five copies of short consensus repeats (SCRs) or sushi domain (complement C3b/C4b binding site), two repeated sequences which were partially similar to the CUB domain or complement C1r/s-like repeat, one transmembrane domain and a short cytoplasmic segment in the C-terminal region. Although many proteins with multiple SCRs or CUB domains other than complement-related proteins have been found, this is the first report about a brain-specific cDNA which encodes membrane protein with both SCRs and CUB domain-like segments. Based on these findings, it is evident that SEZ-6 encodes a novel type of protein which may be related to seizure.

Inhibitory effect of peony root extract on pentylenetetrazol-induced EEG power spectrum changes and extracellular calcium concentration changes in rat cerebral cortex

To elucidate the mechanism of anticonvulsant action of peony root and to determine the relative contributions of pure component substances, the water, water/acetone and methanol extracts of peony roots, paeoniflorin, albiflorin and pentagalloylglucose were studied in rats using the EEG power spectrum changes induced by pentylenetetrazol administration and the extracellular calcium and potassium concentration changes related to seizure activity. The water extract of peony roots, albiflorin and pentagalloylglucose given orally completely inhibited the EEG power spectrum changes as well as the extracellular calcium and potassium concentration changes related to seizure activity. The water/acetone and methanol extracts and paeoniflorin were relatively less potent. These findings suggested that the anticonvulsant action of peony roots is due primarily to albiflorin and the gallotannin fraction. Albiflorin and pentagalloylglucose appear to manifest their anticonvulsant action due to inhibition of the seizure-related decrease of extracellular calcium and consequent intracellular calcium increase.

Pentylenetetrazol-Induced Convulsion and Effect of Anticonvulsants in Mutant Inbred Strain El Mice

Summary: The El mouse is an inbred strain developed from ddY mice and is very susceptible to seizure. In El mice, convulsions could be induced by 18 mg/kg of pentylenetetrazol, which is an inert dose in ddY mice. The features of the convulsions were the same as those induced by the tossing‐up procedure, a common method to evoke convulsions in El mice. Phenytoin, phenobarbital, valproate sodium, and ethosuximide clearly inhibited pentylenetetrazol‐induced convulsions in El mice. These findings suggest that induction of convulsions by pentylenetetrazol in the El mouse is a simple method as compared with induction of convulsions by the tossing‐up procedure, and convulsions evoked by this method are a precise experimental model for the study of hereditary epilepsy and for the evaluation of anticonvulsant drugs.

Protective Effects of Peony Root Extract and Its Components on Neuron Damage in the Hippocampus Induced by the Cobalt Focus Epilepsy Model

Protective effects of peony root extract and its components on neuron damage in the CA1 area of the hippocampus induced by the cobalt focus epilepsy model were examined. Neuron damage in the CA1 area of the hippocampus and frequent spike discharges induced by application of metallic cobalt to the cerebral cortex of rats were completely prevented when peony root extract was continuously administered orally at 1 g/kg/day for 30 days prior to cobalt application. Component crude gallotannin fraction showed marked but incomplete protective action. A combination of crude gallotannin fraction and paeoniflorin showed complete protective action in the same way as peony root extract against neuron damage although use of paeoniflorin alone had no effect. These findings together with our previous reports indicate that peony root extract and its component, gallotannin, have excellent protective effects on neuron damage in addition to anticonvulsant action by prior oral administration.

Effect of phenytoin on intracellular calcium and intracellular protein changes during pentylenetetrazole-induced bursting activity in snail neurons

Effects of phenytoin (PHT) on the intracellular calcium and intracellular protein changes during pentylenetetrazole (PTZ)-induced bursting activity in the neurons of the Japanese land snail Euhadra peliomphala were examined. In the examination with a computer controlled electron probe X-ray microanalyzer, PHT clearly inhibited the intracellular calcium shift induced by PTZ as well as the calcium binding state change near the cell membrane. PHT also clearly inhibited the intracellular protein changes induced by PTZ. PHT, however, did not show any change in the transmembrane ionic currents such as the sodium current, calcium current and potassium current. These findings suggest that one of the main sites of anticonvulsant action of PHT is pathologically changed intracellular calcium movement and intracellular protein changes during seizure discharge.

Intracellular calcium concentration during pentylenetetrazol-induced bursting activity in snail neurons

To clarify the role of intracellular free calcium in the provocation of bursting activity, the intracellular calcium concentration was measured using calcium-sensitive microelectrodes during pentylenetetrazol (PTZ)-induced bursting activity in snail neurons. In the PTZ-sensitive neurons of the snail, Euhadra peliomphala, the intracellular calcium concentration was higher than that in the normal state during PTZ-induced bursting activity, but lower than that which induced the calcium-activated potassium conductance elevation. By application of 50 microM calcium ionophore, A23187, a slight increase in intracellular calcium concentration with a slight depolarization occurred. Then a greater increase of intracellular calcium concentration with bursting activity was observed. By application of 100 microM A23187, a rapid and intense increase of intracellular calcium concentration with hyperpolarization was observed. These findings suggest that, for the provocation of bursting activity, sustained elevation of the intracellular calcium concentration, higher than that in the normal state but lower than that which evokes calcium-activated potassium conductance elevation, is required.

Intracellular protein changes during pentylenetetrazole induced bursting activity in snail neurons

The intracellular protein changes during pentylenetetrazole (PTZ)-induced bursting activity (BA) which is characteristic of seizure discharge were investigated using microdisk electrophoresis with 5 identified neurons. The identified neurons of the snail, Euhadra peliomphala, were used. The PTZ-sensitive neurons which manifest marked BA by application of PTZ were examined. PTZ induced in PTZ-sensitive neurons: (1) a prominent increase of 5-7 kdalton protein and (2) peak separation into 3 peaks of 10-15 kdalton protein. In the 5-7 and 10-15 kdalton protein, a marked increase in radioactivity of 45Ca was observed after PTZ application. PTZ-non-sensitive neurons showed neither these protein changes nor 45Ca incorporation into these proteins. The above findings suggest that during PTZ-induced BA, intracellular protein changes occurred in relation to the intracellular calcium shift.

Characteristics of primary cultured neurons from embryonic mutant El mouse cerebral cortex

To elucidate the differences between neurons of epileptogenic animals and those of normal animals, cellular characteristics of neurons of mutant strain El mice which are highly susceptible to seizures were investigated using immunocytochemical techniques. In neurons of 3-day primary cultures, the control ddY mouse neurons showed dividing stages in about 0.2% of neurofilament (NF)-positive neurons, whereas no dividing neurons were observed among the NF-positive El mouse neurons. In 7-day cultures, localization of GD3 ganglioside in the proliferating control ddY mouse neurons was observed, but there was no GD3 ganglioside in the mutant El mouse neuron. The content of GD3 ganglioside detected by high-performance thin-layer chromatography of El mouse cultured cells was ca. 1/4 of that of ddy mice. These findings suggest that neurons of the El mouse are differentiated earlier than those of the control ddY mouse.