5HT Levels Research Articles

Silencing vesicular neurotransmission in medullary serotonergic (5HT) neurons causes prolonged and failed autoresuscitation during early postnatal life in mice: possible implications for the sudden infant death syndrome (SIDS)

Abnormalities in the medullary 5HT system, including decreased tissue levels of 5HT, have been identified in approximately 70% of SIDS cases studied. Failed autoresuscitation (inability to recover breathing and heart rate following hypoxic apnea) is considered a possible cause of death in SIDS infants. Using 5HT ‘silenced’ mice, we examined the effect of altered neurotransmitter release from 5HT neurons on the ability to autoresuscitate after repeated exposure to intermittent anoxia at postnatal days (P) 5, 8 and 12. In these mice, Cre recombinase activates expression of tetanus toxin light chain in medullary 5HT neurons, which cleaves the VAMP2 protein that anchors vesicles to the synaptic membrane, thus suppressing synaptic vesicular neurotransmission in these neurons. ‘Silenced’ pups took twice as long as controls to initiate gasping at P5 and P8, and had a 22% reduction in gasp frequency at P8. Recovery of heart rate and breathing to 90% of normal was prolonged up to 5 and 2 times, respectively compared to controls at P8. Autoresuscitation failed in 19% of the ‘silenced’ pups at P5 and 44% at P8, compared to no control pup deaths. These data indicate that suppressing vesicular neurotransmission in 5HT neurons delays initiation of gasping and suggest that this may contribute to prolonged or failed autoresuscitation in response to anoxia in early postnatal life. (HD036379)

The role of endogenous serotonin in methamphetamine‐induced neurotoxicity to dopamine nerve endings of the striatum

Methamphetamine (METH) is a neurotoxic drug of abuse that damages the dopamine (DA) neuronal system in a highly delimited manner. The brain structure most affected by METH is the striatum where long-term DA depletion and microglial activation are maximal. Endogenous DA has been implicated as a critical participant in METH-induced neurotoxicity, most likely as a substrate for non-enzymatic oxidation by METH-generated reactive oxygen species. The striatum is also extensively innervated by serotonin (5HT) nerve endings and this neurochemical system is modified by METH in much the same manner as seen in DA nerve endings (i.e., increased release of 5HT, loss of function in tryptophan hydroxylase and the serotonin transporter, long-term depletion of 5HT stores). 5HT can also be modified by reactive oxygen species to form highly reactive species that damage neurons but its role in METH neurotoxicity has not been assessed. Increases in 5HT levels with 5-hydroxytryptophan do not change METH-induced neurotoxicity to the DA nerve endings as revealed by reductions in DA, tyrosine hydroxylase and dopamine transporter levels. Partial reductions in 5HT with p-chlorophenylalanine are without effect on METH toxicity, despite the fact that p-chlorophenylalanine largely prevents METH-induced hyperthermia. Mice lacking the gene for brain tryptophan hydroxylase 2 are devoid of brain 5HT and respond to METH in the same manner as wild-type controls, despite showing enhanced drug-induced hyperthermia. Taken together, the present results indicate that endogenous 5HT does not appear to play a role in METH-induced damage to DA nerve endings of the striatum.

Role of monoamine oxidase, nitric oxide synthase and regional brain monoamines in the antidepressant-like effects of methylene blue and selected structural analogues

Dual action antidepressants have important therapeutic implications. Methylene blue (MB), a charged compound structurally related to tricyclic antidepressants, acts on both monoamine oxidase (MAO) and the nitric oxide (NO)-cGMP pathway, and has demonstrated antidepressant activity in rodents. We investigated the antidepressant properties of MB and selected structural analogues and whether their actions involve MAO, NO synthase (NOS) and regional brain monoamines.Acute imipramine (IMI, 15mg/kg), saline, MB, acriflavine (ACR), methylene green (MG), methylene violet (MV), thionine (THI) and tacrine (TAC) (1-60mg/kg i.p.) were tested for antidepressant activity in the forced swim test (FST), as well as MAO-A/B inhibitory activity. Active antidepressant compounds were subsequently studied at their most effective dose during sub-chronic treatment, followed by behavioural sampling in the FST and assay of cortico-limbic monoamines and hippocampal nitrate (for NOS activity).Only IMI, MB (15, 30, 60mg/kg) and MG (7.5, 25, 40mg/kg) reduced immobility in the acute FST. MB, MG and ACR were potent inhibitors of especially MAO-A. Following sub-chronic treatment, IMI (15mg/kg) increased noradrenergic behaviour in the FST, while MB (15mg/kg) and MG (15mg/kg) enhanced serotonergic behaviour. MB and MG bolstered cortico-limbic serotonin (5HT) levels and to a lesser extent l-norepinephrine (l-NE), but did not significantly alter regional dopamine (DA) levels. MB, and to lesser degree MG, reduced hippocampal nitrate levels.MB and MG present with structure-specific antidepressant-like effects following acute and sub-chronic treatment, possibly involving NOS and MAO-A inhibition and cortico-limbic 5HT and l-NE release. A role for MAO-B and DA appears minimal.

Molecular Mechanisms of SERT in Platelets: Regulation of Plasma Serotonin Levels

The serotonin transporter (SERT) on platelets is a primary mechanism for serotonin (5HT) uptake from the blood plasma. Alteration in plasma 5HT level is associated with a number of cardiovascular diseases and disorders. Therefore, the regulation of the transporter's activity represents a key mechanism to stabilize the concentration of plasma 5HT. There is a biphasic relationship between plasma 5HT elevation, loss of surface SERT, and depletion of platelet 5HT. Specifically, in platelets, plasma membrane SERT levels and platelet 5HT uptake initially rise as plasma 5HT levels are increased but then fall below normal as the plasma 5HT level continues to rise. Therefore, we propose that elevated plasma 5HT limits its own uptake in platelets by down-regulating SERT as well as modifying the characteristics of SERT partners in the membrane trafficking pathway. This review will summarize current findings regarding the biochemical mechanisms by which elevated 5HT downregulates the expression of SERT on the platelet membrane. Intriguing aspects of this regulation include the intracellular interplay of SERT with the small G protein Rab4 and the concerted 5HT-mediated phosphorylation of vimentin.

Alterations in monoamine levels and oxidative systems in frontal cortex, striatum, and hippocampus of the rat brain during chronic unpredictable stress

Stress plays a key role in the induction of various clinical disorders by altering monoaminergic response and antioxidant defenses. In the present study, alterations in the concentrations of dopamine (DA), serotonin (5-HT) and their metabolites, and simultaneous changes in the antioxidant defense system and lipid peroxidation in different brain regions (frontal cortex, striatum, and hippocampus) were investigated immediately and 24 h after exposure to chronic unpredictable stress (CUS). CUS involved subjecting Sprague–Dawley rats to two different types of stressors varying from mild to severe intensity every day in an unpredictable manner, over a period of 7 days. CUS significantly decreased DA and 5-HT concentrations, with increased DA turnover ratios in the selected brain regions. In the frontal cortex and striatum, DA metabolite concentrations were increased; however, in the hippocampus they remained unaltered. Further, a decrease of 5-hydroxyindoleacetic acid content was observed in the frontal cortex and striatum, with no significant alteration in the hippocampus. CUS also reduced the activities of superoxide dismutase and catalase, with increased lipid peroxidation and decreased glutathione levels in the selected brain regions. Glutathione peroxidase activity was increased in the frontal cortex and hippocampus only. The pattern of CUS-induced monoamine and oxidative changes immediately after the last stressor and 24 h later were similar when compared with the control group, indicating that the observed changes were due to the chronic exposure to the various stressors and were not merely acute effects of the last stressor. The altered redox state in the striatum and frontal cortex might be related to the perturbed DA and/or 5HT levels, while the hippocampus seems to be less influenced by CUS in terms of monoamine metabolite changes. These results suggest that the perturbed monoamine levels could interact with the oxidative load during CUS. Hence, the current study has implications for pharmacological interventions targeting both central monoamines and cellular antioxidants as a potential stress management strategy for protecting against central stress-induced disorders.

W1933 Expression of PYY is Unchanged in Rat Small Intestine During a High-Fat Diet

Purpose: Aging is associated with important changes in gastrointestinal function and in the levels of intestinal hormones secreted. Enterochromaffin cells containing serotonin (5-HT) and melatonin may play a major role in maintaining gut function during aging. Our aim was to characterise the mucosal availability of 5-HT and melatonin in the ileum and colon of a mouse model of aging. Methods: Female young mice (2-5 month; n=6), aged mice (~21 months; n=6) and aged mice treated with melatonin (~21 months; n=6; 10mg/kg/day) were examined. Electrochemical methods were used to measure 5-HT and melatonin (MEL) concentrations near the mucosal surface of ileum and distal colon. Amperometry was used to record compression evoked 5-HT while cyclic voltammetry was used to identify peaks for 5-HT and MEL. Differential pulse voltammetry (DPV: 500/800mV) was used to measure 5-HT and MEL steady state levels. Exogenous 5-HT and MEL were used to calibrate the carbon fibre electrodes and convert current into concentration. Paired or unpaired data were compared with a t test or a one way ANOVA (P<0.05) using a Tukey-Kramer post-hoc test. Results: Amperometry studies showed that young mice had peak compression evoked release of 5-HT from combined colon and ileum of 4.9±1.5 μM while aged mice had a enhanced release of 8.1±2.9 μM and aged mice treated with melatonin were significally depressed compared to aged mice (4.3±1.0 μM; P=004). Steady state levels of 5-HT after compression from combined tissue were 5.7±1.8 μM in young tissue while aged mice (4.8±0.6 μM) and aged mice treated with melatonin were no different from young mice (3.2±0.6 μM). DPV studies showed that young mice had concentrations of 5-HT of 4.1±1.1 μM in the ileum and 5.9±2.4 μM in the colon. Concentrations of MEL were 5.3±1.6 μM in the ileum and 4.7±1.5 μM in the colon (all n=6). Compared to young mice, the steady state levels of 5HT and MEL were increased in aged mice (combined ileum and colon: 5-HT=144% and MEL=127% of young mice). In contrast, the melatonin-treated mice had lower steady state levels compared to young mice (5-HT=89% andMEL=74%).When aged mice were compared directly with aged mice treated with melatonin, there was a significant decrease in both 5HT and MEL in the treated combined tissues (P=0.001); however, when ileum and colon were examined separately only MEL was significantly decreased in the ileum (P=0.04; n= 6) and colon (P=0.02; n=6) of treated mice. Conclusions: Our data show that the availability of 5-HT and melatonin are increased in aged mice and that melatonin treatment suppresses natural gastrointestinal production of 5-HT and melatonin in the aged mouse intestine.

W1932 Melatonin Supplementation Suppresses Serotonin and Melatonin Availability in Aged Mouse Intestine

Purpose: Aging is associated with important changes in gastrointestinal function and in the levels of intestinal hormones secreted. Enterochromaffin cells containing serotonin (5-HT) and melatonin may play a major role in maintaining gut function during aging. Our aim was to characterise the mucosal availability of 5-HT and melatonin in the ileum and colon of a mouse model of aging. Methods: Female young mice (2-5 month; n=6), aged mice (~21 months; n=6) and aged mice treated with melatonin (~21 months; n=6; 10mg/kg/day) were examined. Electrochemical methods were used to measure 5-HT and melatonin (MEL) concentrations near the mucosal surface of ileum and distal colon. Amperometry was used to record compression evoked 5-HT while cyclic voltammetry was used to identify peaks for 5-HT and MEL. Differential pulse voltammetry (DPV: 500/800mV) was used to measure 5-HT and MEL steady state levels. Exogenous 5-HT and MEL were used to calibrate the carbon fibre electrodes and convert current into concentration. Paired or unpaired data were compared with a t test or a one way ANOVA (P<0.05) using a Tukey-Kramer post-hoc test. Results: Amperometry studies showed that young mice had peak compression evoked release of 5-HT from combined colon and ileum of 4.9±1.5 μM while aged mice had a enhanced release of 8.1±2.9 μM and aged mice treated with melatonin were significally depressed compared to aged mice (4.3±1.0 μM; P=004). Steady state levels of 5-HT after compression from combined tissue were 5.7±1.8 μM in young tissue while aged mice (4.8±0.6 μM) and aged mice treated with melatonin were no different from young mice (3.2±0.6 μM). DPV studies showed that young mice had concentrations of 5-HT of 4.1±1.1 μM in the ileum and 5.9±2.4 μM in the colon. Concentrations of MEL were 5.3±1.6 μM in the ileum and 4.7±1.5 μM in the colon (all n=6). Compared to young mice, the steady state levels of 5HT and MEL were increased in aged mice (combined ileum and colon: 5-HT=144% and MEL=127% of young mice). In contrast, the melatonin-treated mice had lower steady state levels compared to young mice (5-HT=89% andMEL=74%).When aged mice were compared directly with aged mice treated with melatonin, there was a significant decrease in both 5HT and MEL in the treated combined tissues (P=0.001); however, when ileum and colon were examined separately only MEL was significantly decreased in the ileum (P=0.04; n= 6) and colon (P=0.02; n=6) of treated mice. Conclusions: Our data show that the availability of 5-HT and melatonin are increased in aged mice and that melatonin treatment suppresses natural gastrointestinal production of 5-HT and melatonin in the aged mouse intestine.

Preventive effects of enzyme-treated rice fiber in a restraint stress-induced irritable bowel syndrome model

Irritable bowel syndrome (IBS) is a common health issue that is characterized by abdominal pain, abnormal bowel movements, altered visceral perception, and abnormal metabolism of 5-hydroxy triptamine (serotonin; 5HT). The use of prebiotics or probiotics treatment for IBS has become increasingly important as an adjunct to pharmaceutical options. The aim of this study was to determine the efficacy of enzyme-treated rice fiber (ERF) on an IBS model. We obtained a new prebiotic from defatted rice bran that was developed as an insoluble dietary fiber through amylase and hemicellulase treatment followed by removal of the soluble fraction. Containing approximately 70% hemicellulose, ERF is utilized by lactobacilli and subsequently converted to butyrate using Eubacterium limosum. We employed a restraint stress IBS model which involved the continuous application of stress for 4 h per day for 3 days. Polycarbophil Ca (PC) (500 mg/kg body weight) was used as a positive control and ERF was added to the diet at 4% in diet. During restraint stress, ERF significantly attenuated urgent fecal excretion, colonic mucosal 5HT secretion, and hyperalgesthesia compared with the control. ERF also significantly increased cecal butyrate production as well as total organic acid content. PC was only effective in regard to preventing increases in 5HT levels. Furthermore, there were no significant levels of pro-inflammatory markers CINC-1 and TNF-alpha among the groups. Although more detailed studies are needed, the ERF prebiotic demonstrated potency in attenuating major symptoms of IBS.

The circadian biology of the marbled crayfish

The parthenogenetic marbled crayfish (Procambarus spec.) has recently been introduced as a new preparation for neuroethological studies. Since isogeneity apparently limits inter-individual variation, this otherwise typical decapod species may be especially valuable for circadian studies. Locomotor activity of isolated marbled crayfish and agonistic activity of small social groups maintain circadian rhythmicity in constant darkness. As potential signals of circadian time information, levels of 5HT, N-acetylserotonin and melatonin were determined in brains of marbled crayfish at different daytimes. However, location and structural organization of crustacean circadian pacemakers are still elusive. Immunocytochemical and backfill studies in the marbled crayfish revealed neural structures that may correspond to portions of circadian pacemaker systems in the insect optic lobe. Position and additional chemical contents in two pigment-dispersing hormone-expressing neuron groups resembled insect pigment-dispersing factor-expressing cells in the lamina and the accessory medulla, a neuropil discussed as center for integration of timing information. Here, we discuss new findings about the possible organization of the circadian system of the marbled crayfish in the light of current knowledge about circadian clocks in crustacea.

Preliminary Results of a Study of the Platelet Dense Granule and Serotonin Storage Pool in Autistic Children and Their Families.

Abstract 4456The etiology of autism is thought to be neurobiological, but the mechanisms involved are not known. One possible clue into this disorder can be found in multiple clinical studies which have established that at least 30-40% of individuals diagnosed with autism have significantly elevated levels of serotonin (5HT) in their blood. Serotonin transport by platelets and 5HT plasma levels have been found to be altered in patients with multiple neurobiological disorders, including bipolar disorder, schizophrenia, depression, aggression, autism, and migraine headache. Serotonin has numerous functions, including regulation of vasoconstriction, vasodilatation, and coagulation. A review of the literature reveals few studies of coagulopathy related to autism other than attempts to associate vaccination or infections with the etiology of autism. One recent study, of a limited number of individuals with autism spectrum disorders (ASD) and their families, reported a significant incidence of thrombophilia. All children evaluated (10/10) and 15 of 16 family members in the study were found to be at risk for thrombophilia disorders. We have been evaluating patients for PL dysfunction for many years and recently, have included ELISA tests to determine the 5HT concentration in PLs of patients suffering from migraine headache or syncope. All of our investigations to date have evaluated patients for a deficiency of the PL storage pool (DG SPD) including a decrease of PL DGs and their stored biochemical constituents; we have never conducted a study related to potentially increased numbers of DGs or the concentration of biochemicals contained in these granules. As at least one report exists in the literature suggesting the potential for thrombophilia in patients diagnosed with an autism spectrum disorder and their families, we decided to assess the platelet storage pool in autistic patients and their families as a potential model of platelet dysfunction due to elevated DGs and/or 5HT levels and/or as a potential biomarker for autism. As the major storage pool of 5HT in the circulation is known to exist in platelet PL DGs, it is possible that a PL DG storage pool could be an important mediator of ASD. Our hypothesis is that autistic children may have elevated numbers of DGs and increased levels of PL 5HT. Peripheral blood samples were obtained from children diagnosed with autism as well as their parents and siblings. Platelets were obtained via centrifugation and DG content determined by electron microscopy and an ELISA assay was utilized to determine PL 5HT concentration for all subjects. A questionnaire was also used to evaluate family bleeding history and drug therapy for the autistic children. At present, 4 families have been recruited for the study. Autistic children (n = 5) have an average of 5.42 ± 0.69 DG/PL and a mean 5HT concentration of 447.6.4 ± 85.6 ng/109 PL (PL 5HT normal range = 215-850 ng/109 PL). Their parents have an average of 5.00 ± 0.54 DG/PL and a mean 5HT concentration of 427.6.4 ± 82.66 ng/109 PL and one five year old sibling was found to have 8.17 DG/PL and 2233 ng/109 PL. Control subjects (n=21) have an average of 4.16 ± 0.09 DG/PL and a 5HT concentration of 589.6 ± 56.25 ng/109. There is no statistically significant difference between controls, parents or autistic subjects by ANOVA. Our preliminary data does not support our hypothesis. However, with serotonin being the immediate precursor of melatonin, we do not discount the fact that all of our subjects were receiving drug therapies including melatonin supplementation (n=2) or selective serotonin reuptake inhibitors (n=3) which diminish the PL 5HT concentration. Thus, the therapeutic regimens for these subjects could account for their normal 5HT levels. This project had been initiated just prior to the time of abstract submission; we are currently recruiting study participants and will present additional data at the meeting. Disclosures:No relevant conflicts of interest to declare.

Platelet Delta Granule and Serotonin Concentrations Are Decreased in Patients with Postural Orthostatic Tachycardia Syndrome.

Abstract 2419Poster Board II-396Syncope is generally defined as a state of temporary, partial or complete, loss of consciousness (fainting) with spontaneous recovery. The basis for the phenomenon is complex and includes a variety of orthostatic intolerance conditions including neurocardiogenic syncope (NCS), postural orthostatic tachycardia syndrome (POTS), and other disorders affecting the autonomic nervous system. NCS patients and POTS patients can be differentiated with an upright tilt test because either bradycardia or tachycardia will occur with hypotension, respectively. Both are induced by the inability to maintain adequate systemic blood pressure. Though the two primary types of syncope are different in origin, both conditions are treated similarly. Recent reports suggest that syncope is a low serotonin (5HT) disorder and selective serotonin reuptake inhibitors (SSRIs) are a commonly prescribed treatment for both NCS and POTS patients. The major storage pool of serotonin, independent of the central nervous system, is contained in platelet dense granules (DG). Peripheral serotonin aids in vasoconstriction and blood clotting. Individuals prone to syncope may be deficient in serotonin. Insufficient peripheral serotonin may create conditions that lead to hypotension and excessive bleeding in an individual.We previously reported that patients having episodes of syncope (Blood, 112(11):451, 2008) had statistically significant decreases of DG/PL, consistent with DG storage pool deficiency (δSPD), and PL 5HT concentrations when compared to control subjects. Our test subjects were screened by questionnaire to determine syncope and distinction between NCS and POTS was not considered. These patients had an average of 3.08 ± 0.52 DG/PL and a mean 5HT concentration of 248.40 ± 37.94 ng/109 PL (PL 5HT normal range = 215-850 ng/109 PL). Control subjects (n=24) had an average of 4.22 ± 0.12 DG/PL and a 5HT concentration of 666.54 ± 56.25; both values having a significant difference (p=.003 and p<.001) by a Mann-Whitney Rank Order test respectively. We now report the DG/PL and 5-HT values obtained exclusively from patients diagnosed with POTS by tilt-table assessment and compared with control subjects.Patients for this study were obtained from our cardiology clinic and all were positive for POTS; they were not currently taking or were recently prescribed a SSRI for therapeutic management of their condition. Control subjects did not have syncope or history of bleeding. Peripheral blood was collected and PLs obtained via centrifugation to determine the mean DG/PL by electron microscopy and the mean 5HT concentration extracted from PLs using an ELISA technique. POTS patients (n=14) had a mean serotonin level of 326.6 ± 76.7 ng/109 PL compared to 589.6 ± 56.3 ng/109PL (p<0.004) for control subjects (n=21). POTS patients exhibited a mean of 2.6 ± 0.31 DG/PL compared to the control subject value of 4.16 ± 0.09 DG/PL (p<0.001). To date there is no significant difference in the number of PL DG or 5HT levels POTS patients compared to our previous evaluation of undefined syncope patients. These results support the hypothesis that syncope (NCS and POTS) may be the result of low peripheral serotonin levels. Our preliminary data demonstrates that POTS patients have low serotonin levels and δSPD when compared to control subjects. This study is ongoing to increase the total number of POTS patients enrolled as well as to recruit subjects diagnosed with NCS to determine whether a difference in DG and/or 5HT exists to explain either bradycardia or tachycardia as an early symptom in the development of hypotension. Disclosures:No relevant conflicts of interest to declare.

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Monoamine variability in the chronic model of atypical absence seizures

We studied the variability of the slow-spike-and-wave discharges (SSWDs) derived from AY-9944 (AY) treatment during brain development of Long-Evans hooded (LEh) rats. Although all LEh rats received the standard dose of AY (7.5 mg/kg), we have observed an intersubject variability of the total SSWD duration at postnatal day (P) 55. Therefore, we set out to investigate the underlying brain levels of norepinephrine (NE), dopamine (DA), and serotonin (5-HT) and its metabolite (5-HIAA), as determined by high-performance liquid chromatography (HPLC) analyses from four different brain regions: thalamus (Th), frontoparietal cortex (Cx), hippocampus (Hp), and brainstem (Bs). All brains were obtained after two baseline electrocorticographic (ECoG) recordings with characteristic chronic, recurrent, bilaterally synchronous 4-6 Hz SSWD, at P 55 (336.25 +/- 97.23 s/h) and P60 (494.50 +/- 150.36 s) (r = 0.951, r(2) = 0.904, p < 0.005, Pearson product). The thalamic NE levels and the brainstem NE, DA, and 5HT levels were all significantly correlated with baseline SSWD duration at P55 and P60 (p < 0.01, Pearson product). Our data indicate that brain monoamine levels may determine the intersubject variability of SSWD duration in AY rats with chronic atypical absence seizures.

Platelet Activity In Elevated Plasma Seretonin Level

The serotonin transporter (SERT) on the platelet surface is the major mechanism for the uptake of plasma serotonin (5‐hydroxytryptamine; 5HT), which subsequently is stored in platelet dense granules. The 5HT uptake capacity of the platelets depends on the number of SERT molecules on the plasma membrane.Our earlier studies demonstrated the presence of a dynamic relationship between plasma 5HT elevation, loss of surface SERT, and depletion of platelet 5HT. However it is not clear whether decreased SERT density on the platelet plasma membrane is a cause or consequence of increased plasma 5HT levels.In the current study, we addressed this issue by using two model systems: (a) 5HT‐pretreated platelets isolated from untreated C57BL/6J mice, and (b) platelets isolated from C57BL/6J mice in which plasma 5HT was elevated chronically by osmotic minipump. Our data in both model systems show a profound loss of surface SERT expression in platelets, depletion of platelet 5HT content and enhanced aggregation response to fibrillar collagen, demonstrating that 5HT‐depleted platelets appear to aggregate more readily. Furthermore, the aggregation rate of platelet is rescued by blocking the uptake ability of platelet SERT with serotonin reuptake inhibitor (SSRI), SERT blocker. These studies explicitly indicated that SERT down‐regulation by high extracellular 5HT in isolated platelets occurs in vivo and impacts platelet function against further aggregation in a protective feedback mechanism.Overall, these studies addressed that the intracellular 5HT concentration has a significant role in promoting hypercoagulation than the plasma 5HT‐level. The importance of understanding the structure, function, and regulation of SERT is underscored by the observations that plasma 5HT is elevated in the plasma, either locally or globally, during atherosclerosis, hypertension, stroke, and other cardiovascular diseases.

Sex-specific 24-h profile of extracellular serotonin levels in the medial prefrontal cortex

The medial prefrontal cortex (mPFC) controls emotional responses in many species, receiving serotonergic innervation from the dorsal and median raphe nucleus (DRN and MRN). To examine the sex difference in 24-h profiles of extracellular serotonin (5HT) levels in the mPFC, an in vivo microdialysis study was performed using intact male, diestrous female, and proestrous female rats. Dialysates were automatically collected by a microdialysis probe from the mPFC every 30 min for more than 24 h under freely moving conditions. The levels of 5HT in dialysates were quantified by high performance liquid chromatography. Extracellular 5HT levels exhibited episodic changes in the mPFC of both sexes of rats, with both diestrous and proestrous females exhibiting a clear diurnal change; the 5HT levels were high during the dark phase, but low during the light phase. In contrast, male rats exhibited relatively high 5HT levels throughout the day without significant diurnal changes. At mathematically analyzed trough, males showed higher 5HT levels than diestrous or proestrous females. The overall 24-h 5HT levels in males were significantly greater than proestrous females, but were not different from diestrous females. Further, stereological methods were used to examine the number of tryptophan hydroxylase (TrpH), but no sex differences in the number of TrpH immunoreactive cells in the DRN and MRN were observed. These results suggest that sex and/or the gonadal steroid environment may affect the 24-h profile of extracellular 5HT in the mPFC of rats without changes in the number of 5HT neurons in the DRN and MRN.

Dynamics of platelet mobilisation into lungs in response to 5-hydroxytryptamine (serotonin) in mice

In experimental animals, the lung rapidly removes intravenously injected 5-hydroxytryptamine (5HT), but the mechanism underlying this pulmonary 5HT removal (P-5HT-R) and the responsible cells remains unclear. 5HT reportedly induces rapid pulmonary platelet accumulation (P-PLT-A). Here, we examined the relationship between P-5HT-R and P-PLT-A in mice by comparing the platelet count in the blood with the endogenous 5HT in the tissues (a marker for platelets because the 5HT is largely contained within platelets). 5HT levels in murine blood and tissues were also examined after intravenous injection of 5HT. The data revealed that: (i) 5HT injection (at > or = 0.04 micromol/kg) induced a transient P-PLT-A (occurring within 6 seconds), (ii) platelets rapidly took up injected 5HT, (iii) the P-5HT-R was saturated following injection of 5HT at 1 micromol/kg, (iv) ketanserin (5HT(2)-receptor antagonist) strongly inhibited P-PLT-A, (v) under fluoxetine (5HT-uptake inhibitor), 5HT levels at 6 seconds after 5HT injection were markedly higher in blood, but significantly lower in lung (versus fluoxetine-untreated mice), (vi) P-5HT-R was barely detectable in mutant mice with platelets lacking dense bodies, and was much reduced in platelet-depleted mice, (vii) 5HT injected intravenously at 10 micromol/kg had a half-life in the lung of < 20 seconds, and (viii) unlike 5HT, injected histamine was largely excreted by the kidney. These results demonstrate that platelets rapidly translocate into the lung upon stimulation of 5HT(2) receptors, take up 5HT (and possibly swiftly metabolise it), and then return to the circulation. Hence, pulmonary platelet accumulation plays an important role in pulmonary 5HT removal in mice.

Alteration of Brain Neurotransmitters in Female Rat Offspring Induced by Prenatal Administration of 16 and 64 mg/kg of 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153)

PCB153 (2,2',4,4',5,5'-hexachlorobiphenyl), a non-coplanar PCB and the congener most widely distributed in the environment, was orally administered to pregnant Sprague-Dawley (Crj: CD (SD) IGS) rats from gestation day 10 through 16 at doses of 0 (control), 16 and 64 mg/kg body weight. Female pups were sacrificed at 1, 3, 6, and 9 wk, and at 1 yr of age to evaluate the differences in brain neurotransmitters and their metabolites between PCB153-exposed and control groups. Brain levels of norepinephrine (NE), 3-methoxy-4-hydroxyphenylglycol (MHPG), dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), serotonin (5HT), 5-hydroxyindoleacetic acid (5HIAA), acetylcholine (ACh), and choline (Ch) in discrete brain regions or in whole brain were measured. At 1 to 3 wk after birth, brain levels of DA, DOPAC, HVA, 5HT and 5HIAA in PCB-exposed groups were higher than those of the control group. At 9 wk after birth, DA turnover was reduced in half of the four brain areas examined (forebrain and hindbrain), and 5HIAA levels were increased in all brain areas in the PCB-treated group compared to those of the control group. At 1 yr after birth, the levels of DA, DOPAC, and HVA in the hippocampus, hypothalamus, and medulla oblongata were lower in the PCB-exposed groups than in the control group. Prenatal exposure to PCB153 stimulated the turnover of 5HT neurons in the brain of female offspring at early stages (1 to 9 wk) of development. On the other hand, the turnover of DA neurons in the PCB-exposed groups was reduced in late stages (9 wk to 1 yr) of development compared with that of the control group. The brain neurotransmitters of dams treated with PCB were assayed at 3 wk after delivery (15 wk old), and decreases in DA, DOPAC, and HVA were observed. PCB153 reduced the activity of DA neurons in the brain of dams. These results are discussed in relation to health effects observed in humans exposed to PCBs.

Metabolic state, neurohormones, and vagal stimulation, not increased serotonin, orchestrate postprandial drowsiness

Several theories have arisen to explain postprandial sleepiness. Of these, the most prevalent theory assumes that blood flow is redistributed after a meal. This premise fails, however, because cardiac regulation strictly controls cerebral blood flow (CBF) and brain oxygenation. Another theory proposes that an elevated ratio of free l-tryptophan to large neutral amino acids (LNAA) in the blood increases cerebral serotonin (5HT) levels, which in turn induces drowsiness. But this theory does not explain why fatty meals, which reduce extracellular 5HT, induce more intense sleepiness than meals of carbohydrates, or why protein-rich meals, which actually lower cerebral 5HT, do not differ significantly from carbohydrate meals in promoting sleep. Some studies fail to confirm the presumptive role of 5HT in mood or drowsiness. Reviewing the history of theory and experimentation in this field, we conclude that 5HT is not the principal determinant in postprandial sleepiness. We propose instead that the arcuate nucleus (ARC) modulates satiety in response to metabolic indicators of energy state, postprandial neuropeptide secretion from the gut, and vagus nerve stimulation. The ARC integrates these satiety signals and forwards them to the ventromedial hypothalamus (VMH), which indirectly stimulates the sleep centers (i.e., the ventrolateral preoptic nucleus (VLPO) and median preoptic nucleus (MnPO)) by inhibiting the lateral hypothalamic area (LHA), which coordinates arousal centers. Neuropeptides or satiety signals may activate sleep centers directly to provoke postprandial somnolence. This model may resolve contradictions and inconsistencies in data that previous theories could not explain.

Neurocardiogenic Syncope Patients Often Have Platelet Delta Granule Storage Pool Deficiency and Decreased Platelet Serotonin Levels.

Neurocardiogenic, or vasovagal syncope as it is also known, is a condition that may lead to a temporary loss of consciousness. It is thought to be caused primarily by a lack of blood flow to the brain. The condition, commonly referred to as “fainting” or “passing out”, is reported to be the basis for 3% of emergency room visits and 6% of hospital admissions. The pathogenesis of syncope is yet to be completely understood but it has been postulated that a sudden failure of the autonomic nervous system's ability to maintain blood pressure and heart rate leads to insufficient cerebral perfusion. A “hyper-contractile” cardiac state is created due to excessive venous pooling and a diminished peripheral venous return is thought to activate afferent mechanoreceptors, subsequently causing a paradoxical reflex of bradycardia, that results in a drop of peripheral vascular resistance. It has been postulated that an increase in serotonin (5HT) activity in the central nervous system acts as an inhibitor of sympathetic stimulation, preventing the symptoms of syncope. One of a number of pharmacologic approaches to the management of neurocardiogenic syncope is the use of selective serotonin reuptake inhibitors (SSRIs).We commonly evaluate patients having undiagnosed bleeding symptoms for PL DG SPD, many of which have been diagnosed with neurocardiogenic syncope; we have previously reported laboratory results from a total of 358 subjects having neurocardiogenic syncope (Blood 110(11):945A, 2007). In that study, eighty-two percent (293/358) of the patients had DG SPD, with an average of 2.27 ± 0.06 DG/PL (normal: 4–6 DG/PL). 98% (351/358) of the patients were female. Common bleeding symptoms included 53.8% with menorrhagia (189/351), 43.9% (157/358) with easy bruising, and 15.6% with epistaxsis (56/358). Neurocardiogenic syncope has been reported to be a low 5HT disorder. The major storage pool of 5HT in the circulation is known to exist in platelet (PL) dense granules (DGs) and therefore it is possible that a PL dense granule storage pool deficiency (DG SPD) could be an important mediator of neurocardiogenic syncope. Our ongoing study of neuocardiogenic syncope patients now includes the ELISA results for 5HT concentration extracted from PLs of 38 subjects. These patients had an average of 3.08 ± 0.52 DG/PL and a mean 5HT concentration of 248.40 ± 37.94 ng/109 PL (PL 5HT normal range = 215– 850 ng/109 PL). Control subjects (n=24) had an average of 4.22 ± 0.12 DG/PL and a 5HT concentration of 666.54 ± 56.25. Both DG/PL and 5HT values are significantly different (p=.003 and p<.001 respectively) for syncope and control subjects by a Mann-Whitney Rank Order test. Patients taking SSRIs were excluded from the study (both controls and syncope patients) as the 5HT concentrations found in 7 “normal” subjects using a SSRI were significantly decreased (127 ± 64.51 ng/109 PL).Our results suggest that a significant number of patients having neurocardiogenic syncope have DG SPD and low concentrations of 5HT stored in these granules. These results may be related to the fact that most subjects included in our study have a history or symptom of bleeding. The mechanism of 5HT uptake by the platelet is independent of adenine nucleotide and calcium storage within DGs, thus the low 5HT levels found in the 38 cases described here may/may not be related to pathophysiology of neurocardiogenic syncope.

Effects of a cannabinoid receptor (CB) 1 antagonist AM251 on behavioral sensitization to nicotine in a rat model of novelty-seeking behavior: correlation with hippocampal 5HT

There are marked individual differences in the efficacy of mainstream nicotine cessation agents in preventing relapse. A rat model of novelty-seeking phenotype was reported to have predictive value for psychostimulant taking behavior where locomotor reactivity to novelty is used to rank high (HR, highest 1/3) versus low (LR, lowest 1/3) responsiveness to novelty in outbred rats. We tested the hypothesis that a cannabinoid receptor (CB) 1 antagonist that is in clinical trials for smoking cessation may reverse behaviorally sensitizing effects of nicotine in HRs and repeated nicotine-induced elevations in hippocampal 5HT. Adolescent LRHR rats underwent intermittent behavioral sensitization to nicotine regimen with or without a CB1 receptor antagonist AM251 or bupropion treatment following nicotine training during 1 week of nicotine-free period. Expression of behavioral sensitization to nicotine was assessed in response to a low-dose nicotine challenge. Using the same sensitization regimen and therapeutic treatments, hippocampal 5HT levels were measured via in vivo microdialysis in response to the nicotine challenge. HR but not LR animals showed behavioral sensitization to a low-dose nicotine challenge following intermittent nicotine training and 1 week of injection-free period. AM251 (5 mg/kg, i.p.) but not bupropion administration during injection-free period successfully reversed locomotor sensitization to nicotine challenge in HRs. AM251 treatment also reversed nicotine-induced elevations in extracellular 5HT in the HR hippocampal hilus. These data suggest that CB1 antagonists may prevent locomotor sensitization to nicotine and reverse nicotine-induced elevations in hippocampal 5HT in high novelty seekers.