Dubravka Hranilović

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.

Hyperserotonemia in autism: activity of 5HT-associated platelet proteins

Disturbances in serotonin (5HT) neurotransmission have been indicated as biological substrates in several neuropsychiatric disorders including autism. Blood 5HT concentrations, elevated in about one-third of autistic subjects, are regulated through the action of peripheral 5HT-associated proteins. We have measured the activity of two platelet 5HT-associated proteins: 5HT transporter (5HTT) and monoamine oxidase B (MAOB), and indirectly studied the activity of 5HT2A receptor (5HT2Ar) in 15 hyperserotonemic (HS) and 17 normoserotonemic (NS) autistic subjects, and 15 healthy controls (C). While mean velocities of 5HTT kinetics did not significantly differ among the groups, significant elevation in the mean velocity of MAOB kinetics was observed in NS subjects and was even more pronounced in HS subjects in comparison to controls. Also, a decrease in adenosine 5′-diphosphate-induced platelet aggregation of borderline significance was observed in NS subjects, compared to C subjects. The results suggest a possibility of upregulation of monoaminergic synthesis/degradation and, probably consequential, downregulation of 5HT2Ar in autistic subjects.

Intronic polymorphism of tryptophan hydroxylase and serotonin transporter: indication for combined effect in predisposition to suicide

Summary.Indices of disturbed serotonergic neurotransmission are the most robust biological findings in suicide. Tryptophan hydroxylase (TPH) and 5-hydroxytryptamine transporter (5HTt) are the main regulators of 5HT signaling. Owing to the assumed functionality of intronic polymorphisms of TPH (218AC) and 5HTt (VNTR-2) genes, we investigated frequencies of concurrence of the TPH and 5HTt genotypes containing “lower activity” alleles (CC and 1010, respectively), in 192 suicide victims and 377 controls. Significant differences in frequencies of 5HTt and TPH genotype combinations were found between suicide victims and control subjects (p = 0.0156), with a clear dose-effect of the suspected (“lower activity”) genotypes (p = 0.0046). Concurrent presence of the two, allegedly transcriptionally less active, variants of these genes seems to be in some kind of relation to the increased susceptibility to suicide.

Anxiety-like behavior and cognitive flexibility in adult rats perinatally exposed to increased serotonin concentrations

Serotonin (5-hydroxytryptamine, 5HT) is a biologically active amine that regulates the development of 5HT neurons and target tissues during neurogenesis, while later it assumes the function of a neurotransmitter. Serotonin mediates many essential behaviors common to all mammals, and is held responsible for anxiety-like behavior and cognitive rigidity. Proper serotonin levels, controlled through 5HT synthesis and metabolism, are crucial for normal brain development. In this study we investigated anxiety-like behavior and cognitive flexibility in adult animals after exposing their developing brains to increased 5HT concentrations. Wistar rats were treated subcutaneously from gestational day 12 to post-natal day 21 with the immediate 5HT precursor 5-hydroxytryptophan (5HTP, 25mg/kg), a non-selective MAO inhibitor tranylcypromine (TCP, 2mg/kg), or saline. After reaching adulthood, animals were tested for anxiety-like behavior (exploratory behavior, thigmotactic behavior, social contact, and reaction to stressful stimulus) and cognitive flexibility (ability for reversal learning). Results of the behavioral studies corresponded with our previous neurochemical findings. Treatment with 5HTP, which has induced mild reduction in cortical 5HT concentrations, caused reduction in only one aspect of anxiety-like behavior (increased exploratory activity). Treatment with TCP, which lead to drastic reduction in 5HT concentration/function, resulted in a highly anxiolytic phenotype (reduced thigmotaxis, reaction to stress, and social anxiety) with improved cognitive flexibility. Although further neurochemical, anatomical and gene-expression studies are needed to elucidate the mechanisms underlying the observed behavior, we hope that our results will contribute to the understanding of the role of serotonin in anxiety-like behavior and cognitive rigidity.

Emotional response in dopamine D2L receptor-deficient mice

The dopamine D2 receptor (D2R) system has been implicated in emotional processing which is often impaired in neuropsychiatric disorders. The long (D2L) and the short (D2S) isoforms of D2R are generated by alternative splicing of the same gene. To study differential roles of the two D2R isoforms, D2L-deficient mice (D2L-/-) expressing functional D2S were previously generated. In this study the contribution of D2L isoform to emotional response was investigated by examining behaviors that reflect emotionality (exploratory behavior, anxiety-like behavior and learned helplessness) in D2L-/- and (wild-type) WT mice. While the thigmotactic, locomotor and general components of anxiety in zero maze did not differ among the genotypes, D2L-/- mice displayed significantly lower level of exploration in a hole board and zero maze, and significantly higher increase in latency to escape from a foot-shock after the learned helplessness training, compared with WT mice. These results suggest that D2L may play a more prominent role than D2S in mediating emotional response, such as behavioral reactions to novelty and inescapable stress. Our findings contribute to a better understanding of the molecular and cellular mechanisms underlying emotional responses.

Rats with constitutionally upregulated/downregulated platelet 5HT transporter: differences in anxiety-related behavior.

Serotonin (5-hydroxytryptamine, 5HT) plays important roles in both embryonic development as a mediator of neurogenesis and in the mature brain as a neurotransmitter. Disturbances in serotonergic transmission have been indicated in several psychiatric disorders. In the search for the biological substrates of psychiatric diseases, studies using animal models represent complementary approaches to studies on human subjects. Wistar-Zagreb 5HT rats, with constitutionally upregulated/downregulated platelet 5HT transporter (termed high- and low-5HT rats, respectively), have been developed in our laboratory as a model for studying various aspects of 5HT function. In this work, we have searched for potential behavioral differences between Wistar-Zagreb 5HT rat sublines in three anxiety paradigms: hole-board, zero-maze, and social interaction test. In all three tests, significant differences in behavior between Wistar-Zagreb 5HT sublines have been observed, indicating higher levels of anxiety-related behavior in high-5HT rats. In the social interaction test, high-5HT animals spent less time in active contact with conspecifics and displayed a narrower spectrum of social behaviors than their low-5HT counterparts, while in the zero-maze and hole-board tasks, they showed a lower level of exploratory activity (head dips and nose pokes) in comparison to low-5HT rats. On the other hand, thigmotactic behavior (the percentage of time spent in open quadrants of zero-maze and the percentage of central holes visited in hole-board) did not differ between the sublines. The results suggest that as a result of selection process, a specific component of anxiety-related behavior (i.e. exploratory activity directed towards a novel environment and conspecifics) has been affected in Wistar-Zagreb 5HT rats.

Platelet serotonin measures in migraine

Objective and Background.—Serotonergic mechanisms play an important role in the pathogenesis of headache. To search for potential indicators of altered serotonin homeostasis in migraine, we have investigated three parameters of the platelet serotonin (5HT) system, platelet serotonin level (PSL), platelet serotonin uptake (PSU), and monoamine oxidase (MAO‐B) activity, in a group of 55 patients with migraine and in 81 healthy controls.

Identification of serotonin transporter mRNA in rat platelets

SummaryTotal RNA was isolated from rat platelets by guanidinium — acid —phenol extraction, and mRNA for the serotonin (5-hydroxytryptamine) transporter (5HTt) was identified. From a typical starting sample of 20mL of rat blood (∼9 × 109 platelets), 14 to 17μg of total platelet RNA was obtained. Northern blot analysis, using32P-labeled 5HTt cDNA as a probe, identified ∼3.3 kb long 5HTt mRNA. After rehybridization with cyclophilin cDNA, ∼1kb long mRNA for cyclophilin, which could serve as a reference for 5HTt mRNA quantification, was also identified. Densitometric analysis demonstrated clearly measurable signals for both mRNAs. The possibility of quantification of rat platelet 5HTt mRNA should enable parallel studies on 5HTt gene expression in platelets and brain of the same animal, and the evaluation of the platelet model at the molecular genetic level.

The effects of the perinatal treatment with 5-hydroxytryptophan or tranylcypromine on the peripheral and central serotonin homeostasis in adult rats

Serotonin (5HT) is a biologically active amine present in mammals in the brain and the peripheral tissues. Autism is a neurodevelopmental disorder in which 5HT homeostasis is disturbed both centrally and peripherally, but the relationship between the 5HT disturbances in the two compartments is not understood. In an attempt to explore the relationship between the disturbed peripheral 5HT homeostasis and central 5HT functioning, we exposed the developing rat brain to increased 5HT concentrations, by treatment of rats with subcutaneous injections of the immediate 5HT precursor 5-hydroxy-L-tryptophan (5HTP, 25 mg/kg), or the non-selective MAO inhibitor tranylcypromine (TCP, 2 mg/kg), during the period of the most intensive development of 5HT neurons--from gestational day 13 to post-natal day 21. The effects of the mentioned treatments on peripheral and central 5HT levels were then studied in adult rats. Platelet and plasma 5HT concentrations (measured by ELISA), as well as cortical and midbrain 5HT, tryptophan and 5-hydroxyindoleacetic acid levels (measured by HPLC) were determined in twelve 5HTP treated and eight TCP treated rats, and compared with the values measured in 10 control, saline treated rats. Treatment with 5HTP significantly raised peripheral but not central 5HT concentrations. At adult age, peripheral 5HT homeostasis was re-established, while modest decrease in 5HT concentration was observed in frontal cortex, presumably due to hyperserotonemia-induced loss of 5HT terminals during brain development. Treatment with TCP induced significant 5HT elevations in both compartments. At adult age, permanent changes in 5HT homeostasis were observed, both peripherally (as hyperserotonemia) and centrally (as altered 5HT metabolism with decreased 5HT concentrations). Further studies are planned in order to explore the nature of the different disturbances of 5HT homeostasis induced by the two compounds, and their results are expected to shed some light on the role of hyperserotonemia in autism.