Costorage of serotonin binding protein with serotonin in the rat CNS

Previous studies have identified two neurectoderm-specific serotonin binding proteins (SBP), one with an apparent Mr of 45 kDa, and one of 56 kDa. The current experiments were undertaken to test the hypothesis that these proteins are specific components of serotonergic neurons. Since actin has been found to bind serotonin, the relationship of the 2 forms of SBP to actin was also investigated. Antisera against purified 45 and 56 kDa SBP were raised in rabbits and shown by analyses of immunoblots and differential absorption to be monospecific and not cross-reactive. Neither antiserum reacted with purified actin and none of 3 different anti-actin sera reacted with purified 45 or 56 kDa SBP. The antisera to 45 and 56 kDa were used for immunocytochemical localization of the proteins, which was compared to that of serotonin. SBP immunoreactivity was found in rat brain and spinal cord; however, no significant differences were observed in the pattern of distribution of 45 and 56 kDa SBP-immunoreactive structures. Immunostaining of neuronal perikarya by either SBP antiserum required pretreatment of animals with colchicine. The distribution of neurons and terminals labeled by each antiserum to SBP was similar to that of neurons and terminals labeled by anti-5-HT sera. SBP-immunoreactive neuronal perikarya were present in the nuclei raphe dorsalis, raphe centralis superior, raphe medianus, raphe magnus, raphe obscurus, raphe pallidus, dorsal to the medial lemniscus in the region of the B9 cell group, near the interpeduncular nucleus, in the area postrema, the pars compacta of the substantia nigra, the dorsomedial nucleus of the hypothalamus, and the arcuate nucleus. SBP-immunoreactive fibers and terminals were present in many additional areas of the brain, as well as the spinal cord, where they paralleled those that were immunostained with antibodies to 5-HT. When double-immunostaining was used, serotonin and 45 and 56 kDa SBP immunoreactivities were found to be colocalized in both the brain and spinal cord. Cells and fibers found to be stained by one immunoreagent were also stained by the others; therefore, serotonergic neurons of the CNS probably contain both 45 and 56 kDa SBP. Moreover, it also seems likely that nonserotonergic neurons contain neither form of SBP. These data strongly suggest that SBP is an intrinsic and specific component of serotonergic neurons that can serve as a serotonergic marker.     

The influence of selective serotonin reuptake inhibitors on human platelet serotonin

Clinical depression has been proposed to be an independent risk factor for cardiovascular disease. While it is suggested that selective serotonin reuptake inhibitors (SSRIs) reduce the risk of acute cardiovascular problems of depressed patients, the effect of SSRIs on platelets, the only blood cells committed to serotonin (5-HT) transport, remains largely unknown.The goal of this pilot study was to measure the 5-HT levels in platelets of untreated and SSRI-treated depressed patients and normal subjects and to determine whether the interaction of SSRIs with platelets can explain their possible cardiovascular benefit in patients with depression. Platelet 5-HT was determined by an immunocytochemical assay and high-pressure liquid chromatography with electrochemical detection (HPLC-ECD). In normal control subjects without cardiovascular disease, 78 +/- 8% of platelets were 5-HT-positive (n = 14). Depression caused a significant reduction in platelet 5-HT to 46 +/- 21% in untreated patients (n = 13) and 22 +/- 13% in SSRI-treated patients (n = 14). As a class, all selective serotonin reuptake inhibitors significantly reduced the 5-HT concentration in patient platelets. An inverse relationship of 5-HT level and dose of medication might be suggested.These results correlated well with 5-HT data from HPLC (r = 0.8509, p < 0.001). SSRIs did not affect platelet aggregation and dense granule release in response to thrombin, but significantly reduced ADP-induced platelet aggregation and dense granule release in both patient and normal control samples. The active inhibition of platelet aggregation by SSRIs might explain their cardiovascular benefit.     

Involvement of serotonin in depression: evidence from postmortem and imaging studies of serotonin receptors and the serotonin transporter

Definitive conclusions on the role of serotonin receptors and transporter in suicide and depression have been elusive in studies of postmortem brain tissue. A number of methodological differences in these studies have made it difficult to reach a consensus, but crucial issues are being identified and incorporated into newer studies. This review will follow the evolution of serotonin receptor and transporter studies in postmortem tissues that initially focused on suicide and gradually incorporated depressive disorders as psychiatric assessments were increasingly performed. Studies in postmortem tissues on the serotonin-1A and serotonin-2A receptors and the serotonin transporter will be reviewed and compared with imaging studies of the same sites in depressed subjects. Critical issues to control in future studies of serotonin receptors in postmortem tissues include variables such as the cause of death (i.e. suicide), the specific psychiatric diagnoses of the subjects, whether the disorder was in remission at the time of death, long-term medication histories, psychoactive substance use disorders, the smoking history, the hemisphere from which tissues were dissected, and the specific cytoarchitectonic region to be evaluated. Carefully controlled studies in postmortem tissues will ensure a greater likelihood of reaching a consensus on the involvement of monoamine measures in the etiology of depression.     

Chronic cocaine enhances serotonin autoregulation and serotonin uptake binding.

Repeated cocaine intoxication can result in the development of behavioral sensitization in animals and psychosis in humans, phenomena that have been associated with alterations in dopamine (DA) function. Using electrophysiologic and autoradiographic techniques, modifications of central serotonin (5-hydroxytryptamine; 5-HT) systems were investigated in rats treated with a regimen of cocaine administration that produced behavioral sensitization. The inhibitory response of single 5-HT neurons in the dorsal raphe (DR) to (-)-cocaine, the 5-HT uptake inhibitor fluoxetine or the 5-HT1A agonist 8-hydroxy-2-[di-N-propylamino]tetralin (8-OHDPAT) was significantly enhanced in cocaine-treated rats. Furthermore, several brain areas that contain either cell bodies (DR) or terminals for 5-HT (medial and sulcal prefrontal cortex, frontal cortex) showed cocaine-induced elevations in [3H]imipramine-labeled 5-HT uptake sites, while [3H]-8-OHDPAT-labeled 5-HT1A receptors were decreased only in the central medial amygdala. These results suggest that modifications of autoregulatory mechanisms secondary to alterations of 5-HT uptake processes may contribute to the development of cocaine sensitization.     

Experimental serotonin myopathy

One hundred and eight Wistar rats were injected with serotonin (20 mg per kilogram of body weight intraperitoneally) or imipramine hydrochloride (20 mg per kilogram intraperitoneally), or both, in a single cycle or in multiple (up to 18 weeks) weekly cycles. In contrast to previous reports, a characteristic myopathy was produced with serotonin alone, identical to that produced by serotonin and imipramine in combination. Imipramine alone produced no significant change. The myopathy produced was characterized by (1) preferential damage to myofibers with high oxidative capacity (types I and IIA), (2) prominent regenerative activity occurring as early as 48 hours, and (3) degeneratio of capillary endothelium (thickening, vacuolar degeneration, proliferation of marginal folds, dissolution of mitochondria). Small groups of degenerating fibers and increased connective tissue were not observed. There was no loss of myofibers or fat replacement. The changes suggested repeated acute insults followed the complete recovery. These observations suggest that although the pathogenesis of serotonin and serotonin-imipramine myopathy is primarily ischemic, it is not a satisfactory model of human Duchenne muscular dystrophy.     

Fevarin--a selective serotonin uptake inhibitor. Clinical implications of serotonin selectivity]

The author explains the development of specific inhibitors of serotonin re-uptake. Fluvoxamine differs from classical tricyclic antidepressants by a pharmacological profile of side-effects and safety of overdosage, while the therapeutic effect is comparable. The serotonin selectivity opens perspectives of further indications.     

The serotonin syndrome

The serotonin-syndrome is a possible side-effect in the treatment with serotonergic drugs. There are diagnostic criteria for diagnosis of this syndrome. After discontinuation of administering the serotonergic drug is fully reversible.     

Mirtazapine-induced serotonin syndrome

An 85-year-old woman developed sudden confusion and dysarthria progressing to mutism, orobuccal dyskinesias, generalized tremors worse with activity, ataxia, and rigidity with cog wheeling without high-grade fevers or dysautonomia. These findings were related temporally to the institution of mirtazapine as monotherapy for a major depressive illness with superimposed anxiety disorder. Withdrawal of the agent resulted in early notable clinical resolution with only residual hypertonia after 2 weeks. This is a rare report of serotonin syndrome induced by mirtazapine monotherapy. The hypothesized pathophysiologic mechanism in this case is overstimulation of serotonin (5-hydroxytryptamine or 5-HT) type 1A receptors (5-HT(1A)) in the brainstem and spinal cord in an individual with risk factors for hyperserotoninemia resulting from reduced, acquired endogenous serotonin metabolism.     

The serotonin syndrome

OBJECTIVE AND METHOD: A review of the literature on the serotonin syndrome in animals and human beings was conducted, and 12 reports of 38 cases in human patients were then analyzed to determine the most frequently reported clinical features and drug interactions, as well as the incidence, treatment, and outcome of this syndrome. FINDINGS: The serotonin syndrome is most commonly the result of the interaction between serotonergic agents and monoamine oxidase inhibitors. The most frequent clinical features are changes in mental status, restlessness, myoclonus, hyperreflexia, diaphoresis, shivering, and tremor. The presumed pathophysiological mechanism involves brainstem and spinal cord activation of the 1A form of serotonin (5-hydroxytryptamine, or 5-HT) receptor. The incidence of the syndrome is not known. Both sexes have been affected, and patients' ages have ranged from 20 to 68 years. Discontinuation of the suspected serotonergic agent and institution of supportive measures are the primary treatment, although 5-HT receptor antagonists may also play a role. Once treatment is instituted, the syndrome typically resolves within 24 hours, but confusion can last for days, and death has been reported. CONCLUSIONS: The serotonin syndrome is a toxic condition requiring heightened clinical awareness for prevention, recognition, and prompt treatment. Further work is needed to establish the diagnostic criteria, incidence, and predisposing factors, to identify the role of 5-HT antagonists in treatment, and to differentiate the syndrome from neuroleptic malignant syndrome.     

GABA, serotonin and serotonin receptors in the rat inferior colliculus

GABA and serotonin in the mammalian inferior colliculus both have a restrictive effect on fearful and aversive behavior. Immunohistochemical techniques were used to study the relationship between GABA and serotonin receptors in the central nucleus of the rat inferior colliculus. Neurons positive for 5HT1B are more numerous than those displaying 5HT1A receptors. Approximately two-thirds of GABA-positive neurons are associated with serotonin receptors.     

In vivo electrochemical measurements of serotonin clearance in rat striatum: effects of neonatal 6-hydroxydopamine-induced serotonin hyperinnervation and serotonin uptake inhibitors

Summary Diffusion and clearance of extracellular serotonin (5-HT) was examined using in vivo chronoamperometry with delayed-pulse recordings after pressure ejections of 1 to 60 picomoles 5-HT into rat striatum at a fixed distance from a Nafion-coated carbon fiber electrode. Signals obtained were identified based on the signal characteristics to consist of 5-HT. Clearance times of 5-HT decreased, while amplitudes and rise times increased with serotonergic hyperinnervation induced by neonatal 6-hydroxydopamine (6-OHDA) lesions of dopamine (DA) neurons. Local applications of the 5-HT uptake inhibitors zimelidine or fluoxetine, in conjunction with 5-HT ejections, produced increased clearance times in both normal and 6-OHDA-treated animals. Thus, direct in vivo evidence was obtained for the importance of high affinity nerve terminal uptake as a key mechanism for clearance of 5-HT from the extracellular space. Inhibitors of 5-HT uptake appear to prolong the extracellular presence of 5-HT by increasing its clearance time.     

Organic cation transporter capable of transporting serotonin is up-regulated in serotonin transporter-deficient mice

The serotonin (5HT) transporter (5HTT) regulates serotonergic neurotransmission by mediating the reuptake of 5HT from the synaptic cleft. Although lacking the high affinity and selectivity of the 5HTT, the brain expresses a large number of other transporters, including the polyspecific organic cation transporters (OCTs). OCT1 and OCT3, members of the potential-sensitive organic cation transporter gene family, physiologically transport a wide spectrum of organic cations. In addition, both transporters mediate low-affinity 5HT transport and, therefore, may participate in the clearance of excessive 5HT. Because concentrations of extracellular 5HT are increased in the brain of 5HTT-deficient mice, they are a model for investigating the role of OCTs in 5HT system homeostasis. Here, we analyzed OCT1 and OCT3 gene expression in the brain of 5HTT knockout mice by semiquantitative competitive polymerase chain reaction and in situ hybridization. We demonstrate that, in 5HTT-deficient mice, OCT3 mRNA concentrations were significantly increased in the hippocampus, but not in other brain regions, including cortex, striatum, cerebellum, and brainstem. In contrast, no difference in OCT1 expression was detected between 5HTT knockout and control mice. Up-regulation of OCT3 expression and enhanced low-affinity 5HT uptake may limit the adverse effects of elevated extracellular 5HT and may play a critical role in maintaining 5HT-dependent functions of the hippocampus in the absence of 5HTT.     

Influence of clozapine on platelet serotonin, monoamine oxidase and plasma serotonin levels

The purpose of this study was to investigate the influence of clozapine on plasma serotonin, platelet serotonin and monoamine oxidase (MAO) levels in schizophrenic patients and to compare their results with those of unmedicated healthy controls. Groups of 20 outpatients with schizophrenia and 20 healthy controls matched for age, sex and smoking status were recruited for the study. Psychopathology, neurocognitive functioning, plasma serotonin, platelet serotonin and MAO levels were assessed after 1-week drug free interval, and 8 weeks after initiation of clozapine treatment in an open design. The mean clozapine dose at week 8 was 382.5+/-96.4 (range: 250-600) mg/day. In the patient group, at baseline, plasma serotonin and platelet MAO levels were significantly lower, and platelet serotonin levels were significantly higher than in controls. After 8 weeks of clozapine treatment, plasma serotonin and platelet MAO levels increased significantly, while a significant decrease in platelet serotonin levels was detected compared with baseline values. Baseline platelet MAO levels explained 22% of the variance in Clinical Global Impression - Improvement (CGI-I) and improvement in attention, while baseline platelet serotonin predicted 23% of the variance in the improvement in positive symptoms during clozapine treatment. Our data indicate that clozapine may be reversing or compensating for a pre-existing alteration in serotonergic neurotransmission in schizophrenic patients. The prediction of response to clozapine through peripheral biochemical markers may have important clinical implications if repeated in larger samples.     

Mutational scanning of the human serotonin transporter reveals fast translocating serotonin transporter mutants

The serotonin transporter (SERT) belongs to a family of sodium-chloride-dependent transporters responsible for uptake of amino acids and biogenic amines from the extracellular space. SERT represents a major pharmacological target in the treatment of several clinical conditions, including depression and anxiety. In the present study we have undertaken a mutational scanning of human SERT in order to identify residues that are responsible for individual differences among related monoamine transporters. One mutant, G100A, was inactive in transport. However, ligand binding affinity was similar to wild-type, suggesting that G100A amongst different possible SERT conformations is restrained to a binding conformation. We suggest that the main role of glycine-100 is to confer structural flexibility during substrate translocation. For the two single mutants, T178A and F263C, uptake rates and K(m) values were both several-fold higher than wild-type while binding affinities and inhibitory potencies decreased considerably for several drugs. Ion dependency increased and only at hyperosmotic concentrations were K(m) values partly restored. For the double mutant, T178A/F263C, shifts in uptake kinetics and ligand affinities, as well as ion dependencies, were drastic. Effects were synergistic compared to the corresponding single mutants. In conclusion, we suggest that mutating threonine-178 to an alanine and phenylalanine-263 to a cysteine mainly alter the overall uptake kinetics of SERT by affecting the conformational equilibrium of different transporter conformations.     

Plasticity in serotonin uptake in primary neuronal cultures of serotonin transporter knockout mice

The cross talk between dopaminergic and serotonergic systems in the brain has multiple neurophysiological and behavioral implications. Primary neuronal cultures of embryonic wild type (+/+) and serotonin transporter knockout (-/-) mice were used as a model to elucidate the possibility of plasticity at the level of serotonin uptake. Serotonergic neurons were identified in midbrain-hindbrain cultures of both wild type and knockout mice, using polyclonal anti-serotonin antibodies. Adding serotonin (10 microM) to wild type midbrain-hindbrain cultures increased the intensity of serotonin immunostaining, but did not change the number of serotonergic neurons. This increased intensity of serotonin staining was blocked by the serotonin transporter inhibitors fluoxetine and imipramine, but not with the dopamine transporter inhibitor nomifensine. In serotonin transporter knockout cultures, however, serotonin increased both the intensity of serotonin immunostaining and the number of serotonin positive neurons, and nomifensine decreased the number of serotonin-labeled neurons. Uptake of [3H]serotonin to wild type midbrain-hindbrain cultures was completely blocked by 1 microM fluoxetine, whereas nomifensine had a very small effect. In contrast, [3H]serotonin uptake to serotonin transporter knockout cultures, although very weak, was better inhibited by nomifensine than fluoxetine. The results imply that midbrain-hindbrain neuronal cultures of knockout mice, that do not express serotonin transporters, acquire the capacity to take up serotonin, apparently via dopamine transporters.     

Postnatal changes in serotonin receptors following prenatal alterations in serotonin levels: further evidence for functional fetal serotonin receptors

Recent work by ourselves and others has indicated that serotonin (5-HT) acts as a regulator of neuronal growth in fetal tissue, probably through an action on the high affinity 5-HT receptors known to be present. In order for our hypothesis to be correct, these receptors must be shown to be functional in fetal tissue. Furthermore, since 5-HT has a dual role in neuronal development, these receptors must be functional both in the region of the serotonergic cell body (brainstem) and in projection areas (forebrain). We have tested the functional status of fetal 5-HT receptors by testing their ability to adapt to changes in the level of 5-HT. Pregnant rats were treated with saline, p-chlorophenylalanine (a 5-HT depletor) or 5-methoxytryptamine (a 5-HT agonist) and the characteristics of the high affinity 5-HT receptors in the rat pups determined using a binding assay with [3H]5-HT. Our results show that both forebrain and brainstem receptors respond to alterations in transmitter level in a manner similar to adult brain. Thus, fetal 5-HT receptors are functional and capable of playing a role in neuronal development.     

Effect of destruction of serotonin neurons on basal and fenfluramine-induced serotonin release in striatum

This study examined the relationship between the magnitude of tissue serotonin (5-HT)depletion produced by treatment with the neurotoxin 5,7-dihydroxytryptamine (5,7) and basal and fenfluramine-induced 5-HT release in the striatum. Separate groups of rats were treated with either vehicle or 5,7-DHT (100μ 76% striatal 5-HT depletion; or 200μ 93% styriatal 5-HT depletion). four weeks after treatment 5-HT release was measured in the ventral striatum using in vivo microdialysis in animals anesthetized with chloral hydrate. Basal 5-HT levels were not significantly altered in any lesion group, whereas basal 5-hydroxyindoleacetic acid levels were dosedependently reduced by 5,7-DHT. In contrast, the increase of 5-HT release produced by fenfluramine treatement (10 mg/kg) wa diminished significantly after 5-HT neuronal destruction in correlation with the reduction of striatal tissue 5-HT content. Fractional 5-HT efflux, a measure of the 5-HT release from surviving striatal nerve terminals, was also significantly elevated when tissue depletion of 5-HT exceeded 95%. This study suggests that compensatory mechanisms may enable surviving 5-HT terminals to maintain basal 5-HT levels in th striatum with as little as 5% of the terminals remaining, but those mechanisms are not sufficient to allow the damaged system to respond to a pharmacological challenge. © 1995 Wiley-Liss, Inc.