Branimir Jernej

mRNA expression of serotonin receptors in cells of the immune tissues of the rat.

Serotonin (5-hydroxytryptamine, 5-HT) has been shown to play a role in immunoregulation; however, little is known about specific subtypes of 5-HT receptors involved in peripheral immunomodulation. In the present study we used RT-PCR methods to examine the mRNA expression of 5-HT receptors in the cells of lymphoid tissues of the rat. All 13 rat 5-HT receptor genes cloned so far were examined in ex vivo isolated spleen, thymus, and peripheral blood lymphocytes, as well as in mitogen-stimulated spleen cells. Positive signals were obtained for 5-HT1B, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT6, and 5-HT7 receptor mRNAs in all three compartments. Mitogen (ConA and PWM) stimulated cells additionally expressed mRNA corresponding to the 5HT-3 receptor subtype. In contrast, 5-HT1A, 5-HT1D, 5-HT2C, 5-HT4, 5-HT5A, and 5-HT5B mRNAs were not detected in any of the examined cell populations. These results may be useful as a starting point for future functional studies on immunomodulatory effects of 5-HT and may help to understand conflicting serotonergic effects on immune functions as found in the literature.

Serotonin transporter gene promoter (5-HTTLPR) and intron 2 (VNTR) polymorphisms in Croatian suicide victims

BACKGROUND Disturbances in serotonin (5HT) transmission are the most frequently reported neurobiological substrates of suicidal behavior. Because 5HT transporter plays a central role in the regulation of 5HT synaptic function and its gene contains two functional polymorphisms (5-HTTLPR in the promoter region and VNTR in the second intron), it represents an interesting candidate for association studies in suicidal behavior. METHODS In this study, a possible association of 5-HTTLPR and intron 2-VNTR polymorphisms of the 5HT transporter gene with suicidal behavior was investigated in a sample of 135 suicide victims and 299 healthy control subjects of Croatian/southern Slavic origin. RESULTS There were no significant differences in 5-HTTLPR and intron 2-VNTR genotype- and allele- frequency distributions between suicide victims and healthy control subjects; however, a tendency toward an increase of 5-HTTLPR allele L and VNTR-allele 10 were observed in suicide group. Analysis of distribution of estimated haplotype frequencies revealed differences between suicide victims and control subjects, with an excess of haplotype L10 among suicide victims (p =.0112). CONCLUSIONS Our results provide modest evidence for a possible association of the 5HT transporter gene with a completed suicide. Further studies are needed to determine whether alterations in 5HTt gene expression are involved in suicidal behavior.

Physiological characteristics of platelet/circulatory serotonin: study on a large human population.

The aim of this work was the study of platelet/circulatory serotonin (5-hydroxytryptamine, 5-HT), specifically alternative ways of its measurement and main physiological characteristics. The study was performed on a large human population (N=500) of blood donors of both sexes over the course of a longer time period (17 months). Owing to the heterogeneity in measurement of circulatory serotonin encountered in the literature, three ways of expression were comparatively studied: per unit number of platelets, per unit mass of platelet protein and per unit volume of whole blood. Results demonstrated unimodal distribution of individual frequencies of platelet/circulatory serotonin in the human population with the mean values of 579+/-169 ng 5-HT/10(9) platelets; 332+/-89.9 ng 5-HT/mg protein and 130+/-42.3 ng 5-HT/ml blood (mean+/-S.D.). A progressive decrease of serotonin level with age (18-65 years) was demonstrated, reaching statistical significance between the extreme age groups. No significant differences in the serotonin level between the sexes were observed. No seasonal oscillations in platelet/circulatory serotonin were found. Platelet serotonin demonstrated intra-individual stability over time. Finally, regarding the methodology of measurement, our results demonstrated a good correlation among the above-mentioned ways of expression of platelet/circulatory serotonin. This indicates the possibility of intercomparison of the literature reports expressing this physiological parameter either as 5-HT concentration in platelets or as 5-HT level in the circulation.

Basal and stimulated extracellular serotonin concentration in the brain of rats with altered serotonin uptake.

We examined the relationship between the density of serotonergic (5‐hydroxytryptamine [5‐HT]) uptake sites and extracellular 5‐HT concentration in the rat brain using microdialysis with two different models, lesions with 5,7‐dihydroxytryptamine (50 μg in the dorsal raphe nucleus (DRN) 15 days before) and sublines of rats genetically selected displaying extreme values of platelet 5‐HT uptake. Compared to controls, lesioned rats had a reduced cortical concentration of 5‐hydroxyindoles (45%), unchanged basal extracellular 5‐HT in the DRN and ventral hippocampus (VHPC), and reduced basal 5‐hydroxyindoleacetic acid (5‐HIAA) concentrations (46%, DRN; 22%, VHPC). Yet the perfusion of 100 mmol/L KCl or 1 μmol/L citalopram elevated dialysate 5‐HT significantly more in the DRN and VHPC of controls. In genetically selected rats, platelet 5‐HT content and uptake were highly correlated (r2 = 0.9145). Baseline dialysate 5‐HT (VHPC) was not different between high and low 5‐HT rats and from normal Wistar rats. However, KCl or citalopram perfusion increased dialysate 5‐HT significantly more in high 5‐HT than in low 5‐HT rats, and the former displayed a greater in vivo tissue 5‐HT recovery. Significant but small differences in the same direction were noted in [3H]citalopram binding in several brain areas, as measured autoradiographically. Thus, basal extracellular 5‐HT (but not 5‐HIAA) concentrations are largely independent on the density of serotonergic innervation and associated changes in uptake sites. However, marked differences emerge during axonal depolarization or reuptake blockade. The significance of these findings for the treatment of mood disorders in patients with neurological disorders is discussed. Synapse 28:313–321, 1998.

Serotonin 1B (5HT-1B) receptor polymorphism (G861C) in suicide victims: Association studies in German and Slavic population

Serotonergic dysfunction is believed to be involved in the susceptibility to suicide due to functional alternations in the serotonin‐related genes. Serotonin 1B (5HT‐1B) receptor mediates aggressive behavior in mice models and was proposed to be involved in the control of aggression and impulsivity in humans. In this study we have investigated the association of G861C polymorphism of the 5HT‐1B receptor gene with suicide commitment. Study was based on two independent samples, one of German (245 suicide victims vs. 248 controls) and the other of Slavic/Croatian (118 suicide victims vs. 192 controls) ethnicity. No significant differences in allele or genotype frequencies between victims and controls were demonstrated either in German or Croatian sample. There were no differences in allele frequencies between German and Croatian population, and the combined sample, having high statistical power, also did not demonstrate significant differences between victims and controls. Results provide evidence that the investigated 5HT‐1B receptor gene variants are not implicated in the susceptibility to suicide.

Platelet serotonin level in rats is under genetic control

It has recently been shown that platelet serotonin (5-hydroxytryptamine, 5HT) levels (PSL) in Wistar rats represent an individually stable biological parameter (neither subject to periodic oscillations nor markedly influenced by sex and age) that shows a unimodal frequency distribution within the population (range: 1.2-2.2 micrograms 5HT/mg platelet protein). To investigate the genetic background of PSL, selective breeding for the extreme values of this trait was performed. In the fourth generation, two discrete sublines of animals (statistically different from the unselected population) with constitutionally high or low PSL could be discerned: one with congenitally low PSL (1.1-1.6 micrograms 5HT/mg platelet protein; approximately normal distribution) and the other with congenitally high PSL (1.6-2.9 micrograms 5HT/mg platelet protein; irregular distribution). No difference in the pattern of inheritance between sexes could be discerned. The results demonstrated a marked heritable component underlying the expression of individual values of PSL in rats, suggesting that this parameter is a trait characteristic.

Epilepsy and serotonin (5HT): Variations of 5HT-related genes in temporal lobe epilepsy

Several lines of evidence point to the role of serotonin (5HT) neurotransmission in the epileptogenesis. The present preliminary study investigated possible association of the temporal lobe epilepsy (TLE) with the polymorphisms in several 5HT-related genes, including serotonin transporter (5HTT), monoamine oxidase A (MAO-A) and serotonin receptors 5HT-1A, 5HT-1B and 5HT-2C. All participants (101 TLE patients and 170 healthy controls) were unrelated individuals of Croatian origin. 5HT-1B allele 861G was found to be slightly overrepresented in the patient group (p=0.0385). No significant differences between groups were observed for the other tested polymorphisms. Within the limitations imposed by the size of our sample, negative findings suggest that the respective loci do not make considerable contribution to the etiopathogenesis of TLE. Further examination of 5HT-1B gene, which yielded positive result at a trend level, is possibly warranted.

Monoamine oxidases A and B gene polymorphisms in migraine patients

Abnormal cortical activity and brainstem functioning are considered the possible etiopathogenetic factors of migraine. Monoamine oxidase A and B (MAO-A and -B) regulate the levels of monoamine neurotransmitters, so changes in their activity could participate in migraine pathogenesis. We have investigated the possible association of MAO-A and -B alleles and haplotypes with two common types of migraine, i.e. migraine without aura (MO) and migraine with aura (MA), on the sample of 110 migraineours (80 MO and 30 MA) and 150 controls. MAO-A promoter and MAO-B intron 13 polymorphisms were genotyped by the PCR-based methods. In addition, we have reevaluated the reported association between MAO-B intron 13 polymorphism and platelet MAO-B activity. The platelet MAO-B activity was determined fluorimetrically using kynuramine as a substrate. We have found a tendency toward association of the shorter variant of MAO-A gene promoter with migraine without aura in male subjects. Regarding investigated MAO-B polymorphism, no association with migraine or with platelet MAO-B activity was found. The suggestive association of the variant in MAO-A gene with migraine is considered worthy of independent replication. On the other hand, further studies on MAO-B polymorphism in migraine do not seem promising.

Serotonin and Immune Response: Effect of the Amine on in vitro Proliferation of Rat Lymphocytes

Objective: The effect of serotonin (5-hydroxytryptamine; 5HT) on the in vitro proliferation of mitogen-stimulated lymphocytes was studied in primary cultures of rat spleen cells. Methods: 5HT was added to the cultures 1 h prior to the mitogen, at final concentrations from 10–13 up to 10–2M. T and B cell mitogens (concanavalin A, pokeweed mitogen and lipopolysaccharide) were used at suboptimal and optimal concentrations. The cell proliferation was measured 24–72 h after the addition of mitogen. The effect of each 5HT concentration was studied on a group of 6–12 animals and was expressed as a percentage of the control values obtained with mitogen alone. Results: No significant effect of 5HT at concentrations from 10–13 to 10–5M was found. At concentrations of ≧10–4M, a regular dose-dependent inhibition of the lymphocyte proliferation appeared, the concentration producing the half-maximal effect being 6 × 10–4M. The observed suppression was not due to 5HT cytotoxicity toward spleen cells. Conclusion: With the experimental system used, we failed to confirm an immunostimulatory effect of 5HT in the range of concentrations of its receptor sensitivities or lower, but found a clear-cut immunoinhibitory effect at higher concentrations.

5HT-2C receptor polymorphism in suicide victims. Association studies in German and Slavic populations.

Abstract.Sustainable observations suggest that suicidal behaviour by itself may have biological correlates, among which those related to the serotonergic synapse hold the key position. Based on the association of suicide and serotonergic dysfunction, it was proposed that genetic mechanisms affecting suicidal behaviour could be related to the alterations of the genes encoding the elements of 5HT synapse. The present study tested the association of the polymorphism in the serotonin 2C (5HT-2C) receptor coding region (Cys23Ser) with suicide commitment. Study was based on two independent samples, one of German (284 suicide victims versus 297 controls) and other of Slavic/Croatian (118 suicide victims versus 275 controls) ethnicity. No significant differences in allele or genotype frequencies between victims and controls were demonstrated. Results did not provide supporting evidence for the potential involvement of the investigated variants of 5HT-2C receptor in the susceptibility to suicide.