Distinct haplogenotypes of the dopamine D2 receptor gene are associated with non-smoking behaviour and daily cigarette consumption.

INTRODUCTION: Dopamine systems in the CNS are decisively implicated in the motivational and rewarding properties of nicotine. The dopamine D2 receptor (DRD2) plays a pivotal role by promoting these properties, making this gene a good candidate for association studies. Several single nucleotide polymorphisms (SNPs) have been described to influence the expression of DRD2. The amount of expressed DRD2 will finally be the result of the sum and/or interaction of several functional polymorphisms located at the respective DNA strand forming a distinct haplotype. Thus, the knowledge about the distribution of the haplotypes in groups of subjects, differing by their smoking behaviour, would result in a better understanding of the putative associations compared to single SNP investigations. METHODS: 218 healthy subjects grouped for being never smokers, former smokers, and current smokers, were genotyped for the following polymorphisms: -141 ins(I)/del(D), STRPi2 (intron 2), C957T (exon 7), A1385G (exon 8), and TaqIA. Regular immoderate alcohol consumption was an exclusion criterion. RESULTS: In the total study group four haplotypes represented 90% of the haplotypes, with I-T-A-A2, I-C-G-A2, I-C-A-A1, and D-C-G-A2 accounting for around 50%, 20%, 10%, and 10%, respectively. I-C-G-A2 homozygosity was significantly higher in never smokers compared to ever smokers (current+former smokers) (chi2=36.585, df=1, p<0.001). There was a significant difference in the daily cigarette consumption of current smokers with respect to the haplogenotype (chi2=3211.9, df=18, p=0.003). Current smokers with a haplogenotype containing at least one I-T-A-A2 allele showed a significant smaller daily cigarette consumption (15.1+/-7.93) compared to subjects with a genotype not bearing this allele (20.1+/-6.79; T=-2.06, df=61, p=0.044). CONCLUSION: We have demonstrated an association of the distinct haplogenotype I/I-C/C-G/G-A2/A2 of the DRD2 gene with a reduced risk to become a smoker in Caucasians of German origin. This protection may result from an association of this haplotype with a reduced activation of the dopaminergic neurotransmission by nicotine. Moreover, in current smokers, higher daily cigarette consumption is associated with those haplogenotypes that do not contain the I-T-A-A2 haplotype.

The influence of acute and chronic administration of 1,2-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline on the function of the nigrostriatal dopaminergic system in rats.

The contribution of (R)-enantiomer of N-methyl-salsolinol (1,2-dimethyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline; NMSal) to the degeneration of dopaminergic neurons in the course of Parkinson's disease (PD) has been predominantly suggested by in vitro experiments in cell culture and by an in vivo study in which this compound has been directly injected into the rat striatum. The aim of the present study was to examine the effects of racemic NMSal (50 and 100 mg/kg) administered systemically, acutely and chronically for 21 days to rats, on the neurochemical and behavioral markers of PD. Our results showed that racemic NMSal easily penetrated the blood-brain barrier. Its brain level was relatively high 2-6 h after a single injection than gradually decreased. NMSal was quickly eliminated from the rat brain, its concentration 24 h after withdrawal from chronic treatment was very low. NMSal at both examined doses did not affect striatal and nigral levels of dopamine (DA) 2 h after the first and last chronic injections, however, it markedly changed DA catabolism. In the striatum both its doses evoked a significant acceleration of the total and oxidative, monoamine oxidase (MAO)-dependent DA catabolism without affecting the catechol-O-methyltransferase (COMT)-dependent O-methylation. In the substantia nigra (SN), only the higher dose of NMSal produced such effect. DA catabolism in either structure was the same as in control 24 h after cessation of chronic treatment. The second characteristic marker of PD, the number of tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the SN, was not affected by chronic NMSal treatment as revealed by the stereological counting. In the behavioral study, it was found that racemic NMSal significantly suppressed spontaneous locomotor activity and effectively prevented that stimulated by apomorphine. Our results suggest that NMSal may play an important role in the regulation of dopaminergic activity rather than in inducing changes of parkinsonian type.

Functional cortical effects of novel allelic variants of the serotonin transporter gene-linked polymorphic region (5-HTTLPR) in humans.

INTRODUCTION: Genetic variations of the serotonin transporter-linked polymorphic region (5-HTTLPR) have been implicated in the pathogenesis of several psychiatric disorders. Recent evidence indicates that the biallelic polymorphic region (S and L allele) contains additional variations affecting the mRNA expression. METHODS: According to recent preclinical and clinical studies, the loudness dependence of auditory evoked potentials (LD) was investigated as surrogate parameter for the central serotonergic activity in 185 healthy subjects subdivided according to newly identified 5-HTTLPR genotypes. RESULTS: Individuals homozygous for the L (A) allele showed the lowest LD of all genotypes suggesting a high serotonergic neurotransmission. The other observed genotypes (L (A)/L (G), S/L (A), S/L (G), S/S) had an LD which was similar to each other but higher compared to the L (A)/L (A) genotype. DISCUSSION: The data provide a rationale to subdivide the L allele of the 5-HTTLPR into L (A) and L (G) alleles in terms of their serotonin activity as indicated by the LD. The present IN VIVO measurements provide a basis for grouping the L (G) and S alleles for further investigations.

Degeneration of dopaminergic mesocortical neurons and activation of compensatory processes induced by a long-term paraquat administration in rats: implications for Parkinson's disease.

A deficiency of the dopaminergic transmission in the mesocortical system has been suggested to contribute to cognitive disturbances in Parkinson's disease. Therefore, the aim of the present study was to examine whether the long-term administration of a commonly used herbicide, paraquat, which has already been found to induce a slowly progressing degeneration of the nigrostriatal neurons, influences mesocortical dopaminergic neurons in rats. Paraquat at a dose of 10 mg/kg i.p. was injected either acutely or once a week for 4, 8, 12 and 24 weeks. Acute treatment with this pesticide increased the level of homovanillic acid (HVA) and HVA/dopamine ratio in the prefrontal cortex. After 8 weeks of administration paraquat increased the number of stereologically counted tyrosine hydroxylase-immunoreactive (TH-ir) neurons and their staining intensity in the ventral tegmental area (VTA), which is a source of the mesocortical dopaminergic projection. At the same time, few TH-ir neurons appeared in different regions of the cerebral cortex: in the frontal, cingulate, retrosplenial and parietal cortices. Chronic paraquat administration did not influence the level of dopamine in the prefrontal cortex but increased the levels of its metabolites: 3,4-dihydroxyphenylacetic acid (after 8-12 weeks), HVA (after 4 and 12 weeks) and HVA/dopamine ratio (4 weeks). After 24 weeks this pesticide reduced the number of TH-ir neurons in the VTA by 42% and of the Nissl-stained neurons by 26%, and induced shrinkage of this structure by ca. 25%. Moreover, TH-ir neurons in the cortex were no more visible after such a long period of administration and levels of dopamine metabolites returned to control values. The present results suggest that the long-term paraquat administration destroys dopaminergic neurons of the VTA. However, compensatory activation of the VTA neurons and cortex overcomes progressing degeneration and maintains cortical dopaminergic transmission.

2,9-Dimethyl-beta-carbolinium, a neurotoxin occurring in human brain, is a potent inducer of apoptosis as 1-methyl-4-phenylpyridinium.

The causes of neurodegeneration are not well understood. However, the role of environmental and endogenous toxins is receiving much attention. In this study, we compared the synthetic neurotoxin 1-methyl-4-phenyl-pyridinium with beta-carbolines occurring in human brain. Methylation of both nitrogens is necessary to convert a beta-carboline into a potent inhibitor of mitochondrial complex I. The respective beta-carboline, 2,9-dimethyl-beta-carbolinium ion is neurotoxic in rats. To investigate the underlying mechanisms, we incubated mouse neuroblastoma 2A cells with 2,9-dimethyl-beta-carbolinium ion, and compared the findings with effects of norharman, the precursor beta-carboline of methylated derivatives, and with 1-methyl-4-phenyl-pyridinium. 2,9-Dimethyl-beta-carbolinium ion caused a significant increase of reactive oxygen species (higher efficiency than 1-methyl-4-phenyl-pyridinium) and of mitochondrial membrane potential within the first minutes. After 60 min, the membrane potential dissipated. Concomitantly, the levels of glutathione increased in 2,9-dimethyl-beta-carbolinium ion but not in 1-methyl-4-phenyl-pyridinium treated cells. After 24 h effector caspases 3 and 7 were activated and the number of apoptotic cells increased as revealed by fluorescence-activated cell sorting cytometry. When incubated longer (48 h), cells underwent late apoptosis/secondary necrosis as shown by fluorescence-activated cell sorting analysis and confirmed qualitatively by an electron microscopy study. The effects of 2,9-dimethyl-beta-carbolinium ion on apoptotic changes were similar to those induced by 1-methyl-4-phenyl-pyridinium(,) while norharman showed only a weak potency at the very high doses. To investigate whether 2,9-dimethyl-beta-carbolinium ion is neurotoxic under in vivo conditions and whether only dopaminergic neurones are affected we conducted a dose-response study. Three weeks after injection of 2,9-dimethyl-beta-carbolinium ion in the substantia nigra we found a dose-dependent decrease of dopamine and its metabolites in the striatum of rats. The levels of 5-hydroxytryptamine were diminished although the decrease was less. The levels of noradrenaline increased after some doses. The findings strongly suggest an important role of endogenous beta-carbolines in neurodegeneration with apoptosis as the predominant mechanism.

Serotonergic effects of smoking are independent from the human serotonin transporter gene promoter polymorphism: evidence from auditory cortical stimulus processing.

BACKGROUND: Cigarette smoking has been associated with mood enhancing properties and modulating effects on serotonin activity. The loudness dependence (LD) of the auditory-evoked N1/P2-component has been related to serotonergic neurotransmission, i. e. the allelic variants in the promoter of the 5-hydroxytryptamine-transporter (5-HTT) gene (SCL6A4). Moreover, smoking behavior has been associated to the 5-HTT-genotype. It was hypothesized that cigarette smoking modulates the LD and this effect was expected to interact with the 5-HTT-genotype. METHODS: 5-HTT-genotype and LD were determined in 63 healthy smokers and 114 nonsmokers. RESULTS: LD was significantly affected by smoking status (p = 0.008) and 5-HTT-genotype (p = 0.045) but not by smoking*genotype-interaction or daily cigarette consumption. Current smokers exhibited a significantly weaker LD compared to nonsmokers. 5-HTT-genotype showed no significant effect on smoking behavior. DISCUSSION: The results indicate a higher serotonergic activity in smokers as compared to nonsmokers independent of 5-HTT-genotype. Since former smokers and never smokers showed similar LDs, the serotonin enhancing effect of smoking seems to be a characteristic state, which may contribute to the maintenance of smoking behavior.

The strong inhibition of triosephosphate isomerase by the natural beta-carbolines may explain their neurotoxic actions.

The natural beta-carbolines (BC) closely resemble the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in structure. The N-methylated beta-carbolinium ions (BC+) are potent inhibitors of mitochondrial respiration and are nigrostriatal neurotoxins. Utilizing [3H]BC, we have identified several proteins to which BC binds with high affinity (e.g. the chaperone member glucose regulated protein 78, the enzyme carboxylesterase, the cytochrome P450 2E1, the enzyme monoamine oxidase B and a small G-protein of the Rho subfamily). In the present study we isolated a protein from bovine brain to which [3H]BC binds with high affinity and identified it being the enzyme triosephosphate isomerase (TPI; EC 5.3.1.1.). 2,9-Dimethyl-BC+ was the most potent inhibitor of TPI, clearly more potent than the known inhibitors. TPI deficiency is a rare disorder in humans characterized by a severe progressive extrapyramidal course. Thus, TPI inhibition could contribute to neurodegeneration observed after injection of BCs into substantia nigra. Furthermore, the findings fit into the hypothesis of BCs as endogenous toxins responsible for neurodegeneration.

Impaired immunoglobulin M production by incubation of hybridoma cells with ethanol.

Several reports have presented results that demonstrate suppression of the immune system by ethanol. Using a hybridoma cell model, we studied the effects of ethanol on cell proliferation and on the production of immunoglobulin M (IgM) antibodies. The number of cells decreased while incubated with as little as 25 mM ethanol but not in a clonal subline incapable of IgM production, indicative of an increased vulnerability associated with the antibody-producing machinery. Levels of antibodies in cell culture supernatants were monitored by -heavy-chain-specific and -light-chain-specific enzyme-linked immunosorbent assays. We found a significant decrease in antibody concentration at 200 mM ethanol compared with findings for nonexposed cells. In addition, lower -chain compared with -chain values were monitored at ethanol concentrations of 50 mM and higher. This difference suggests irregular composition of the antibodies in the supernatant. Determination of IgM levels within the hybridoma cells revealed a linear increase in antibody concentrations by as much as three times the control levels with increasing ethanol concentrations when correlated with cell numbers. Analysis of the mRNA levels of two ethanol-inducible stress proteins, the 78-kilodalton glucose-regulated protein (GRP78) and the 70-kilodalton heat-shock protein (HSC70), by quantitative Northern hybridization yielded increased mRNA in a nonlinear fashion. The results demonstrate that ethanol impairs IgM composition, whereas antibody production within hybridoma cells is increased and the assembling machinery is activated, indicating compensating processes.

Acute and chronic actions of a dry methanolic extract of Hypericum perforatum and a hyperforin-rich extract on dopaminergic and serotonergic neurones in rat nucleus accumbens.

Dry hydroalcoholic extract of Hypericum perforatum L. is effective in the treatment of mild-to-moderate depression. Neither the mechanism of action nor the component or components of the extract responsible are known to date. In several in vitro and ex vivo models, the extract and hyperforin, one of its active components, cause changes similar to those of tricyclic antidepressants. Little is known about effects on a complex neuronal system relevant to antidepressant actions such as the mesolimbic system in the brain. In animal models of depression, the levels of dopamine were reduced in the nucleus accumbens, which is an important part of the mesolimbic system. These and other deficits were compensated by imipramine. We investigated the actions of the methanolic Hypericum extract LI 160 and a hyperforin-rich research extract in the shell region of the nucleus accumbens under in vivo conditions after acute administration, and after application repeated fourteen times. Both extracts induced an increase of dopamine (DA) and 5-hydroxytryptamine (5-HT) levels measured by in vivo microdialysis. The dose-response curve followed an inverse U-shape. Repeated application caused a rapid tolerance of DA but not of 5-HT neurones. Similar changes were observed after acute and repeated applications of imipramine. Several lines of evidence have suggested other active components in the Hypericum extract. The potency of hyperforin surpassed that of imipramine in the acute release of both DA and 5-HT by the nucleus accumbens. The effect of hyperforin correlated well with the inhibiting potency on high-affinity DA and 5-HT uptake. The missing effect of relatively high doses on DA and 5-HT levels as found in dose-response experiments may be explained by self-inhibition caused by the activation of autosomal receptors and by the inhibition by GABA. The inhibition of neuronal GABA transport occurs at somewhat higher concentrations of hyperforin. In conclusion, the findings demonstrate an imipramine-like effect of methanolic Hypericum extract LI 160 and of hyperforin on mesolimbic, dopaminergic and serotonergic neurones in acute and chronic experiments, which probably contributes to the antidepressant action of the medication. The methanolic Hypericum extract contains active compounds other than hyperforin.

Genetic analysis of the mu-opioid receptor in alcohol-dependent individuals.

On the basis of various study results, it is suggested that the ethanol-induced activation of the endogenous opioid system may play an important role in mediating the reinforcing effects of ethanol. The mesolimbic dopamine reward system is activated by both ethanol and opioids, and genetic differences in the sensitivity of the endogenous opioid system to alcohol may be an important factor determining the risk for the development of excessive alcohol consumption. Thus, variants of the mu-opioid receptor (muOR) gene may confer vulnerability to alcohol dependence. Five exon 1 variants of the muOR were investigated in 327 alcohol-dependent and 340 healthy control subjects. The Val6 variant of the +17C/T polymorphism and the Asp40 variant of the +118A/G polymorphism showed a trend to an increased allele frequency in alcohol-dependent subjects. The latter polymorphism was investigated in more detail. The dopamine receptor agonist apomorphine causes an increase in growth hormone (GH) levels in the blood by stimulating the release of growth hormone-releasing hormone. beta-endorphin also activates this regulatory circuit. We found a blunted response in intoxicated alcohol-dependent subjects, but no difference in GH response between the groups of alcohol-dependent subjects with and without the variant Asp allele. However, alcohol-dependent subjects with the Asp allele showed a significantly higher GH response at day 7 after alcohol withdrawal and a tendency to lower novelty seeking. These results suggest to us that there is reduced dopaminergic neuronal activity in alcohol-dependent subjects with the muOR Asp40 allele, along with a compensating increase in dopamine receptor activity. The difference between the two groups of alcohol-dependent subjects can be demonstrated only under certain conditions such as alcohol withdrawal, which necessitates the adaptation of the neurones to a new homeostasis.

Ethanol and gene expression in brain.

This article represents the proceedings of a symposium at the 2000 ISBRA Meeting in Yokohama, Japan. The chairs were Izuru Matusmoto and Peter A. Wilce. The presentations were (1) GABA receptor subunit expression in the human alcoholic brain, by Tracey Buckley and Peter Dodd; (2) NMDAR gene expression during ethanol addiction, by Jorg Puzke, Rainer Spanagel, Walther Zieglgansberger, and Gerald Wolf; (3) Differentially expressed gene in the nucleus accumbens from ethanol-administered rat, by Shuangying Leng; (4) Expression of a novel gene in the alcoholic brain, by Peter A. Wilce; and (5) Investigations of haplotypes of the dopamine D2-receptor gene in alcoholics, by Hans Rommelspacher, Ulrich Finckh, and Lutz G. Schmidt.

Five exon 1 variants of mu opioid receptor and vulnerability to alcohol dependence.

The human mu opioid receptor (hMOR) gene is a prime candidate gene responsible for addictive disorders. The present association study tested the hypothesis that hMOR exon 1 variants elicit susceptibility to alcohol dependence. We have analyzed five nucleotide changes in exon 1 of the hMOR gene. Three of them are in the 5'untranslated region of exon 1 at positions -172G/T,-111C/T and -3 8C/A, the remaining two variants cause amino acid substitutions: +17C/T (Ala6Val) and +118A/G (Asn40Asp). Our population-based association study included 327 German alcohol-dependent subjects and 340 ethnically matched controls. The lack of an allelic association suggests that the analyzed hMOR exon 1 variants do not contribute a common and substantial effect to the genetically determined vulnerability of alcohol dependence.

Different allele distribution of a regulatory MAOA gene promoter polymorphism in antisocial and anxious-depressive alcoholics.

Heritable factors account for approximately 40-60% of the total variance of liability to alcohol dependence. The present study tested whether a novel functional polymorphism in the promotor region of the X-chromosomal monoamine oxidase A gene (MAOA) was related to antisocial and anxious-depressive traits in alcoholics. Due to the X-chromosomal localization of the MAOA gene, psychobiological traits were compared separately for both genders of 298 male and 66 female alcoholics. In males, 30 of 59 alcoholics with antisocial personality disorder carried the low-activity 3-repeat allele in contrast to only 7 of 31 anxious-depressive alcoholics (51% vs. 23%; p = 0.02). Likewise, female anxious-depressive alcoholics showed a trend towards a low frequency of genotypes with the 3 repeat allele compared to female alcoholics without these symptoms (29% vs. 53%; p = 0.09). Taken together, these findings suggest that the 3-repeat allele of the MAOA polymorphism contributes modestly to the dimension of overand underreactive behaviors as possible antecedents of alcoholism.

Transition to alcohol dependence: clinical and neurobiological considerations.

The transition to alcohol dependence is supposed to occur during a critical period that begins with increased drinking and ends with a loss of control. This process may last about 3 to 4 years, and is modified by gender and accelerated by premorbid traits (e.g., novelty-seeking) and comorbid disorders (e.g., dissocial personality disorders according to ICD-10). Genetic disposition, environmental influences (e.g., stress), and sensitization by exposure are factors implicated in dependence that alter brain functions, some possibly in an irreversible way. Underlying neurobiological mechanisms that may have different time patterns are beginning to be characterized on a systemic, cellular, and molecular level. Repeated free choices of the rewarding compound seem to be necessary for the transition to dependence.

Association analysis of GABAAbeta2 and gamma2 gene polymorphisms with event-related prefrontal activity in man.

Gamma-aminobutyric acid (GABA)A-receptors play a crucial role in the generation of electroencephalogram (EEG) oscillations and evoked potentials (ERPs). The present association study was designed to test whether EEG and ERPs are modulated by genetic variations of the human GABAA beta2 (GABRB2) and gamma2 (GABRG2) genes on chromosome 5q33. The genotypes of two nucleotide substitution polymorphisms of the GABRB2 and GABRG2 genes were assessed in 95 psychiatrically healthy subjects of German descent. Neurophysiological phenotyping was performed with four factorized EEG/ERP parameters: EEG activation, anterior and posterior EEG synchronization, and event-related activity (N100/ P200-complex). No genotypic association was found for the GABRB2 nucleotide exchange polymorphism with any electrophysiological parameter. A significant association was found between the genotype of the intronic GABRG2 G-->A nucleotide exchange and the event-related N100/P200 (ANOVA: F=3.81; df=2; P=0.026). A comparison of homozygous subjects carrying either the G/G or A/A genotype of the GABRG2 polymorphism consistently revealed an even stronger difference in the effect-size (ANOVA: F=11.13; df=1; P=0.002). Post hoc analysis of this association with current density analysis in three-dimensional neuroanatomic Talairach space-time showed a reduction in the event-related signal power after 120 ms in the right dorsolateral prefrontal cortex. Taking into account the risk of false-positive association findings attributable to multiple testing, our results encourage further replication studies to examine the phenotype-genotype relationship of GABRG2 gene variants and event-related prefrontal activity.

Evaluation of an allelic association of the serotonin 5-HT1B G681C polymorphism with antisocial alcoholism in the German population.

Our study tested whether an association of the 861C allele of the serotonin 5-HT1B gene (HTR1B) with antisocial alcoholism exists in the German population. The HTR1B G861C polymorphism was genotyped in 588 subjects of German descent, comprising 250 non-alcoholic controls and 338 alcohol-dependent subjects, of whom 56 exhibited a dissocial personality disorder (DSPD). The Tridimensional Personality Questionnaire was assessed in 109 alcohol-dependent males to explore an effect of the 861C allele to risk-taking behaviour. Our results revealed no evidence for an association of the 861C allele with antisocial alcoholism (p > 0.63). There were no significant differences in the personality traits, novelty-seeking, harm avoidance and reward dependence between 46 male alcoholics carrying the 861C allele compared to those 63 alcoholics lacking it (p > 0.52). Our results do not provide evidence that the 861C allele contributes a substantial vulnerability effect to antisocial behavior in German alcohol-dependent subjects.

Effect of ethanol on (R)- and (S)-salsolinol, salsoline, and THP in the nucleus accumbens of AA and ANA rats.

Tetrahydroisoquinolines (TIQ) are active metabolites of dopamine. Intracerebral application stimulates the voluntary ethanol intake. In the present study, the levels of several TIQ's [(S)- and (R)-salsolinol, salsoline and tetrahydropapaveroline (THP)] were measured in the extracellular space of the nucleus accumbens of alcohol-preferring AA and alcohol-avoiding ANA rats. Ethanol (2 g/kg i.p.) caused an increase in dopamine levels in ANA but not in AA rats. Neither (R)- nor (S)-salsolinol concentrations changed after ethanol application, though (S)-salsolinol concentrations were higher in ANA than in AA rats. Ethanol caused an increase in salsoline concentrations in ANA but not in AA rats. THP increased following ethanol, which tended to be stronger in ANA rats. The study revealed differences in the TIQ levels of the nucleus accumbens between AA and ANA rats. In case of changes following ethanol application (dopamine, salsoline, THP), the AA rats were less sensitive. The findings resemble observations in high-risk sons of alcoholics with reduced sensitivity to ethanol in young age and increased risk to become alcoholic.