Identification of a novel variant of the human NR2B gene promoter region and its possible association with schizophrenia.

N-methyl-D-aspartate (NMDA) receptor dysfunction is involved in the pathogenesis of schizophrenia. We determined the nucleotide sequence of the 5'-upstream region of the human NMDA receptor 2B (NR2B) subunit gene and identified a novel T-200G variant located in one of the Sp1 binding sites. To investigate the effect of this variant on the transcriptional activity of the hNR2B gene, we performed gene reporter assays using PC12 pheochromocytoma cells transiently transfected with luciferase reporter plasmids. In the absence of nerve growth factor (NGF), luciferase activities did not significantly differ between the two alleles and the control plasmid. However, luciferase reporter activity of the T allele was significantly up-regulated compared to that of the G allele in the presence of NGF (P = 0.0013), indicating that this polymorphic site is a critical region for NR2B gene regulation through NGF-induced Sp1-binding. A case control study showed that the frequency of the G allele (P = 0.0164) was significantly higher in 100 schizophrenics than in 100 controls. These findings suggest that the T-200G variant causes dysfunction of NMDA receptors consisting of the NR2B subunit and may be involved in the development of schizophrenia. Replication studies of independent samples and family-based association studies are necessary to further evaluate the significance of our findings.

Recent research on alcohol tolerance and dependence.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Hiroshi Suwaki and Harold Kalant. The presentations were (1) Influence of ADH genotypes on acute alcohol withdrawal syndrome in Japanese, by Susumu Higuchi; (2) Use of genetic analyses to refine phenotypes related to alcohol tolerance and dependence, by John C. Crabbe; (3) Neurochemical basis for alcohol dependence, by Seitaro Ohkuma and Masashi Katsura; (4) Adenylyl cyclase and development of tolerance to addictive drugs, by Masami Yoshimura; (5) Tolerance in rat lines selectively bred for alcohol preference, by Robert C. Stewart and Ting-Kai Li; and (6) Ethanol reinforcement, dependence, and vulnerability to relapse: Interactions between neuroadaptive and conditioning factors, by Friedbert Weiss.

Immunohistochemical study on acetaldehyde adducts in alcohol-fed mice.

BACKGROUND: Acetaldehyde binds to some proteins, which results in Schiff base formation. It is assumed that acetaldehyde binds to the proteins after the consumption of ethanol, to form an adduct. Such acetaldehyde adducts are related to organ disease. METHODS: We examined 8-week-old male BALB/c mice, which were given a liquid diet for 7 days. The diet consisted of vitamins, minerals, amino acids, and a 5% (v/v) ethanol solution. After the 7 days, we took tissue samples from the brain, liver, and adrenal cortex to investigate the distribution of acetaldehyde adducts. We performed immunohistochemical staining of the cerebral cortex, liver, and adrenal cortex from the mice by using antibodies against acetaldehyde adducts. RESULTS: Our study showed that acetaldehyde adducts formed in the cerebral cortex in the early phase in alcohol-fed mice. CONCLUSIONS: Because acetaldehyde in the liver has been shown to cause liver damage, our study suggests a relationship between acetaldehyde adducts in the brain and brain damage.

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.

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.

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

It is assumed that the acetaldehyde binds to the proteins after the consumption of ethanol to form an adduct. Such acetaldehyde adducts are related to organ diseases. We examined 8-week-old female BALB/cAJcl mice which had been on a liquid diet for six months. The alcohol-fed mice's liquid diet comprised vitamins, minerals, amino acid and a ethanol solution. The five groups of mice examined were 1.5% ethanol-fed, 2% ethanol-fed, 2.5% ethanol-fed, 3% ethanol-fed, and 3.5% ethanol-fed mice respectively. All mice fed by 2.5% or more ethanol died within a month. Tissue samples were taken from the 1.5% and 2% ethanol-fed mice to investigate the distribution of acetaldehyde adducts in various organs. We demonstrated the immunohistochemical staining of the cerebral cortex and liver from the ethanol-fed mice with antibodies against acetaldehyde adducts. In the 2% ethanol-fed mice, the protein epitope related to acetaldehyde was found in the cerebral cortex and the liver. There was a significant difference in the amount of cell degeneration between the 1.5% and 2% ethanol-fed mice. The results suggest that the neurotoxicity of ethanol starts to cause serious cell degeneration in concentrations of more than 1.5%. The neurotoxicity of ethanol in the cerebral cortex was found to be more prominent than in the liver, resulting in more cell degeneration.

[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.

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.

Positive and negative syndromes, and Borna disease virus infection in schizophrenia.

The relationship between Borna disease virus (BDV) infection and positive and negative syndromes in schizophrenia was investigated. By nested RT-PCR and Western blotting, BDV-specific RNA and anti-BDV antibodies were examined in blood from 67 schizophrenic patients (DSM-III-R) in Japan, and the psychometric properties of the Positive and Negative Syndrome Scale (PANSS) were analyzed. There were significant (p < 0.05) differences in the composite index denoting the positive minus negative difference indicating a dominant contribution by negative items, and the proportion of negative type (positive minus negative value below zero) patients, between patients positive and negative for anti-BDV p24 antibodies. It is possible that BDV infection with induction of BDV p24 antibodies may be associated with negative syndromes in schizophrenic patients.

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.

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.

Ethanol metabolism, toxicity and genetic polymorphism.

The relationships between the individual (and racial) differences in alcohol metabolism and toxicity, and the genetic polymorphism of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH), and cytochrome P-4502E1(CYPIIE1) were reviewed. In recent studies involving DNA analysis, it was found that a deficiency of the ALDH2 isozyme (ALDH2*2) was responsible for the flushing symptoms as well as other vasomotor symptoms caused by a higher acetaldehyde level after alcohol consumption. Deficiency of ALDH2 activity has been found prevalently only among people of Mongoloid origin, and the deficiency of ALDH2 prevents them from developing alcohol dependence due to the unpleasant physical effects of the flushing symptom. It was reported that Mongoloids such as Japanese and Chinese people carry the enzymatically active (ALDH2*1) subunit and/or the inactive (ALDH2*2) one, and that a low proportion of ALDH2 deficiency (ALDH2*2 allele frequency) was found in alcoholics compared with healthy controls. It was also reported that polymorphism of ALDH2 and/or CYP2E1 may be associated with the susceptibility to alcohol-induced liver injury. Concerning blood ethanol elimination kinetics, it was reported that the c2 gene of CYP2E1 and the ALDH2*1 gene may have greater effects on ethanol and acetaldehyde elimination than the other genotypes, when the blood ethanol level is below 20 m M.

[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.

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.

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.