Tissue-specific alternative splicing of mouse brain type ryanodine receptor/calcium release channel mRNA.

We detected alternative splicing of the mouse brain type ryanodine receptor (RyR3) mRNA. The splicing variant was located in the transmembrane segment. The non-splicing type (RyR3-II) included a stretch of 341 bp, and that of the 13th codon was stop codon TAA. Reverse transcription-polymerase chain reaction (RT-PCR) analysis shows that RyR3-II mRNA was expressed in various peripheral tissues and brain at all developmental stages. However, interestingly, the splicing type (RyR3-I) mRNA was detected only in the cerebrum. These findings suggest that the splicing variants RyR3-I and RyR3-II may generate functional differences of RyR3 in a tissue-specific manner.

Time-dependent and regional expression of GABA transporter mRNAs following amygdala-kindled seizures in rats.

To investigate the role played by GABA transporters in epileptic seizures, we examined time-dependent and regional changes in expression of GAT-1 and GAT-3 GABA transporter mRNA in amygdala-kindled rat brain using an in situ hybridization method. GAT-1 mRNA was significantly increased bilaterally in the hippocampal dentate gyrus (111-116%) at 1 h after kindled generalized seizures. GAT-1 mRNA was also significantly increased bilaterally in the hippocampal subfields (CA1-4 and dentate gyrus [110-117%]) at 4 h after kindled seizures. There were no significant changes in GAT-1 mRNA level in the amygdalar nuclei, pyriform cortex or cerebral cortex either ipsilaterally or contralaterally at any time after kindled seizures. In contrast, GAT-3 mRNA was significantly increased bilaterally in the amygdalar nuclei and in the contralateral pyriform cortex and cerebral cortex 1 h after seizures. Since all these changes returned to control levels by 8 or 24 h after kindled seizures, the increases in GABA transporter mRNA appeared to be transient responses to seizure activity. These findings indicate that GAT-1 subtype transporter is specifically involved in seizure activity in the hippocampus, while GAT-3 subtype transporter is mainly involved in seizure activity in the amygdalar nuclei and pyriform cortex following amygdala-kindled generalized seizures.

A localization study of the cytochrome P-450(21)-linked monooxygenase system in adult rat brain.

Immunohistochemical studies were performed to investigate the localization of the cytochrome P-450(21)-linked monooxygenase system in rat brain using a specific antibody against bovine adrenal cytochrome P-450(21) (P-450XXIA1), which was purified electrophoretically as a single protein band and with a heme content of 18.0 nmol/mg protein from adrenocortical microsomes. The cytochrome P-450(21) was found to be mainly localized in the tractus reticulothalamicus and other ascending fibers in adult rat brains. This finding indicated that deoxycorticosterone or its derivatives could be implicated in the regulation of consciousness and the induction of an anesthetic effect.

Developmental changes of cyclin dependent kinase 5 subcellular localization in the rat cerebellum.

Cyclin dependent kinase 5 (Cdk5) phosphorylates tau protein, a microtubule-associated protein, at pathological sites in vitro as well as in Alzheimer's disease brain. The enzyme is therefore regarded as an important candidate responsible for the progression of Alzheimer's disease. We and others have suggested that the enzyme has physiological roles in brain development and maturation. In this study, we investigated the exact distribution and developmental changes of the enzyme in cerebellum by immunohistochemistry and non-radioactive in situ hybridization. Immunohistochemistry demonstrated that Cdk5 was consistently expressed in the cerebellum at all developing stages. However, the subcellular localization of Cdk5 dramatically changed during maturation of the cerebellum. In the early neonatal stage, Cdk5 was strongly expressed in the cell bodies of neurones. With neuronal maturation Cdk5 immunoreactivity changed its subcellular localization from the cell body to axon. In terminally differentiated neurons, the immunoreactivity was only detected in the axon. These results suggest that subcellular localization of Cdk5 is strictly regulated and may play an important role in neuronal maturation.

Modification of behavioral responses induced by electrical stimulation of the ventral tegmental area in rats.

To investigate the role of the ventral tegmental area (VTA), a source of the mesolimbic dopaminergic pathway, in paranoid psychosis, a detailed analysis of the behavioral responses induced by electrical stimulation of the VTA was made. Abnormal behavior induced by bilateral high-frequency stimulation of the VTA consisted of two components: forward locomotion and exploration. Similar responses were obtained when the nucleus accumbens (NAC) or prefrontal cortex (PFC) were stimulated. The expression of behavioral responses to stimulation was significantly attenuated by dopamine (DA) receptor or antagonists, such as haloperidol, YM-09151-2 and SCH23390. These results indicate that VTA stimulation causes a transient hyperdopaminergic state in the brain, that resembles psychostimulant-induced abnormal behavior. The effects of chronic administration of methamphetamine (MAP) on the behavioral responses to electrical stimulation of the VAT were also investigated. Although an acute administration of MAP did not affect the behavioral responses to electrical stimulation of the VTA, chronic treatment with MAP (for 2 weeks) caused a long-lasting reduction in the electrical threshold for the induction of abnormal behavior, compared with chronic saline-treated rats. It is suggested that a lasting enhancement in the behavioral response to stimulation of VTA neurons may contribute to the etiology of paranoid schizophrenia and amphetamine psychosis.

Amygdala-kindled and pentylenetetrazole-induced seizures in glutamate transporter GLAST-deficient mice.

The glutamatergic system has been shown to be important for the induction of epileptiform activity and the development of epileptogenesis. To investigate the role of the astroglial glutamate transporter GLAST in epileptogenesis, we examined amygdala (AM)-kindled and pentylenetetrazole (PTZ)-induced seizures in GLAST-deficient mice (GLAST(-/-)) and compared them to those observed in wild-type mice (GLAST(+/+)) and maternal C57Black6/J (C57) mice. AM-kindling resulted in no significant differences in afterdischarge threshold or in the seizure responses induced by first stimulation between these groups. In addition, although no significant differences were seen in kindled seizure development, the generalized seizure duration of AM-kindled seizures in GLAST(-/-) mice was significantly prolonged (approximately 35%) compared with that of C57 mice. Furthermore, GLAST(-/-) mice showed more severe stages of PTZ-induced seizures than GLAST(+/+) mice, and the latency to the onset of seizures was significantly shorter for the mutant mice. These results indicate that GLAST is one of factors determining seizure susceptibility.

Effect of embryonic hippocampal transplantation in amygdaloid kindled rat.

Embryonic neural tissue was transplanted into previously kindled rats. A thirteen- to fourteen-day embryonic hippocampal cell suspension was grafted in the stratum oriens near the CA2 area of the hippocampus. Almost 80% of the animals had a good recovery and became seizure-free. Injection of neocortical cells or saline did not show any positive effect on the kindling susceptibility. Although 20 day embryonic cell transplantation was also effective, the effect did not last as long as the 13- to 14-day embryonic transplantation. These observations open the possibility that the neural grafts may be used for therapy of medically intractable epilepsies.

Effect of ceruletide on epileptogenesis in amygdaloid kindled rats.

The inhibitory effects of ceruletide (CLT), a cholecystokinin-8 (CCK)-like peptide, were investigated in the epileptogenesis in the amygdaloid kindled rats. Lower doses of CLT (20-80 micrograms/kg) inhibited the progression of kindling process. After acquiring C5 stage, a higher dose (160 micrograms/kg) was required to suppress the seizure susceptibility. These results, in light of several previous studies showing no serious side effects, suggest that CLT might be useful as an anti-epileptogenic agent for clinical usage.

Correspondence of increased debrisoquine 4-monooxygenase activity with seizure-susceptibility in Mongolian gerbils.

Four drug-metabolizing activities in Wistar rat and Mongolian gerbil liver microsomes were investigated to clarify the biochemical basis of convulsions. No significant associations were observed between the susceptibility to the tonic-clonic seizures in Mongolian gerbils and the drug-metabolizing enzymatic activities of p-nitroanisole O-demethylase, aminopyrine demethylase and benzo[a]pyrene-3-monooxygenase. However, a significant association was observed with the debrisoquine 4-monooxygenase (cytochrome P-450db1-linked monooxygenase system) activity, which is known to metabolize exogenous and endogenous parkinsonism-inducing neurotoxins such as MPTP and TIQ, and to activate carcinogens during the detoxication of xenobiotics. The mean activity of debrisoquine 4-monooxygenase in the seizure-sensitive group (male, 380; female, 350 pmol/h/mg protein) remained significantly higher (about 4-5 times) than that in the seizure-resistant group (male, 90; female, 80 pmol/h/mg protein). More Mongolian gerbils were extensive metabolizers of debrisoquine in the seizure-sensitive group than in the seizure-resistant group. These results suggest a possibility that the cytochrome P-450db1-linked monooxygenase system may activate the potential neurotoxins which are associated with tonic-clonic seizures in the Mongolian gerbil.

No association between the neuroleptic malignant syndrome and mutations in the RYR1 gene associated malignant hyperthermia.

The neuroleptic malignant syndrome (NMS) is a drug-induced disease caused by neuroleptics, but the pathogenesis of NMS is unknown. Since NMS is similar to malignant hyperthermia (MH) in clinical features and treatment, 6 mutations in the skeletal muscle ryanodine receptor (RYR1) gene, which were associated with MH, were investigated in unrelated NMS patients by single-strand conformation polymorphism analysis (SSCP). As a result, MH-susceptible RYR1 mutations were not detected in our NMS patients. A single base substitution, C7278T, was detected in one patient whose serum CPK level was repetitively elevated, but his other major symptoms did not fulfil the clinical criteria for NMS. Our results do not support the association between the neuroleptic malignant syndrome and mutations in the RYR1 gene associated with malignant hyperthermia.

Morphological changes in the hippocampus in amygdaloid kindled mouse.

To clarify the origin and maintenance of epileptogenesis, morphological changes in the hippocampus of amygdaloid-kindled mice were analyzed at different stages of kindling. The granule cell size in dentate gyrus and the pyramidal cell size in CA1 were clearly decreased depending on seizure stage. The cell size in CA2 was increased and density in dentate gyrus and CA2 was reduced, significantly. The morphological changes in hippocampus associated with kindling must be closely related to the acquisition and the maintenance of epileptogenesis. The results support the hypothesis that seizure-induced damage of neurons may lead to formation of new synaptic connections that produce abnormal hyperexitability and result in seizures.

CYP2E1 genotypes and serum LAP in Japanese alcoholics.

The genotypes of the ALDH2 and CYP2E1 loci of Japanese alcoholic patients were determined to investigate the susceptibility to alcoholic liver injury. In alcoholics with a liver-function disorder, a significant association was observed between the genotypes of the CYP2E1 loci and the serum level of a liver-derived enzyme, LAP. However, there was no significant association between the ALDH2 genotypes and liver dysfunction.

The relationship between phenytoin pharmacokinetics and the CYP2C19 genotype in Japanese epileptic patients.

The relationship between the phenytoin pharmacokinetics, expressed by the mean of the Michaelis-Menten equation and the CYP2C19 genotype was investigated in 16 Japanese epileptic patients treated with phenytoin. Between genetically (S)-mephenytoin poor and extensive metabolizers, there were no differences in the Michaelis-Menten parameters. But divided into genotype groups, Vmax values were 3.9 +/- 0.4, 5.3 +/- 0.7, and 5.7 +/- 1.4 mg/kg/day for the patients with the m2 allele, with the m1 allele, and with neither the m1 or m2 allele, respectively. In the patients with the m2 allele of CYP2C19, the Vmax value was significantly lower than in those without the m2 allele. It is possible that the m2 allele of CYP2C19 may be one of the factors of slow phenytoin metabolism, and its frequency may underlie the ethnic difference in phenytoin metabolism between Japanese and white individuals.

Phenytoin kinetics in Japanese adult epileptics: phenotype of phenytoin slow metabolizers.

Phenytoin pharmacokinetics in 18 of 126 Japanese epileptic patients were investigated using the Michaelis-Menten equation. Five of these (4% of total) patients, who showed significantly high plasma concentrations of phenytoin even when administered a relatively low daily dose of phenytoin, were classified as slow metabolizers; 13 of these, who showed lower plasma concentrations, were classified as normal metabolizers. Comparison of slow and normal metabolizers revealed that the maximum rate of metabolism, Vmax, differed significantly between the two groups, the borderline Vmax value between the two groups being approximately 4.5-4.8 mg/kg/day. The mean Vmax value of slow metabolizers was calculated to be 70% that of normal metabolizers. It is possible that one means of phenotyping slow and normal phenytoin metabolizers is by analysis of phenytoin pharmacokinetics, with estimation of Vmax values.

The drug-drug interaction effects of haloperidol on plasma carbamazepine levels.

The metabolic interaction between carbamazepine (CBZ) and haloperidol (HP) was studied in Japanese schizophrenic patients treated with HP but not with CBZ and with both CBZ and HP. The serum CBZ concentrations in patients treated without HP were significantly decreased (p < 0.05), on average approximately 40%, as compared to those in patients treated with both CBZ and HP, whereas the serum HP concentrations in patients treated with both HP and CBZ were significantly decreased (p < 0.05), as compared to those in patients treated with HP but not with CBZ. The effect of HP, which prevents the serum CBZ level from decreasing, was shown in this study.

Characteristics of Japanese alcoholics with inactive aldehyde dehydrogenase: clinical features of alcoholics with ALDH2*2.

Abstract In a person with inactive ALDH2 (ALDH2*2) the blood aldehyde concentration tends to rise faster and higher and there are flushing responses which are considered to be a restraint against excessive alcohol drinking. The subjects in this study comprised 71 Japanese alcoholics. Psychiatrists interviewed the patients concerning the clinical features. Alcoholics homozygous (n = 59) for ALDH2*1/ALDH2*1 (Group I) and those heterozygous (n = 12) for ALDH2*1/ALDH2*2 (Group II) were compared. Group II alcoholics included significantly more cases of guilt or personality disorder. These findings indicate that alcoholics with the ALDH2*2 genotype showed generally typical clinical features.

Alcohol-induced facial flushing and drinking behavior in Japanese men.

The drinking behavior and alcohol-induced facial flushing of 1646 Japanese men (50.9% flushers and 48.0% nonflushers) were analyzed from questionnaires completed by their wives. The results indicate a relationship between flushing and various indices of sensitivity to alcohol and low rates of alcohol-related problems. It is proposed that alcohol-induced flushing acts as an inhibitory factor against excessive alcohol use and consequent problems due to drinking.

No linkage of the cytochrome P-450IIE1 (CYP2E1) C1/C2 polymorphism to schizophrenia.

We investigated, using PCR-SSCP analysis, the relationship between schizophrenia and the polymorphism of d-benzphetamine N-demethylase (cytochrome P-450j or CYP2E1), which metabolizes psychotropic substances such as d-benzphetamine and alcohols. Among 41 patients with schizophrenia, no statistically significant change in the frequency of the mutant (C2) allele relative to in controls was found, and no novel structural mutation in the CYP2E1 gene, which would be expected to alter the CYP2E1 protein, was found. This could be explained by no linkage of the CYP2E1 gene (mutations in the exon 1-9, and C1/C2 polymorphism) to schizophrenia.

[A clinical study of substance dependence patients combined with other psychiatric disorders].

The present study was conducted for clarifying the comorbidity of substance dependence and other psychiatric disorders in outpatients of four psychiatric hospitals in May, 1995. The results were as follows; 7.4% (N = 234) of the total 3155 psychiatric outpatients were diagnosed as substance dependence. Among those substance dependence patients, alcohol dependence accounted for 82.5% and the percentage of the other substance dependence were very small, i.e., methamphetamine dependence 6.4%, solvent dependence 1.7%, multiple substance dependence 9.4%, respectively. The percentage of comorbidity of substance dependence and psychiatric disorders was 23.9% (N = 56) of 234 substance dependence patients. The percentage of co-morbid alcohol dependence patients with affective disorder in all affective disorder patients was 5.0%; the percentage of comorbidity of alcohol dependence in neurotic patients 4.1%; the percentage of alcohol dependence comorbidity in schizophrenic patients 0.7%. In many cases, onsets of substance dependence and psychiatric disorders were within 2 years, which suggests the common backgrounds for substance dependence and psychiatric disorders, such as disruption of family and occupational life, stress and individual vulnerability, and substance use for self-medication. The study indicates that the percentages of diagnosed comorbidity of substance dependence and psychiatric disorders are generally smaller in Japan than in the U.S., which may be based on the differences of diagnostic standards between the two countries. Further studies are needed on the comorbidity of substance dependence and psychiatric disorders in other general hospital and psychiatric clinic patients.

[Acetaldehyde adducts in the cerebral cortex of ethanol-fed mice].

To investigate the neurotoxicity of acetaldehyde covalent adducts, immunohistochemical staining for acetaldehyde adducts using the antibody against acetaldehyde adducts, was performed in the cerebral cortex of ethanol-fed (withdrawal) mice. In the ethanol-fed mice, the degeneration in the cerebral cortex was found, while the protein epitope related to acetaldehyde was found in the cerebral cortex, liver and adrenal cortex. No histochemical and immunohistochemical changes in the tissues from the control mice were found. It is possible that acetaldehyde adducts may effect on the cerebral cortex as the neurotoxicity which cause psychosis such as delirium and hallucination after alcohol drinking.