Tatjana Bordukalo-Niksic

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.

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.

5HT-1A receptors and anxiety-like behaviours: studies in rats with constitutionally upregulated/downregulated serotonin transporter.

Altered activity of brain serotonergic (5HT) system has been implicated in a wide range of behaviours and behavioural disorders, including anxiety. Functioning of 5HT-1A receptor has been suggested as a modulator of emotional balance in both, normal and pathological forms of anxiety. Here, we studied serotonergic modulation of anxiety-like behaviour using a genetic rat model with constitutional differences in 5HT homeostasis, named Wistar-Zagreb 5HT (WZ-5HT) rats. The model, consisting of high-5HT and low-5HT sublines, was developed by selective breeding of animals for extreme activities of peripheral (platelet) 5HT transporter, but selection process had affected also central 5HT homeostasis, as evidenced from neurochemical and behavioural studies. Anxiety-like behaviour in WZ-5HT rats was evaluated by two commonly used paradigms: open field and elevated-plus maze. The involvement of 5HT-1A receptors in behavioural response was assessed by measuring mRNA expression in cell bodies (raphe nuclei) and projection regions (frontal cortex, hippocampus) by use of RT-PCR and in situ hybridization, and by measuring functionality of cortical 5HT-1A receptors by use of [(3)H]8-OH-DPAT radioligand binding. Animals from the high-5HT subline exhibit increased anxiety-like behaviour and decreased exploratory activity when exposed to novel environment. No measurable differences in constitutional (baseline) functionality or expression of 5HT-1A receptors between sublines were found. The results support contribution of increased serotonergic functioning to the anxiety-like behaviour. They also validate the high-5HT subline of WZ-5HT rats as a potential model to study mechanisms of anxiety, especially of its nonpathological form, while the low-5HT subline may be useful to model sensation seeking phenotype.

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.

Functional promoter polymorphism of the neuronal isoform of tryptophan hydroxylase (Tph2) in suicide

The association between suicide and G-703T polymorphism of the tryptophan hydroxylase 2 (TPH2), the rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, was studied in a sample of 291 suicide victims and 280 healthy subjects of Croatian origin. No significant differences were found between the groups. Obtained results do not support involvement of the investigated polymorphism in the susceptibility to suicide completion.

Expression of brain and platelet serotonin transporters in sublines of rats with constitutionally altered serotonin homeostasis

By selective breeding, two sublines of rats, termed Wistar-Zagreb 5HT rats, with constitutionally high or low values of platelet serotonin (5HT) level and activity of platelet serotonin transporter (5HTt) have been developed. Previous studies demonstrated significant differences between the sublines in the expression of platelet 5HTt at the level of both mRNA and protein. Neurochemical and behavioural studies demonstrated differences in functional activity of brain 5HTt indicating that, similarly to platelets, differences in mRNA level might be expected in brains of selected animals. In this work, semi-quantitative RT-PCR method for measuring the 5HTt expression in rat tissues was described and then used to quantify the 5HTt mRNA in brains and platelets of animals from high-5HT and low-5HT sublines. Three different housekeeping genes: GAPDH, beta-actin and cyclophylin B, were used as internal standards to normalise 5HTt signals. Significant differences in platelet 5HTt mRNA between the sublines were confirmed, as contrasted to only a tendency toward higher 5HTt mRNA levels in midbrain of animals from the high-5HT subline. Results indicate differences in transcriptional regulation of central and peripheral 5HT transporters, suggesting that homeostatic control in the brain counteract more efficiently the selection pressure than in the periphery.

Serotonin level and serotonin uptake in human platelets: A variable interrelation under marked physiological influences☆

BACKGROUND Although it is known that platelet serotonin level (PSL) depends directly on platelet serotonin uptake (PSU) through the plasma membrane, reports on their interrelation are inconsistent. The aim of this study was to systematically explore the relationship between these two platelet serotonin parameters in large human population. METHODS PSL and full-kinetics of PSU were determined on 318 blood donors (276 males, 42 females; 20-67 years). RESULTS The overall correlation coefficient between PSL and maximal velocity of PSU was highly significant but unexpectedly low (r=0.269). Further analyses revealed lack of correlation among females, and variable association among males, depending on the subject age and season of measurements. Highly significant correlations were observed in spring-winter, while association was absent during summer-autumn. Lowering of PSL-PSU correlation with increased age was also demonstrated, showing modest interrelation among younger men and no interrelation in older population. By multiple regression analyses season was identified as the only independent predictor of PSL-PSU relationship. CONCLUSIONS The results show prominent influence of biological (sex, age) and, especially, environmental (seasons) physiology on the intraindividual relationship between PSL and PSU. Although serotonin transporter activity plays an important role in determining PSL, the observed correlations indicate that other factors may predominate.

Constitutively Elevated Blood Serotonin Is Associated with Bone Loss and Type 2 Diabetes in Rats

Reduced peripheral serotonin (5HT) in mice lacking tryptophan hydroxylase (TPH1), the rate limiting enzyme for 5HT synthesis, was reported to be anabolic to the skeleton. However, in other studies TPH1 deletion either had no bone effect or an age dependent inhibition of osteoclastic bone resorption. The role of 5HT in bone therefore remains poorly understood. To address this issue, we used selective breeding to create rat sublines with constitutively high (high-5HT) and low (low-5HT) platelet 5HT level (PSL) and platelet 5HT uptake (PSU). High-5HT rats had decreased bone volume due to increased bone turnover characterized by increased bone formation and mineral apposition rate, increased osteoclast number and serum C-telopeptide level. Daily oral administration of the TPH1 inhibitor (LX1032) for 6 weeks reduced PSL and increased the trabecular bone volume and trabecular number of the spine and femur in high-5HT rats. High-5HT animals also developed a type 2 diabetes (T2D) phenotype with increased: plasma insulin, glucose, hemoglobin A1c, body weight, visceral fat, β-cell pancreatic islets size, serum cholesterol, and decreased muscle strength. Serum calcium accretion mediated by parathyroid hormone slightly increased, whereas treatment with 1,25(OH)2D3 decreased PSL. Insulin reduction was paralleled by a drop in PSL in high-5HT rats. In vitro, insulin and 5HT synergistically up-regulated osteoblast differentiation isolated from high-5HT rats, whereas TPH1 inhibition decreased the number of bone marrow-derived osteoclasts. These results suggest that constitutively elevated PSL is associated with bone loss and T2D via a homeostatic interplay between the peripheral 5HT, bone and insulin.

Opioid system genes in alcoholism: A case–control study in Croatian population

Due to their involvement in dependence pathways, opioid system genes represent strong candidates for association studies investigating alcoholism. In this study, single nucleotide polymorphisms within the genes for mu (OPRM1) and kappa (OPRK1) opioid receptors and precursors of their ligands - proopiomelanocortin (POMC), coding for beta-endorphin and prodynorphin (PDYN) coding for dynorphins, were analyzed in a case-control study that included 354 male alcohol-dependent and 357 male control subjects from Croatian population. Analysis of allele and genotype frequencies of the selected polymorphisms of the genes OPRM1/POMC and OPRK1/PDYN revealed no differences between the tested groups. The same was true when alcohol-dependent persons were subdivided according to the Cloningers criteria into type-1 and type-2 groups, known to differ in the extent of genetic control. Thus, the data obtained suggest no association of the selected polymorphisms of the genes OPRM1/POMC and OPRK1/PDYN with alcoholism in Croatian population.

Platelet serotonin transporter (5HTt): physiological influences on kinetic characteristics in a large human population.

The present study had two goals: first, to give a detailed description of a reliable method for full kinetic analysis of serotonin transporter (5HTt) on the membrane of human platelets, and second, as a main issue, to report on physiological influences on kinetic characteristics of this transmembrane transport on a large population of healthy individuals. Full kinetic analyses of platelet serotonin uptake were performed on 334 blood donors of both sexes by the use of 14C-radioisotopic method, which was first optimized according to assumptions of enzyme kinetic analyses, with regard to platelet concentration, duration of uptake, concentration of substrate as well as important technical parameters (underpressure of filtration, blanks, incubating temperature, etc). Kinetic parameters of platelet serotonin uptake in the whole population were for Vmax: 142 ± 25.3 pmol 5HT/108 platelets/minute and for Km: 0.404 ± 0.089 µM 5HT. Besides the report on kinetic values of 5HT transporter protein, we have also described major physiological influences on the mentioned parameters, Vmax, Km and their derivative, Vmax/Km (transporter efficiency): range and frequency distribution of normal values, intraindividual stability over time, lack of age influence, gender dependence and seasonal variations. The report on kinetic values and main physiological influences on platelet serotonin transport kinetics, obtained by the use of thoroughly reassessed methodology, and on by far the largest human population studied until now, offers a reliable frame of reference for pathophysiological studies of this parameter in various clinical fields.