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Background Sez4 identified as a seizure-activated gene shows a similarity to the yeast REV3 that encodes a catalytic subunit of the nonessential DNA polymerase ζ which is involved in error-prone translesion synthesis. Although yeast REV3 homologues in mouse and human have recently been identified and characterized, their precise roles remain elusive. Results Here we investigated the role of mouse pol ζ by targeted inactivation of the Sez4 gene. The homozygous Sez4 mutants died around embryonic day (E) 10.5. This lethal effect was the result of developmental defects and apoptotic cell death within the embryo proper at the gastrulation stage, and it was partially rescued at E12.5 by the expression of a Sez4-transgene. In wild-type embryos, Sez4 transcripts were up-regulated within the embryo proper from E7.5, correlating well with the lethal stage of Sez4-inactivation. Conclusion Our findings indicate that Sez4 is essential for epiblast lineage-specific development and suggests a requirement of mammalian DNA polymerase ζ in the survival of certain subcellular populations which are indispensable to normal embryogenesis.

To elucidate the role of the large conductance calcium-activated potassium channel (BK(Ca) channel) in the production of bursting activity, which is characteristic of convulsions, effects of iberiotoxin (IbTX), a selective blocker of the BK(Ca) channel, on bursting activity, induced by various procedures were examined using primary cultured neurons from the cerebral cortex of mice. IbTX completely inhibited bursting activity induced by pentylenetetrazol (PTZ), caffeine, 1,4,5-inositol triphosphate (IP3) and direct forced increase of intracellular calcium. Inherent spontaneous bursting activity in the cerebral cortical neurons of the El mouse, which shows a high susceptibility to convulsions was also completely inhibited by IbTX. Apamin, a specific blocker of the small conductance calcium-activated potassium channel (SK(Ca) channel) showed no inhibition of bursting activity. These findings suggest that the BK(Ca) channel is essential for the production of bursting activity, and also suggest the possibility of clinical use of blocking agents of the BK(Ca) channel against intractable epilepsy.

Gene mapping of the newly discovered SEZ genes (seizure-related genes) in the mouse was performed by linkage analysis. SEZ6 was on chromosome 11, SEZ12 on chromosome 16, SEZ15 on chromosome 3 and SEZ17 (PTZ17) on chromosome 18. The mouse chromosomal locus related to high susceptibility to pentylenetetrazol (PTZ) was also determined by linkage analysis using the recombinant inbred mouse, BXD (C57BL×DBA). A significant level of PTZ susceptibility was found on chromosome 2. Chromosomal loci of the newly discovered SEZ genes were not coincident with the significant chromosomal loci to PTZ susceptibility. Since epilepsy is assumed to be a disease syndrome which is probably manifested by abnormal expression of multifocal genes, determination of the role of each chromosomal locus in the provocation of seizure activity is important.

To determine the molecular mechanism of regulation of pentylenetetrazol (PTZ)-induced calcium entry by the seizure-related gene, PTZ-17, the role of the 3'-untranslated region (3'UTR) and also interaction between 3'UTR and intracellular factors were investigated. PTZ-induced calcium inward current in Xenopus oocytes injected with PTZ-17 RNA varied in magnitude among strains of mice: RNA derived from the DBA/2 mouse, which has a high susceptibility to convulsions, showed the largest current and that from the BALB/c mouse with a low susceptibility to convulsions showed no PTZ response. The sequence of 3'UTR showed alterations among mouse strains: 3'UTR of BALB/c showed a sequence alteration from T to G and that of DBA/2 showed a GTG insertion compared with that of B6. The 3'UTR also regulated the translation of chloramphenicol acetyltransferase (CAT) RNA depending on its sequence. A particular region within the 3'UTR demonstrated interaction with 60- and 47-kDa proteins. Sequence alterations in this region corresponded to disappearance or increase in PTZ-induced calcium entry. These findings suggest that a particular region within 3'UTR of the seizure-related gene, PTZ-17, is involved in PTZ-induced calcium entry via interaction between mRNA and specific RNA-binding proteins.

Doctors who learned exclusively western medicine probably understand a priori Kampo (Japanese herbal) medicine merely as a kind of folk medicine which is not so effective and only a supplementary therapy to western medicine. We have been performing experiments on the mechanism of epileptogenesis mainly at the cellular level for a long time. During the research process, we unexpectedly encountered Kampo (Japanese herbal) medicine, and also performed research on the mechanism of action of a herbal mixture prescription, saiko-keishi-to-ka-shakuyaku (SK, TJ-960). Recently we discovered that SK acts to induce the best functional state of neurons and consequently intractable nervous symptoms disappear. SK has protective effects against neuron damage, normalizing effects on developmental defects of El mouse neurons, complete preventive effects on stress-induced increased c-fos and HSP 72 expression, complete suppression effects on the Reilly syndrome, complete normalizing effects on expression of the seizure-related gene, PTZ-17, and also, surprisingly, complete suppression effects on amyloid beta protein-induced neuron death. Such wide ranging effects which are preferable to functional maintenance and development of neurons can not be obtained by pure chemical drugs. These findings suggest that we should effectively use such ancient herbal prescriptions which show excellent preventive effects against neuron damage, enforcing action on natural healing forces and even regulatory action against adverse expression of genes, at least to prevent intractable nervous diseases, such as epilepsy, Alzheimer's disease and developmental defects of neurons during pregnancy and after birth. We should also create a future medicine, the 'third medicine', which is situated in a higher dimension than that of contemporary oriental and western medicines. For this purpose, it is necessary to perform research on the mechanism of action of Kampo (Japanese herbal) medicine.

SEZ-12 is one of the seizure-related cDNAs which was isolated by differential hybridization from primary cultured neurons from the mouse cerebral cortex with or without pentylenetetrazol (PTZ). SEZ-12 expression is transiently down-regulated in the mouse brain by injection of PTZ. To characterize SEZ-12, isolation of full-length cDNA and nucleotide sequence analysis were performed. The deduced amino acid sequence of SEZ-12 revealed that it encodes membrane-bound C-type lectin and has a significant homology to that of human cDNA, DGCR2 and IDD, which were cloned from a balanced translocation breakpoint associated with the DiGeorge syndrome. The isolated cDNA was about 4 kb in length and the message was expressed ubiquitously in various organs with low-abundance. Previously, we also cloned a transmembrane protein which is probably involved in cell-cell interaction by the differential hybridization technique. These findings suggest that transmembrane signaling in neuronal cells may have an important role in PTZ-induced seizure.

To isolate seizure-related genes, we applied differential screening technique to the cDNA library which constructed from primary cultured cerebral cortical cells treated with pentylenetetrazol (PTZ). Northern blotting analysis of mRNA levels in the cerebra after systemic administration of PTZ confirmed the results of differential screening procedure: 6 clones showed increased mRNA level and 3 clones showed decreased expression with PTZ. Interestingly, 4 genes which were isolated by this technique were related to intracellular calcium action. They were cytosolic phospholipase A2, 78 kDa glucose regulated protein, SEZ-15 which has an EF hand motif and PTZ-17 that causes calcium current in Xenopus oocyte with PTZ application. These data and our previous results suggest that intracellular calcium may play an important role for seizure-related pathophysiological changes in neuronal cells.

To elucidate the mechanism of the invigorating and antisenile action of the dried herb of Boschniakia rossica (Boschniakiae Herba), the free radical scavenging activity of its 50% ethanol extract (BR) was examined using an electron spin resonance spectrometer. The scavenging activity of plasma from Fisher-334 rats with continuous administration of BR was also examined. The concentrations showing 50% inhibition of the free radical of BR on the l,l-diphenyl-2-picrylhydrazil (DPPH) radical, Superoxide radical and hydroxyl radical were 0.003%, 0.06% and 9.67%, respectively. Plasma from the rats with BR administered clearly showed higher free radical scavenging activity compared with that of normal control rats. These findings suggest that Boschniakia rossica has strong free radical scavenging activity and consequently it has inhibitory effects on the disorders caused by free radical production in living tissue.

In the cobalt focus experimental epilepsy model, severe hippocampal neuron damage occurs with marked EEG changes. The effects of TJ-960, a herbal medicine formulation, were studied on neuron damage in the CA1 area of rat hippocampus. Continuous oral administration of TJ-960 from one month prior to the cobalt application showed almost complete protection against hippocampal neuron damage induced by cobalt application to the cerebral cortex. TJ-960 also completely inhibited the EEG changes as well as the brain edema induced by cobalt application.

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.

Cobalt focus is a seizure focus model in which cerebral neurons exhibit long-lasting severe spike discharges, followed by neuronal death. However, the neuronal death is prevented when peony root extract (PR) is administered prior to cobalt application. We tested the hypothesis that PR modulates the expression of neuroprotective proteins in the cerebrum of mouse cobalt focus by proteomic analysis using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry to screen for differentially expressed proteins. Analyses revealed that transthyretin, a carrier protein for thyroid hormones and retinoids, and the brain form of phosphoglycerate mutase, a glycolytic enzyme, were upregulated in the cobalt-treated mouse cerebrum and further increased by PR administration in association with upregulation of neurogranin/RC3, a target of the transcriptional activation by thyroid hormones and retinoids. These findings suggest that PR-induced protection of mouse cerebral neurons involves neurotrophic events caused by thyroid hormones and/or retinoids and enhanced glycolysis.

To elucidate the mechanism of inhibitory action of peony root extract on pentylenetetrazol-induced bursting activity, effects of peony root extract on the iberiotoxin-sensitive large conductance calcium-activated potassium (BKCa) current that plays an essential role in the production of bursting activity were investigated. Peony root extract showed a clear inhibitory effect on the iberiotoxin-sensitive calcium-activated potassium current. Peony root extract also showed clear inhibitory effects on spontaneous bursting activity and BKCa current in the cerebral cortical neurons of the EL mouse, a hereditary epilepsy animal model. These results together with our previous studies, including the protective effect against neuron damage, indicate that peony root extract is a promising herbal drug for inhibition of convulsions.

The molecular mechanism of the protective effects of peony root extract and its component substances on neuron damage induced by the cobalt focus epilepsy model and the EL mouse was investigated. Long-term administration of peony root extract for 30 days prior to metallic cobalt powder application to the cerebral cortex of mice resulted in increased expression of A20, an inhibitor gene of cell death. In the EL mouse, a hereditary epilepsy animal model with vulnerable neurons, increased expression of A20 was observed even without administration of peony root extract. Long-term administration of peony root extract to the EL mouse resulted in a marked increase of expression of A20. These results suggested that an increase in A20 expression is the main molecular mechanism of protective action of peony root extract on neuron damage.

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.

SEZ-6 is a brain-specific cDNA. Its expression is increased by convulsant drug and it encodes membrane protein with five copies of short consensus repeat (SCR; complement C3b/C4b binding site) and two repeated sequences which are partially similar to CUB domain (complement Clr/s-like repeat). In this study, we analyzed the gene product of SEZ-6 by antibody raised against SEZ-6 peptide. In vitro translation and immunoblot revealed that SEZ-6 protein is modified post-translationally and expressed in the cerebrum and the cerebellum. To analyze the SEZ-6 expression in detail, we searched for other types of SEZ-6 cDNA by the polymerase chain reaction. Two additional types of cDNAs were isolated and sequence analysis showed that one cDNA encodes membrane protein with a different C-terminal region and the other encodes secreted protein with two SCRs and one CUB-like domain.

cDNAs related to pentylenetetrazol-induced bursting activity in neurons were screened by a differential hybridization method using normal and pentylenetetrazol-treated primary cultured neurons from the cerebral cortex of mice. Twenty clones of candidate cDNA with expression increased or decreased by treatment with pentylenetetrazol were obtained. One of them, PTZ-17, was sequenced. Injection of PTZ-17 derived RNA into Xenopus oocytes showed a large calcium inward current with extracellular application of pentylenetetrazol.

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.

The dried herb or stem of Boschniakia rossica (Boschniakiae Herba) has been used as an antisenile agent in the Jilin Province of China. To elucidate the effects of Boschniakiae Herba on decreased learning ability and memory weakness, a 50% ethanol extract of Boschniakiae Herba was administered to rats whose nucleus basalis of Meynert had been destroyed by the injection of ibotenic acid. This administration showed a significant improvement in the decrease of the ratio of correct responses caused by destruction of the nucleus basalis of Meynert. These results suggest that B. rossica would be therapeutic in senility.

Epilepsy is one of the most frequently occurring nervous diseases. One out of 200 persons suffers from epilepsy. Nevertheless, there is still no definite radical therapy against epilepsy. Actual therapy against epilepsy is merely the expectation of a spontaneous cure by long-term inhibition of convulsions that facilitate the next convulsions using so-called anticonvulsant drugs, even when patients are tortured with side effects. To achieve a reasonable radical therapy, it is dispensable to elucidate the process of intracellular change of normal regular firing neurons into seizure-evoking neurons; and the differences between neurons manifesting bursting activity and those showing normal regular firing. We have hitherto investigated the intracellular events during bursting activity in a single neuron. I would like to talk about our experiments on such cellular mechanisms of epileptogenic phenomena, especially intracellular events during seizure activity. I will also describe anticonvulsant effects of a herbal mixture,. TJ-960, which shows a different mechanism of action from pure chemical anticonvulsants; and finally I will give some hints for future therapy against epilepsy.

Irritation of the trigeminal nerve provokes not only severe pain but can also lead to the Reilly syndrome (irritation syndrome). To elucidate the mechanism of the Reilly syndrome, we investigated the interaction between the trigeminal nerve and neurons in the vagus nucleus and sympathetic neurons in the lateral horn. We also examined the effect of an herbal mixture, TJ-960, on the Reilly syndrome. The neurons in the three nuclei of the vagus (ambiguous, dorsal, and solitary nuclei), neurons in the sacral parasympathetic center, and also neurons in the lateral horn showed clear evoked unit discharges produced by stimulation of the trigeminal nerve. These unit discharges showed summation effects for stimulation of the infra-alveolar, infraorbital, and lingual nerves. The spontaneous unit discharges in the above autonomic center. The irritation to the trigeminal nerve provoked extraordinary pathological discharge in the neurons in the autonomic centers showed excitatory and inhibitory modification after repetitive stimulation of the trigeminal nerve. There were also neurons unaffected by repetitive trigeminal stimulation. These three types of neurons were scattered randomly in each nucleus. An herbal mixture, TJ-960 completely suppressed the manifestations of the Reilly syndrome when administered 2 weeks before trigeminal irritation. These data indicate that there is a clear direct connection between the trigeminal nerve and neurons in the autonomic center. The irritation to the trigeminal nerve provoked extraordinary pathological discharge in the neurons in the autonomic center, and this in turn disturbed the microcirculation in various abdominal and thoracic organs and consequently provoked the Reilly syndrome. Administration of TJ-960 effectively suppressed the Reilly syndrome without any disturbances in normal nervous function.

The actions of the Chinese medicine “Hange-Koboku-To” (HK) and its component crude drugs on the laryngeal reflex, spontaneous locomotor activity and other neuropharmacological actions were studied. The extracts of HK, the barks of MAGNOLIA OBOVATA and aerial parts of PERILLA FRUTESCENS var. ACUTA clearly inhibited the laryngeal reflex in cats. The HK extract also inhibited the locomotor activity in rats, and prolonged the sleeping time induced by hexobarbital sodium in mice. These findings suggest that HK action of removing the foreign body sensation in the throat is mainly due to inhibition of the laryngeal reflex.

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 peliomphada , 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 45 Ca was observed after PTZ application. PTZ-non-sensitive neurons showed neither these protein changes nor 45 Ca incorporation into these proteins. The above findings suggest that during PTZ-induced BA, intracellular protein changes occurred in relation to the intracellular calcium shift.

The relationship between membrane potential and extracellular potassium concentration [K + ] 0 of primary cultured glial cells showed a complete Nernstian slope (10-fold change of [K + ] 0 corresponding to 58 mV). The C 6 cell line glia, however, showed a more gradual membrane potential/[K + ] 0 slope (10-fold [K + ] 0 change corresponding to 40 mV).

The effect of anticonvulsants on pentylenetetrazol (PTZ)-induced EEG power spectrum changes was examined. When the minimum dose of PTZ (15 mg/kg) was administered intravenously twice, with an interval of 80 min, a clear EEG power spectrum change was observed after the second PTZ administration, irrespective of whether or not the first PTZ administration evoked marked EEG changes. We defined the values F/N, S/N and S/F. The value F/N, obtained by dividing the power spectrum area after the first PTZ administration by the normal power spectrum area, was 1.06 +/- 0.05 (mean +/- S.E.). The value S/N, obtained by dividing the power spectrum area after the second PTZ administration by the normal power spectrum area, was 2.00 +/- 0.21. The value S/F, obtained by dividing S/N by F/N, was 1.86 +/- 0.16. The S/F value was almost constant regardless of whether or not the first PTZ administration could evoke marked EEG changes. The effect of anticonvulsants was examined by S/F value changes, and 100 mg/kg of PHT completely inhibited the double PTZ effect. Phenobarbital, ethosuximide and sodium valproate also inhibited the double PTZ effect. Using the S/F value of the EEG power spectrum with minimum dose double PTZ administration, quantitative evaluation is possible for anticonvulsant drugs.

To elucidate the mechanism of the Reilly syndrome (irritation syndrome), direct current (DC) potential changes in the medulla oblongata were investigated. Ipsilateral and contralateral trigeminal stimulation induced a DC potential shift above the vagal nuclear area in the medulla. Maxillary and mandibular nerve stimulation showed a summation effect. Continuous stimulation of the tooth pulp cavity by croton oil induced a progressively developed DC shift. DC potential change by trigeminal stimulation was accompanied by the same process of elevation of extracellular potassium concentration. These findings suggest that trigeminal stimulation increases the excitability of vagal nuclear neurons and consequently provokes the Reilly syndrome.

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.

Effects of phenytoin (PHT) on the intracellular calcium reservoir, lysosome-like granules (LLG), and calcium-related intracellular events during pentylenetetrazole (PTZ)-induced bursting activity in the neurons of the Japanese land snail, Euhadra peliomphala, were examined. PTZ-induced morphological change of LLG was inhibited by PHT. Calcium release from LLG was inhibited by PHT. PHT also inhibited the cyclic AMP increase by PTZ. PHT inhibited the increase in calcium-dependent protein kinase activity during PTZ-induced bursting activity. These findings suggest that PHT inhibits, as a first step, cyclic AMP increase which is one of the trigger factors of bursting activity as well as subsequent calcium-related intracellular pathological changes during seizure activity.

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 μM 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 μM 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.

The vago-vagal reflex plays an important role in the occurrence of “Irritation syndrome”. Electrophysiological properties of the vago-vagal reflex were studied in connection with the mechanism of “Irritation syndrome”. 1. The vago-vagal reflex augmented by strychnine provoqued a remarkable disturbance in EKG as in the state of irritation. 2. The region responsible for vago-vagal reflex involved not only the vagal nuclei but also other structures in the midbrain such as the reticular formation. 3. Moderate hypothermia evoqued a marked augmentation of the vago-vagal reflex response. Attempt was made to explain the troublesome complications of hypothermia-therapy such as hemorrhage of digestive canal. 4. The mechanism of “Irritation syndrome” was discussed in connection with these observations.

The autonomic irritation from the digestive organs and the urinary tract influences upon the cardiac function. Using the electrophysiological technique, this mechanism was revealed. 1. The authors presented two patients with arrhythmia, the one suffering from dysuria and the other suffering from cholelithiasis. These arrhythmias appeared with overdistension of the urinary bladder and operative procedure, and disappeared promptly by urination and cholecystectomy. 2. Experimental overdistension of the urinary bladder in dogs, rabbits and guinea pigs resulted in transitory fall of blood pressure, pictures of coronary insufficiency in EKG and/or appearance of arrhythmias. These changes disappeared by relieving the bladder of overdistension. 3. In guinea pigs it was shown that the EKG changes produced by overdistension of the urinary bladder were erased by transection of the spinal cord between the lumbo-sacral segment. 4. Electrophysiological study showed that the nervous transmission from the bladder wall to the heart was related to, vago-vagal reflex. 5. It was known that the irritation of the gallbladder and gastro-enterical tract sometimes produced the myocardial changes.

Pentylenetetrazole induces a bursting activity accompanied by intracellular movement of calcium toward the cell membrane in the snail neuron. The calcium appears to originate from the dense lysosome-like granules in the cytoplasm. Pentylenetetrazole markedly depletes the granules of calcium and alters them ultrastructurally from dense granules to lamella type granules.

Localization of ganglioside GM1 in cholinergic neurons from the septal area of a primary culture newborn rat brain was studied with a double avidin-biotin complex system. Cholinergic neurons were identified by double immunolabeling techniques that use choline acetyltransferase (ChAT) and neurofilament (NF) protein-antibodies. ChAT-positive neurons also were stained for ganglioside GM1 by using an avidin-biotin complex technique.

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 weigth. 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.

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.

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. RESUMEN El raton El es una cepa natural desarrollada a partir del raton ddY y muy susceptible a crisis. En el raton El, peuden inducirse convulsiones mediante 18 mg/kg de pentilentetrazol, que es una dosis inerte en el raton ddY. Las caracteristicas de las convulsiones fueron las mismas que las inducidas por el procedimiento de lanzamiento hacia arriba del animal que es un metodo habitual de producir convulsiones en el raton El. Fenitoina, fenobarbital, valproato sodico y etoxusimida claramente inhiben las convulsiones inducidas por pentilentetrazol en el raton El. Estos hallazgos sugieren que la induccion de convulsiones por pentilentetrazol en el raton El es un metodo sencillo comparado con la induccion de convulsiones mediante el procedimiento de lanzamiento hacia arriba, y que las convulsiones evocadas por este metodo son un modelo experimental preciso para el estudio de la epilepsia hereditaria y para la evaluacion de las drogas anticonvulsi vantes. ZUSAMMENFASSUNG Die El-Mause sind ein Inzuchtstamm der sich von den ddY-Mausen ableitet und sehr empfanglich fur Anfalle ist. Bei EL-Mausen konnten Konvulsionen durch 18 mg/kg Pentylentetrazol, eine ungefahrliche Dosis fur ddY-Mause, erzeugt werden. Das Aussehen der Krampfe entsprach dem, das durch Hochwerfen, einer ublichen Methode bei EL-Mausen Krampfe hervorzurufen, ausgelost werden konnen. Phenytoin, Phenobarbital, Natrium-Valproat und Ethosuximid inhibierten die Pentylentetrazol-induzierten Krampfe bei EL-Mausen. Diese Befunde deuten darauf hin, das die Induktion von Krampfen durch Pentylentetrazol bei der EL-Maus eine einfache Methode ist, verglichen mit der Induktion von Krampfen durch das Hochwerfen der Tiere und das Konvulsionen, die durch diese Methode hervorgerufen werden, ein gutes experimentelles Modell fur die Untersuchung der hereditaren Epilepsie und fur die Prufung der antikonvulsiven Medikamente darstellen.

The Chinese herbal medicine “Saiko-Keishi-To” (SK), which is a mixture of nine crude drugs, shows a dramatic anticonvulsant effect in some difficult cases of epilepsy which had long been unsuccessfully treated by established anticonvulsant drugs. The anticonvulsant mechanism of SK was investigated from the viewpoint of intracellular calcium and protein behavior during bursting activity, which is characteristic of seizure discharge. SK clearly inhibited the intracellular calcium shift toward the cell membrane as well as the calcium binding state change near the cell membrane during bursting activity induced by pentylenetetrazole (PTZ). SK also inhibited the intracellular protein changes during PTZ-induced bursting activity together with the incorporation of calcium into these proteins. These findings suggest that the mechanism of anticonvulsant action of SK is closely related to pathologically induced intracellular calcium and protein changes during seizure activity.

Effects of the Japanese Kampo medicine ‘Shosaiko-to-go-keishika-shyakuyaku-to’ (TJ-960), which is a mixture of 9 herbal drugs and is practically equivalent to ‘Saiko-keishi-to’ (SK), on the pentylenetetrazol (PTZ)-induced EEG power spectrum changes were examined. The EEG power spectrum change with 2 PTZ administrations at an 80 min interval was clearly inhibited by oral administration of 1.0 mg/kg of TJ-960. By separate single administrations of the main component herbal drugs, Bupleuri radix, Cinnamomi cortex, Paeoniae radix and Zingiberis rhizoma, only Paeoniae radix showed statistically significant inhibition of PTZ-induced EEG power spectrum changes at a proportional dose of 1.0 mg/kg of TJ-960. The other main component herbal drugs showed no statistically significant inhibitory effect although they had a tendency to inhibit. These findings suggest that the Japanese Kampo medicine, TJ-960 (SK), has an inhibitory effect on PTZ-induced power spectrum changes and one of the component herbal drugs, Paeoniae radix, is the important component drug.

The effect of the Chinese herbal medicine "Saiko-Keishi-To" (SK) on transmembrane ionic current was examined to elucidate the mechanism of the anticonvulsant action of SK. SK caused a decrease in tramsmembarane sodium, calcium and potassium current. SK was also found to inhibit the negative resistance characteristics in an experiment using a voltage clamp with triangular command.