Constitutive Serotonin Tone Modulates Molecular and Behavioral Response to Chronic Fluoxetine Treatment: A Study on Genetic Rat Model.

Selective serotonin reuptake inhibitors (SSRIs) are the most commonly prescribed medications for the treatment of mood disorders. Yet, individual response to SSRIs is highly variable, with only a portion of patients showing the desired therapeutic effect. To better understand the molecular basis underlying individual variability in response to SSRIs, here we comparatively studied behavioral and molecular consequences of chronic treatment with fluoxetine, a widely used SSRI, in two sublines of rats with constitutionally different serotonin (5HT) homeostasis: the high-5HT and low-5HT sublines. Platelet 5HT levels, a recognized indicator of SSRI efficacy, were decreased by fluoxetine treatment in both 5HT-sublines. On the other hand, biologically active plasma 5HT levels were reduced only in high-5HT rats. The anxiolytic effect of fluoxetine was also evident only in high-5HT rats, as supported by spatio-temporal and ethological behavioral measures in the elevated plus maze (EPM) test and exploratory behavior measures in the open field (OF) test. None of the behavioral EPM or OF measures were significantly altered by fluoxetine treatment in low-5HT rats. Unexpectedly, 5HT levels in cerebral cortices tended to be reduced only in low-5HT rats. Moreover, the effects of fluoxetine on cortical expression levels of 5HT-related proteins were also present only in low-5HT rats, with serotonin transporter (5HTT) and serotonin receptor type 1a (Htr1a) being down-regulated, while serotonin receptor type 4 (Htr4) was up-regulated by fluoxetine treatment. The obtained results support a role of individual 5HT tone as an important influencing factor on the biological actions of SSRI antidepressants.

Th2-TRMs Maintain Life-Long Allergic Memory in Experimental Asthma in Mice.

Allergic asthma is a chronic inflammatory remitting-relapsing disease affecting the airways. Long-lived allergen-specific memory CD4+ T helper 2 (Th2) cells in mice persist in lungs for more than 2 years after the induction of experimental allergic asthma (EAA). To further understand lung Th2 memory cells, we tracked CD4+ T cells in spleen and lungs from healthy mice, through the initiation of acute EAA, recovery (remission), and allergen-induced disease relapse. We identified a lung CD3+CD4+ cell subset that expresses CD44hiCD62L-CD69+ST2+, produces Th2 cytokines, and mediates allergen-induced disease relapse despite treatment with FTY720 and anti-CD4 antibody. These cells reside in the lung tissue for the lifetime of mice (>665 days) and represent long-lived pathogenic Th2 tissue resident memory cells (TRMs) that maintain "allergic memory" in lung. We speculate that these data implicate that human Th2-TRMs sentinels in lungs of patients are poised to rapidly respond to inhaled allergen and induce asthma attacks and that therapeutic approaches targeting these cells may provide relief to patients with allergic asthma.

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, ß-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.

Platelet serotonin in primary Sjögren's syndrome: level and relation with disease activity.

Primary Sjögren's syndrome (pSS) is chronic autoimmune disorder of unknown ethiopathogenesis. In line with the concept of neuroimmunohormonal dysregulation in inflammatory rheumatic diseases, the aim of this study was to investigate platelet serotonin level (PSL) in patients with pSS and its relation with the activity and duration of the disease. Significantly lower PSL in pSS patients (N=61) was shown as compared to healthy controls (N=103). No correlation was found between PSL and the actual disease activity assessed by the recently developed EULAR Sjögren's Syndrome Disease Activity Index (ESSDAI). Results suggest involvement of the serotonin system in the pathogenesis of pSS.

Combination of polymorphic variants in serotonin transporter and monoamine oxidase-A genes may influence the risk for early-onset alcoholism.

The combinatory effect of polymorphisms in serotonin transporter and monoamine oxidase-A genes on the aetiopathogenesis of alcoholism was investigated in a sample of 714 individuals. Increased frequency of subjects having three 'suspected' genotypes (5-HTTLPR-LL, STin2-1010 and MAO-A 3-repeat allele) was found among type-2 alcoholic patients (P=0.0189). Results highlight serotonergic/genetic contribution to early-onset alcoholism.

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.

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.

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.

Expression of 5HT-1A and 5HT-1B receptor genes in brains of Wistar-Zagreb 5HT rats.

By selective breeding, two sublines of rats with high or low activity of platelet serotonin (5HT) transporter (5HTt) have been developed (Wistar-Zagreb 5HT rats). Previous studies demonstrated significant differences between the sublines in the expression of platelet 5HTt at the level of both, mRNA and protein. Pharmacological studies showed marked alterations in brain 5HTt function, indicating differences in central serotonin homeostasis, although analysis of regional brain 5HTt gene expression did not show analogous differences. In this study, we searched for possible changes in the expression of the two central 5HT receptor subtypes: 5HT-1A and 5HT-1B, both participating in the regulation of brain 5HT transmission. Semi-quantitative RT-PCR, with three different housekeeping genes as internal standards, showed no differences in the levels of 5HT-receptor expression between the sublines. Results suggest that constitutional alteration of 5HT homeostasis, induced by selective breeding for the extremes of platelet 5HTt activity, did not cause measurable changes in the expression of central 5HT-1A (hippocampus) and 5HT-1B (striatum) receptors in the mentioned rat sublines under physiological conditions.

Alcohol dependence and polymorphisms of serotonin-related genes: association studies.

Variations in 5HT-related genes contribute to the alterations of serotonergic neurotransmission, which is implicated in the etiopathology of alcoholism. In this preliminary study we have tested polymorphisms of genes involved in 5HT transport and turnover for their association with alcohol dependence. A case group of males with type 2 alcoholism (N=59) and a control group of healthy males (N=282), both of Croatian origin, were analyzed for the frequency distribution of polymorphisms in 5HT transporter (5HTT-VNTR2, 5HTT-LPR), monoamine oxidase A (MAOA-uVNTR) and B (MAOB-A/G) and tryptophan hydroxylase 1 (TPH1 A218C) and 2 (TPH2 G-703T) genes. An increase in the frequencies of 10-repeat allele (p = 0.010; OR = 1.73; 95% CI = 1.14-2.60) and 10/10 genotype (p = 0.006; OR = 2.57; 95% CI = 1.32-5.00) of the 5HTT-VNTR2 polymorphism was found in alcoholic patients. No differences between case and control groups were observed for the other tested polymorphisms. Present results support earlier studies implicating the role of 5HTT gene in alcoholism. The increase of sample size (in progress) is expected to enable search of more subtle differences, as well as re-evaluation of these preliminary findings.

Platelet monoamine oxidase kinetics, alcoholism subtypes and cigarette smoking.

In trying to dissociate the effect of alcohol and tobacco use on platelet monoamine oxidase-B (MAO-B) activity, we compared the enzyme kinetics in controls (n = 66) and alcohol-dependent patients (n = 81), subdivided according to the severity of both, alcohol and tobacco use. Platelet MAO-B kinetics was measured spectrophotofluorimetrically in chronic alcohol intoxication and after 3 weeks abstinence. In alcoholic patients, an increased Michaelis-Menten constant (16%, p < 0.01) was shown, notwithstanding smoking status. Maximal velocity did not differ between patients and controls when adjusted for smoking. In cigarette smokers, a highly significant dose-dependent reduction of platelet MAO velocity (40%, p < 0.001) was demonstrated, with a similar degree of reduction in patients and controls. Tobacco use itself had no influence on MAO affinity. No differences were shown between subtype 1 and 2 alcoholics, or between the day of admission and the 21st day of abstinence. In conclusion, it seems that both, alcohol and tobacco consumption, may contribute to the lowering of overall platelet MAO-B activity. The effect of alcohol is small, due to interference with substrate binding, and not alteration of catalytic activity. In contrast, the effect of cigarette smoking is pronounced and relates to the dose-dependent reduction of platelet MAO velocity, with no influence on its affinity.

The effect of dietary supplementation with calcium salts on skeletal calcium in suckling rats.

This study aimed at identifying a calcium compound which could serve as an effective and safe dietary supplement in suckling rats over the period of intense growth and development. The main objective was to assess the effect of additional calcium intake on skeletal calcium in suckling pups. Suckling Wistar rats were fed using a pipettor with one of the following calcium salts from day 6 to 14 after the birth: gluconate, hydrogenphosphate, carbonate (each suspended in cow's milk), or chloride (in demineralized water). Control rats received only cow's milk. Calcium in the carcass (body without organs and skin) was analysed by atomic absorption spectrometry. The only effective dietary supplement that produced no risk for the suckling pups' growth was calcium hydrogenphosphate in cow's milk in the total amount of 340 mg. That dose increased the daily calcium intake 3 to 4 times compared to non-supplemented controls, increasing carcass calcium content by about 16 per cent. Other calcium compounds were either inefficient (carbonate) or had adverse effects on pups' growth (chloride and gluconate).