Risk factors for the onset of dependence and chronic psychosis due to cannabis use: Survey of patients with cannabis-related psychiatric disorders.

AIM: The objective of the current study was to identify risk factors that affect the onset of dependence and chronic psychosis due to cannabis use. METHODS: We examined clinical genetic factors, psychiatric disorders prior to cannabis use, starting age of cannabis use, duration and frequency of cannabis use, types of cannabis products used, combined use of other psychoactive substances, and the psychiatric diagnosis of 71 patients with cannabis-related psychiatric disorders who underwent treatment at nine mental health hospitals in Japan. Information was collected from cross-sectional interview surveys conducted by each patient's attending psychiatrist. RESULTS: For the diagnosis of dependence syndrome due to the use of cannabis, we found associations with the number of years of cannabis use and the use of cannabis products with a high Δ9-tetrahydrocannabinol (THC) content. However, we found no association between diagnosis of residual and late-onset psychotic disorders and clinical genetic factors, presence of preceding psychiatric disorders, duration and frequency of cannabis use, starting age of cannabis use, or combined use of other psychoactive substances; an association was found only for the absence of use of cannabis products other than dried cannabis. CONCLUSION: The onset of cannabis dependence was related to long-term cannabis use and the use of cannabis products with a high THC content. However, chronic psychosis was not associated with total THC intake or psychiatric vulnerability. Thus, unknown factors appear to be involved in the onset of chronic psychosis.

Association analysis of the dopamine receptor D2 (DRD2) SNP rs1076560 in alcoholic patients.

The dopamine system plays a well-established role in alcoholism. In this study, we examined the association between the single-nucleotide polymorphism (SNP) rs1076560 of the dopamine receptor D2 (DRD2) gene and susceptibility to alcoholism. SNP rs1076560 (C/A) is located in intron 6 of DRD2, where it is 1.4 kb downstream from alternative exon 6 and 83 bp upstream from exon 7. A total of 248 alcoholic patients and 322 healthy controls, all Japanese males, were genotyped for rs1076560 polymorphism by direct sequencing and allele-specific PCR. Data were analyzed using standard chi(2) statistics and a backwards logistic regression approach to adjust for the contribution of aldehyde dehydrogenase-2 (ALDH2) genotype status. The DRD2 risk allele A was more prevalent in the alcoholic patients (40.1%) than in the healthy controls (34.0%) (P=0.034, odds ratio=1.300, 95% confidence interval=1.020-1.657). These data identify SNP rs1076560 as a potentially important variable in the development of alcoholism.

Two-month stability of hyperbolic discount rates for delayed monetary gains in abstinent inpatient alcoholics.

OBJECTIVES: Alcohol dependence has been associated with disrupted neuroendocrine systems, impulsivity in intertemporal choice (delay discounting). However, little is known regarding stability of discount rates in alcoholics. This study examined both differential stability (stability of individual differences) and absolute stability (stability of group mean) of hyperbolic discount rates for monetary gains in severe alcoholic inpatients (diagnosed with DSM-IV) over a 2-month period during abstinence. METHODS: We estimated male alcoholics' discount rates for delayed monetary rewards on the basis of their pattern of choices between smaller immediate rewards (1,100-8,000 yen) and larger, delayed rewards (2,500-8,500 yen; at delays from 1 week to 6 months), two times at 2-month time-interval during hospitalized alcohol withdrawal. RESULTS: It was observed that the alcoholics' mean hyperbolic discount rates for gains had both differential and absolute stability over 2 months, although a slight non-significant decrease in the group mean of the discount rates was observed. CONCLUSIONS: The results indicate that abstinent alcoholic's discount rates are stable over a relatively long-term period. The usefulness of assessing discount rates of addicts in psychoneuroendocrinology and neuroeconomics of addiction is discussed.

Hippocampal cytochrome P450s synthesize brain neurosteroids which are paracrine neuromodulators of synaptic signal transduction.

Hippocampal pyramidal neurons and granule neurons of adult male rats are equipped with a complete machinery for the synthesis of pregnenolone, dehydroepiandrosterone, 17beta-estradiol and testosterone as well as their sulfate esters. These brain neurosteroids are synthesized by cytochrome P450s (P450scc, P45017alpha and P450arom) from endogenous cholesterol. Synthesis is acutely dependent on the Ca(2+) influx attendant upon neuron-neuron communication via N-methyl-D-aspartate (NMDA) receptors. Pregnenolone sulfate, estradiol and corticosterone rapidly modulate neuronal signal transduction and the induction of long-term potentiation via NMDA receptors and putative membrane steroid receptors. Brain neurosteroids are therefore promising neuromodulators that may either activate or inactivate neuron-neuron communication, thereby mediating learning and memory in the hippocampus.

Functional polymorphisms in the sigma1 receptor gene associated with alcoholism.

BACKGROUND: Sigma1 receptors are involved in the pathogenesis of drug abuse. Two polymorphisms (GC-241-240TT and Gln2Pro) in the sigma1 receptor gene (SIGMAR1) have been identified. To investigate the role of SIGMAR1 in conveying susceptibility to alcoholism, we performed a functional analysis of polymorphisms in the SIGMAR1 and a case-control study. METHODS: We initially screened for polymorphisms in the 5'-upstream region. The effects of the polymorphisms on transcriptional activity were determined using a gene reporter assay. The distribution of SIGMAR1 polymorphisms was analyzed in 307 alcoholic and 302 control subjects. RESULTS: A novel T-485A polymorphism was identified. The transcriptional activity of the A-485 allele and the TT-241-240 allele was significantly reduced compared with that of the T-485 allele and the GC-241-240 allele. The frequencies of the A-485 allele (chi2=5.575, df=1, p=.0205) and the TT-241-240/Pro2 haplotype (chi2=21.464, df=1, p<.0001) were significantly higher in control subjects compared with alcoholic subjects. The T-485A and the GC-241-240TT may be functional polymorphisms, and the A-485 allele and TT-241-240/Pro2 haplotype are possible protective factors for the development of alcoholism.

Association between a novel polymorphism in the promoter region of the neuropeptide Y gene and schizophrenia in humans.

Hypoactivity of neuropeptide Y (NPY) is thought to be involved in the pathophysiology of schizophrenia, because post-mortem brain studies revealed a decrease of the NPY in schizophrenia, and antipsychotic treatments increase the NPY in animal brains and in cerebrospinal fluid of patients. We performed genetic analysis of the NPY gene in schizophrenia. Mutation screening of the gene detected nine single nucleotide polymorphisms, of which we typed a -485C>T variation from potential functional relevance. The -485T allele was overly represented in the disease group (P=0.0043). An in vitro promoter assay revealed that a C to T change at nt -485 significantly reduced transcriptional activity. These results suggest that the -485T allele in NPY may confer susceptibility to schizophrenia by decreasing the neuropeptide in brains.

Enhanced carbonyl stress in a subpopulation of schizophrenia.

CONTEXT: Various factors are involved in the pathogenesis of schizophrenia. Accumulation of advanced glycation end products, including pentosidine, results from carbonyl stress, a state featuring an increase in reactive carbonyl compounds (RCOs) and their attendant protein modifications. Vitamin B(6) is known to detoxify RCOs, including advanced glycation end products. Glyoxalase I (GLO1) is one of the enzymes required for the cellular detoxification of RCOs. OBJECTIVES: To examine whether plasma levels of pentosidine and serum vitamin B(6) are altered in patients with schizophrenia and to evaluate the functionality of GLO1 variations linked to concomitant carbonyl stress. DESIGN: An observational biochemical and genetic analysis study. SETTING: Multiple centers in Japan. PARTICIPANTS: One hundred six individuals (45 schizophrenic patients and 61 control subjects) were recruited for biochemical measurements. Deep resequencing of GLO1 derived from peripheral blood or postmortem brain tissue was performed in 1761 patients with schizophrenia and 1921 control subjects. MAIN OUTCOME MEASURES: Pentosidine and vitamin B(6) concentrations were determined by high-performance liquid chromatographic assay. Protein expression and enzymatic activity were quantified in red blood cells and lymphoblastoid cells using Western blot and spectrophotometric techniques. RESULTS: We found that a subpopulation of individuals with schizophrenia exhibit high plasma pentosidine and low serum pyridoxal (vitamin B(6)) levels. We also detected genetic and functional alterations in GLO1. Marked reductions in enzymatic activity were associated with pentosidine accumulation and vitamin B(6) depletion, except in some healthy subjects. Most patients with schizophrenia who carried the genetic defects exhibited high pentosidine and low vitamin B(6) levels in contrast with control subjects with the genetic defects, suggesting the existence of compensatory mechanisms. CONCLUSIONS: Our findings suggest that GLO1 deficits and carbonyl stress are linked to the development of a certain subtype of schizophrenia. Elevated plasma pentosidine and concomitant low vitamin B(6) levels could be the most cogent and easily measurable biomarkers in schizophrenia and should be helpful for classifying heterogeneous types of schizophrenia on the basis of their biological causes.

Synaptotagmin XI as a candidate gene for susceptibility to schizophrenia.

Synaptotagmin XI (Syt11) is a member of the synaptotagmin family, which is localized in cells either in synaptic vesicles or the cellular membrane, and is known to act as a calcium sensor. The Syt11 gene is located on chromosome locus 1q21-q22, which was previously reported as a major susceptibility locus of familial schizophrenia. Here, we present evidence for an association between the number of 33-bp repeats in the promoter region of the Syt11 gene and schizophrenia. We found that the transcriptional activity of the gene is affected by the number of 33-bp repeats, which include an Sp1 binding site, suggesting that the excessive expression of Syt11 can be associated with schizophrenia. Another (single nucleotide) polymorphism in the Syt11 5'UTR region, where the potent transcription factor YY1 can bind, also affects the transcriptional activity of Syt11.

Rapid modulation of long-term depression and spinogenesis via synaptic estrogen receptors in hippocampal principal neurons.

Rapid modulation of hippocampal synaptic plasticity by estrogen has long been a hot topic, but analysis of molecular mechanisms via synaptic estrogen receptors has been seriously difficult. Here, two types of independent synaptic plasticity, long-term depression (LTD) and spinogenesis, were investigated, in response to 17beta-estradiol and agonists of estrogen receptors using hippocampal slices from adult male rats. Multi-electrode investigations demonstrated that estradiol rapidly enhanced LTD not only in CA1 but also in CA3 and dentate gyrus. Dendritic spine morphology analysis demonstrated that the density of thin type spines was selectively increased in CA1 pyramidal neurons within 2 h after application of 1 nm estradiol. This enhancement of spinogenesis was completely suppressed by mitogen-activated protein (MAP) kinase inhibitor. Only the estrogen receptor (ER) alpha agonist, (propyl-pyrazole-trinyl)tris-phenol (PPT), induced the same enhancing effect as estradiol on both LTD and spinogenesis in the CA1. The ERbeta agonist, (4-hydroxyphenyl)-propionitrile (DPN), suppressed LTD and did not affect spinogenesis. Because the mode of synaptic modulations by estradiol was mostly the same as that by the ERalpha agonist, a search was made for synaptic ERalpha using purified RC-19 antibody qualified using ERalpha knockout (KO) mice. Localization of ERalpha in spines of principal glutamatergic neurons was demonstrated using immunogold electron microscopy and immunohistochemistry. ERalpha was also located in nuclei, cytoplasm and presynapses.

Role of cytochrome p450 in synaptocrinology: endogenous estrogen synthesis in the brain hippocampus.

In the hippocampus, the center for learning and memory, cytochrome P450s (P450scc, P450(17alpha), and P450arom) as well as 17beta-, 3beta-hydroxysteroid dehydrogenases, and 5alpha-reductase participate in the synthesis of brain steroids from endogenous cholesterol. These brain steroids include pregnenolone, dehydroepiandrosterone, testosterone, dihydrotestosterone, and 17beta-estradiol. Both estrogens and androgens are synthesized in the adult male hippocampal neurons. Although the expression levels of steroidogenic enzymes are as low as 1/200 to 1/50,000 of those in testis or ovary, the levels of synthesized steroids are sufficient for the local usage within small neurons (i.e., intracrine system). This intracrine system contrasts with the endocrine system in which high expression levels of steroidogenic enzymes are necessary in endocrine organs in order to supply steroids to many other organs via blood circulation. Endogenous synthesis of sex steroids in the hypothalamus is also discussed. Rapid modulation by estrogens and xenoestrogens is discussed concerning synaptic plasticity such as the long-term potentiation, the long-term depression, or spinogenesis. Synaptic expression of P450(17alpha), P450arom, and estrogen receptors suggests "synaptocrine" mechanisms of brain steroids, which are synthesized at synapses and act as synaptic modulators.

Lack of association between sigma receptor gene variants and schizophrenia.

Several pharmacological studies suggest the possible involvement of sigma(1) receptors in the pathogenesis of schizophrenia. An association has been reported between schizophrenia and two variants (GC-241-240TT and Gln2Pro) in the sigma(1) receptor gene (SIGMAR1). We also previously reported that, along with T-485 A, these two variants alter SIGMAR1 function. To investigate the role of SIGMAR1 in conveying susceptibility to schizophrenia, we performed a case-control study. We initially screened for polymorphisms in the SIGMAR1 coding region using PCR-single strand conformation polymorphism analysis. The distribution of SIGMAR1 polymorphisms was analyzed in 100 schizophrenic and 104 control subjects. A novel G620A variant was detected in exon4. G620A was predicted to alter the amino acid represented by codon 211 from arginine to glutamine. Our case-control study showed no significant association between the T-485 A, GC-241-240TT, Gln2Pro, and G620A (Arg211Gln) variants and schizophrenia and clinical characteristics. These findings suggest that these SIGMAR1 variants may not affect susceptibility to schizophrenia.

Phosphoinositide-3-kinase-independent contractile activities associated with Fcgamma-receptor-mediated phagocytosis and macropinocytosis in macrophages.

Previous studies have shown that Fcgamma receptor (FcR)-mediated phagocytosis and macropinocytosis in macrophages consist of two dissociable activities: a phosphoinositide 3-kinase (PI3K)-independent extension of phagocytic cups and a PI3K-dependent contractile mechanism that closes phagosomes and ruffles into intracellular organelles. Here, we identify an additional contractile activity that persists in the presence of the PI3K inhibitor wortmannin. ML-7, an inhibitor of myosin-light-chain kinase (MLCK), inhibited FcR-mediated phagocytosis, macropinocytosis and cell movements associated with ruffling. Scanning electron microscopy demonstrated a striking difference in morphology between phagocytic cups in the different inhibitors: whereas phagocytic cups of control cells and wortmannin-treated cells conformed closely to particles and appeared to have constricted them, the phagocytic cups in cells treated with ML-7 were more open. Video microscopy of macrophages expressing green-fluorescent-protein (GFP)-actin fusions revealed that bound IgG-opsonized erythrocytes were squeezed during phagosome formation and closure. In ML-7, GFP-actin-rich protrusions extended outward but failed to squeeze particles. Moreover, in contrast to the effects of PI3K inhibitors, ML-7 markedly reduced ruffle movement, and perturbed circular ruffle formation. These PI3K-independent myosin-II-based contractile activities that squeeze phagocytic cups and curve ruffles therefore represent a third component activity of the actin cytoskeleton during phagocytosis and macropinocytosis.

Acetaldehyde adducts in the brain of alcoholics.

Acetaldehyde binds to some proteins and becomes a Schiff base. It is assumed that after the consumption of ethanol the acetaldehyde binds to the proteins to form adducts, and such acetaldehyde adducts are associated with organ diseases. We investigated the detection of acetaldehyde adducts in the brain region of a human alcoholics. Brain samples collected from an alcoholic autopsied case were used. Determination of acetaldehyde adducts was performed using a fluorescence immmunohistochemical staining method with antibodies against acetaldehyde adducts. We demonstrated acetaldehyde adducts in the frontal cortex and the midbrain of an alcoholics. Our studies showed that an acetaldehyde adduct was produced in the brain of alcoholics.

Adult male rat hippocampus synthesizes estradiol from pregnenolone by cytochromes P45017alpha and P450 aromatase localized in neurons.

In adult mammalian brain, occurrence of the synthesis of estradiol from endogenous cholesterol has been doubted because of the inability to detect dehydroepiandrosterone synthase, P45017alpha. In adult male rat hippocampal formation, significant localization was demonstrated for both cytochromes P45017alpha and P450 aromatase, in pyramidal neurons in the CA1-CA3 regions, as well as in the granule cells in the dentate gyrus, by means of immunohistochemical staining of slices. Only a weak immunoreaction of these P450s was observed in astrocytes and oligodendrocytes. ImmunoGold electron microscopy revealed that P45017alpha and P450 aromatase were localized in pre- and postsynaptic compartments as well as in the endoplasmic reticulum in principal neurons. The expression of these cytochromes was further verified by using Western blot analysis and RT-PCR. Stimulation of hippocampal neurons with N-methyl-d-aspartate induced a significant net production of estradiol. Analysis of radioactive metabolites demonstrated the conversion from [(3)H]pregnenolone to [(3)H]estradiol through dehydroepiandrosterone and testosterone. This activity was abolished by the application of specific inhibitors of cytochrome P450s. Interestingly, estradiol was not significantly converted to other steroid metabolites. Taken together with our previous finding of a P450scc-containing neuronal system for pregnenolone synthesis, these results imply that 17beta-estradiol is synthesized by P45017alpha and P450 aromatase localized in hippocampal neurons from endogenous cholesterol. This synthesis may be regulated by a glutamate-mediated synaptic communication that evokes Ca(2+) signals.

T cell-specific disruption of arylhydrocarbon receptor nuclear translocator (Arnt) gene causes resistance to 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced thymic involution.

The arylhydrocarbon receptor nuclear translocator (ARNT) is a member of the basic helix-loop-helix, PER-ARNT-SIM family of heterodimeric transcription factors, and serves as a dimerization partner for arylhydrocarbon receptor (AHR) and hypoxia-inducible factor-1alpha. To assess the function of ARNT in T cells, we disrupted the Arnt gene specifically in T cells of mice by conditional gene targeting using T cell-specific p56(lck)-Cre (Lck-Cre) transgenic Arnt-floxed mice. Thus generated, T cell-specific Arnt-disrupted mice (Lck-Cre;Arnt(flox/Delta) transgenic mice) exhibited complete loss of the expression of ARNT protein only in T cells, and were viable and appeared normal. The Arnt-disrupted T cells in the thymus were phenotypically and histologically normal. The Arnt-deficient T cells in the spleen were capable of responding to TCR stimulation in vitro. However, unlike normal mice in which exposure to the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an AHR ligand, resulted in thymic involution, the thymus of Lck-Cre;Arnt(flox/Delta) mice were resistant to TCDD treatment in vivo. In contrast, benzo(a)pyrene, another AHR ligand, still caused thymic involution in Lck-Cre;Arnt(flox/Delta) mice. Finally, fetal thymus organ culture using Lck-Cre;Arnt(flox/Delta) and K5-Cre;Arnt(flox/Delta) (epithelial cell-specific Arnt-disrupted mice) showed that thymocytes rather than thymic epithelial cells are predominantly responsible for TCDD-induced thymic atrophy. Our results indicate that ARNT in T lineage cells is essential for TCDD-mediated thymic involution.