Motoi Okamoto

Anticonvulsant action of a non-competitive antagonist of NMDA receptors (MK-801) in the kindling model of epilepsy

Anticonvulsant action of MK-801, a novel non-competitive antagonist of N-methyl-D-aspartate (NMDA) receptors, was investigated in the kindling model of epilepsy in rats. The results obtained were as follows. (1) Both the seizure stage and afterdischarge duration of previously kindled seizures from the amygdala were significantly suppressed following systemic injection of MK-801 (0.25-4 mg/kg) in a dose-dependent manner. The maximum effects were observed between 2 and 4 after the injection. (2) The MK-801 also showed significant anticonvulsant effects on kindled seizures from the frontal cortex and the ventral and dorsal hippocampus. The efficacy, however, significantly differed between these kindled sites. (3) Daily treatment of MK-801 (0.25 and 1 mg/kg) prior to each electrical stimulation of the amygdala significantly retarded kindling seizure development and increased the total amount of afterdischarge (accumulated AD) required to reach the first stage 5 seizure. During drug sessions of 1 mg/kg MK-801 for 19 days, all rats showed only partial seizures and the growth of afterdischarge was strongly prevented. (4) Pretreatment with reserpine did not antagonize the anticonvulsant effects of MK-801 on previously kindled seizures from the amygdala, suggesting that the effects may not be mediated by catecholaminergic systems. These results indicate that MK-801 has potent anticonvulsant actions on kindled seizures from both limbic and cortical foci, the NMDA system may play a critical role in the seizure-triggering mechanism of kindling. The possible application of NMDA antagonists in clinical epilepsy is suggested.

A protective action of chondroitin sulfate proteoglycans against neuronal cell death induced by glutamate

The role of chondroitin sulfate proteoglycans (CSPGs) on excitotoxic cell death and long-term survival of neurons were investigated in primary cultured neurons of the rat cortex. Soluble CSPGs were prepared from 10-day-old and adult rat brains by the ion-exchange chromatography on DEAE-Sephacel. CSPGs were added to the culture medium on culture day 4, and glutamate neurotoxicity was examined on culture day 7 by both microscopic cell count and measurement of lactate dehydrogenase activity in culture media. The effect on long-term survival was evaluated by counting viable neurons until culture day 28. CSPGs and core proteins, but not glycosaminoglycan chains (GAGs), protected cultured neurons from excitotoxic cell death induced by 24 h exposure to 1 mM glutamate, but CSPGs did not promote the long-term survival of neurons. The neuroprotective effect of CSPGs and core proteins was dose-dependent with ED50 about 10 microM hexuronate and 2 micrograms/ml protein respectively. This effect was not considered to be due to adsorption of glutamate by CSPGs because [3H]glutamate was not adsorbed by CSPGs added to the culture medium. Based on these findings, we suggested that CSPGs may exert their neuroprotective action through molecular interactions with the binding sites on neuronal membrane, neurotrophic factors, or other extracellular matrix molecules and may be involved in the pathogenesis of neuronal cell death in acute pathological conditions and chronic degenerative diseases of the brain.

Chondroitin sulfate proteoglycans protect cultured rat's cortical and hippocampal neurons from delayed cell death induced by excitatory amino acids

Protective effects of chondroitin sulfate proteoglycans (CSPGs) from rats brain against delayed cell death induced by excitatory amino acids were examined in cultured neurons of the rat. CSPGs reduced delayed neuronal death induced by 10 min exposure to glutamate at a concentration between 100 microM and 1 mM when lactate dehydrogenase activity of culture medium was assayed 24 h after the exposure. CSPGs also protected neuronal death induced by 200 microM N-methyl-D-aspartate (NMDA), kainate or 100 microM alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA). CSPGs reduced death of cortical and hippocampal neurons even when they were administered at 2 h, but not 6 and 12 h, after the exposure to glutamate. These results indicate that CSPGs may have a neuroprotective action against acute noxious conditions in the brain.

Kainic acid-induced convulsions cause prolonged changes in the chondroitin sulfate proteoglycans neurocan and phosphacan in the limbic structures

Systemic administration of kainic acid induces repeated convulsive seizures (KA convulsions) that result in neuropathological changes similar to temporal lobe epilepsy and the appearance of spontaneous recurrent seizures (SRS). The appearance of SRS is considered a result of the remodeling of neuronal networks following neuronal degeneration. We investigated the changes in chondroitin sulfate proteoglycans (CSPGs) in the limbic structures after KA convulsions in the rat using monoclonal antibodies 1G2, which recognizes full-length neurocan and the C-terminal half of neurocan, neurocan C, and 6B4, which recognize phosphacan and protein tyrosine phosphatase zeta. After KA convulsions, full-length neurocan appeared by 24 h and reached a peak by 48 to 72 h, whereas phosphacan decreased within 24 h in the hippocampus. In immunohistochemistry, neurocan increased in the limbic structures coincident with the appearance of reactive astrocytes. Phosphacan decreased coincident with pyramidal cell loss in the hippocampus, and the number of phosphacan-positive perineuronal nets around parvalbumin neurons decreased, whereas parvalbumin neurons were relatively conserved. In contrast, phosphacan increased in the entorhinal and piriform cortices in correlation with the severity of neuronal loss. Both neurocan and phosphacan recovered to the control level by 8 weeks after KA convulsions in some rats, but the changes in neurocan and phosphacan described above still persisted in more than half the rats. The results indicate that KA convulsions induce prolonged changes in neurocan and phosphacan similar to those in the developing rat brain and suggest a role of these CSPGs in the remodeling of neuronal networks related to the establishment or enhancement of epileptogenesis.

Chlorogenic Acid and Its Metabolite m-Coumaric Acid Evoke Neurite Outgrowth in Hippocampal Neuronal Cells

We evaluated the neurotrophic activity of dietary polyphenols by using primary cultures of fetal rat hippocampal neurons in a serum-free medium. Among the tested compounds, chlorogenic acid and its metabolite, m-coumaric acid, together with catechins and flavanone, were found to promote neuronal differentiation comparable to the phytochemical, honokiol, which has been reported to show potent neurotrophic activity. The present findings may contribute to the development of further neurotrophic studies on dietary polyphenols and their metabolites.

Increase in Prefrontal Cortex Blood Flow during the Computer Version Trail Making Test

Background/Aims: Previous studies using near-infrared spectroscopy (NIRS) have reported increases in both oxygenated hemoglobin (oxyHb) and deoxygenated hemoglobin (deoxyHb) during the paper version Trail Making Test (TMT), a neuropsychological test for evaluating cognitive and executive functions. We measured oxyHb and deoxyHb in the prefrontal cortex during the computer version TMT. Methods: Fifteen healthy students first performed TMT-A and then TMT-B; another 7 students first performed TMT-B and then TMT-A. The mean concentrations of Hb (10 s before TMT, during TMT and 30–40 s after TMT) were determined and analyzed by ANOVA. Results: oxyHb increased while deoxyHb decreased during the TMT. There was a significant order effect on the change in deoxyHb, but not in oxyHb. oxyHb significantly increased in the bilateral prefrontal cortices both in TMT-A and TMT-B. The increase tended to be more prominent in TMT-B than in TMT-A. deoxyHb significantly decreased in the bilateral prefrontal cortices, both in TMT-A and TMT-B. Conclusion: The results suggest that blood flow increases in the prefrontal cortex during the performance of TMT, and that the bilateral prefrontal cortices are involved in the performance of the computer version TMT.

Expression of brain specific chondroitin sulfate proteoglycans, neurocan and phosphacan, in the developing and adult hippocampus of Ihara's epileptic rats.

Iharas epileptic rats (IER) is an animal model of temporal lobe epilepsy with mycrodysgenesis, that exhibit abnormal migration of hippocampal neurons and recurrent spontaneous seizures. As an attempt to elucidate the roles of extracellular matrix molecules in the epileptogenecity and mossy fiber sprouting, immunohistochemical localization of brain specific chondroitin sulfate proteoglycans (CSPGs), neurocan and phosphacan, was examined in the hippocampus of postnatal IER and Sprague-Dawley (SD) rats using monoclonal antibodies 1G2 against neurocan and 6B4 against phosphacan. There was no difference in the expression of these two CSPGs between IER and SD rats in the 1st postnatal week. However, the expression of neurocan was poor in the hippocampus of IER in the 2nd and 3rd weeks whereas intense labeling of neurocan was present throughout the hippocampus of SD rats. Labeling of neurocan was almost absent in the hippocampus, while phosphacan was diffusely expressed in the stratum oriens and radiatum of Ammons horn, and in the hilus and inner one-third molecular layer of the dentate gyrus at the 2nd month after birth. There was no difference in the expression of neurocan and phosphacan between IER and SD rats at the 2nd month after birth. By contrast, phosphacan was reduced in the inner molecular layer of the dentate gyrus in 8-month-old IER, while neurocan was reexpressed in the outer molecular layer and hilus in 3- and 8-month-old IER. It was suggested that the insufficient expression of neurocan may affect the development of neuronal organization in the hippocampus, and that the remodeling of extracellular matrix in the dentate gyrus may contribute to the mossy fiber sprouting into the inner molecular layer.

An analysis of anticonvulsant actions of GABA agonists (progabide and baclofen) in the kindling model of epilepsy

The anticonvulsant action of progabide, an agonist of gamma-aminobutyric acid (GABA)A and GABAB receptors, was investigated in the kindling model of epilepsy in rats. Progabide shortened afterdischarge durations and attenuated the severity of the accompanying convulsive responses in previously kindled rats from the amygdala (AM), frontal cortex (FC), ventral and dorsal hippocampus (HIPP), in a dose-dependent manner. Although progabide was less effective in the dorsal HIPP kindled seizures, the efficacy was potent in AM, FC and ventral HIPP kindled seizures. On the other hand, the anticonvulsant action of baclofen, a selective agonist of GABAB receptors, was relatively weak in terms of the measurement of the afterdischarge duration of AM and HIPP kindled seizures even at toxic doses, compared with progabide. In addition, the anticonvulsant effects of progabide were partially reversed by treatment with the antagonist of benzodiazepine receptors, Ro 15-1788, whereas Ro 15-1788 administration alone did not alter AM kindled seizures. We concluded that the action of progabide may be mediated via the GABA/benzodiazepine receptor complex. These results support the hypothesis that a failure of GABAA-mediated inhibition is one of the bases of induction and generalization of seizures.

Purification and characterization of alkaline phosphatase from Bacillus stearothermophilus.

Soluble alkaline phosphatase from the thermophilic bacterium Bacillus stearothermophilus was purified by a combination of chromatographic methods, and its properties were examined. The purified enzyme had specific activity of 4.43 μmol p‐nitrophenol/min per mg of protein and seemed to be a single band on SDS/PAGE with a molecular mass of 32 kDa. Its apparent Km for p‐nitrophenyl phosphate was 1.114 mM. The enzyme exhibited an optimal pH of approx. 9.0 and exhibited its highest activity at 60–70 °C. It also showed a bivalent cation requirement for activity, with maximal enhancement in the presence of Mg2+. In addition, significant thermal stability was observed in comparison with counterparts from mesophiles. Its partial N‐terminal sequence was T1FSIVAFDPATGELGIAVQ19 as estimated by automated Edman degradation method. A search on the SwissProt database did not reveal any similar protein sequences from other sources.

Developmentally regulated expression of brain-specific chondroitin sulfate proteoglycans, neurocan and phosphacan, in the postnatal rat hippocampus.

Abstract. Developmental changes in the distribution of brain-specific chondroitin sulfate proteoglycans, neurocan and phosphacan/RPTPζ/β, in the hippocampus of the Sprague-Dawley rat were examined using monoclonal antibodies 1G2 and 6B4. The 1G2 immunoreactivity was predominant in the neonatal hippocampus while the 6B4 immunoreactivity was predominant in the mature hippocampus. Moderate 1G2 immunoreactivity was detected in the dentate gyrus and subiculum immediately after birth. Immunoreactivity reached a peak on postnatal days 7–10 (P7–P10) when intense 1G2 labeling was present throughout the neuropil layers of the hippocampus except the mossy fiber tract. 6B4 immunoreactivity was limited in the stratum lacunosum moleculare of CA1 in the neonatal hippocampus. It gradually increased by P21 when diffuse 6B4 immunoreactivity was detected in the stratum oriens and radiatum of Ammons horn, and in the hilus and inner one-third molecular layer of the dentate gyrus, while 1G2 immunoreactivity decreased after P21. In the adult hippocampus, moderate 6B4 immunoreactivity was present in the stratum oriens and radiatum of Ammons horn, and in the hilus and inner one-third molecular layer of the dentate gyrus, but not in the mossy fiber tract. In addition, strong 6B4 labeling appeared around a subset of neurons after P21. The results suggest that neurocan may have a role in the development of neuronal organization, while phosphacan/RPTPζ/β may contribute to the maintenance and plasticity of synaptic structure and function. Furthermore, the absence of 1G2 and 6B4 immunoreactivities in the stratum lucidum suggests that neurocan and phosphacan/RPTPζ/β may function as a barrier for the extension of mossy fibers and provide an environment permissive for fasciculation of the mossy fibers.