Serotonin Transporter Immunoreactivity Research Articles

Chronic stress enhances methamphetamine-induced extracellular glutamate and excitotoxicity in the rat striatum.

Striking parallels exist between the neurochemical and toxic effects of stress and methamphetamine. Despite these similarities, no studies have examined how stress may promote the toxic effects of methamphetamine (METH). The current study tested the hypothesis that chronic stress enhances METH toxicity by augmenting glutamate (GLU) release and excitotoxicity in response to METH administration. Adult male Sprague-Dawley rats were exposed to 10 days of unpredictable stress and then received either saline or METH (7.5 mg/kg, i.p., once every 2 h x four injections). Prior exposure to unpredictable stress acutely enhanced the striatal extracellular GLU concentrations in response to METH, and eventually caused proteolysis of the cytoskeleton protein spectrin. Administration of the corticosterone synthesis inhibitor, metyrapone (25 mg/kg, i.p., prior to each stressor), during unpredictable stress attenuated the enhanced striatal GLU release in response to METH, blocked spectrin proteolysis, and attenuated METH-associated toxicity measured by long-term depletions in the dopamine and serotonin tissue content as well as depletions in dopamine and serotonin transporter immunoreactivity of the striatum. In summary, prior exposure to unpredictable stress enhances METH-induced elevations of GLU in the striatum, resulting in long-term excitotoxic damage and an augmentation of damage to dopamine and serotonin terminals. These studies provide a neurochemical basis for how stress contributes to the deleterious effects of METH abuse.

Expression of the Serotonin Transporter (SERT) in the Choroid Plexuses from Buffalo Brain

Choroid plexuses (CPs) play pivotal roles in a wide range of processes that establish, survey, and maintain the biochemical and cellular status of the central nervous system. Mammalian CPs contain a very high density of serotonin receptors, and serotonin has been shown to affect CP functions. The serotonin transporter (SERT) regulates the entire serotonergic system, including serotonin receptors by means of modulation of serotonin concentration in the extracellular fluid. In this study, the expression of SERT in the CPs from the brain of a mammalian species, Bubalis bubalis, was established. By immunogold labeling in scanning electron microscopy, SERT immunoreactivity was found to be localized on the apical surface of the choroid epithelium. In particular, SERT positivity was detected on the apical portion of villi, and both on the membrane and in the cytoplasm of grouped cells on the surface of the choroid epithelium. Significantly, no SERT was detected in blood vessels irrigating the CPs. The expression of SERT mRNA transcripts of 440 bp in the CPs was detected by reverse-transcription polymerase chain reaction, and Western blotting analysis revealed the presence of three isoforms of the protein with molecular masses of approximately 70, 80, and 140 kDa, respectively, probably corresponding to differently glycosylated SERT. Our findings provide the first report of SERT detection in the CPs of buffalo brain and indicate that this protein is locally synthesized from the choroid epithelial cells. We suggest that SERT might have an important role in mammalian CPs, possibly regulating the serotonin flow between brain and rest of the body.

Preferential loss of serotonin markers in caudate versus putamen in Parkinson's disease

Interest in serotonergic involvement in Parkinson's disease (PD) has focussed recently on the possibility that the remaining serotonin neurons innervating striatum (caudate and putamen) might release dopamine as a 'false transmitter'--an action that could have both beneficial and harmful (e.g. promotion of levodopa-induced dyskinesias) consequences. Evidence for a brain serotonergic disturbance in PD is derived in large part from findings of decreased binding of different radioligands to the serotonin transporter (SERT), one 'marker' of serotonin neurons. However, it is not known whether the reported changes in SERT binding reflect actual changes in levels of SERT protein or whether concentrations of all serotonin markers are similarly and markedly decreased in the two striatal subdivisions. We measured levels of SERT immunoreactivity, and for comparison, protein levels of tryptophan hydroxylase (TPH; the marker synthetic enzyme) using a Western blot procedure, as well as concentrations of serotonin, its metabolite 5-hydroxyindoleacetic acid (5-HIAA), and dopamine by HPLC in post-mortem striatum of patients with PD and normal controls. Whereas concentrations of dopamine were severely decreased (caudate, -80%; putamen, -98%) and showed little (caudate) or no (putamen) overlap between individual control and patient values, levels of all four serotonin markers were less markedly reduced (-30% to -66%) with some patients having distinctly normal levels. Unlike the preferential loss of dopamine in putamen, the caudate was affected more than putamen by loss of all serotonin markers: serotonin (-66% versus -51%), 5-HIAA (-42% versus -31%), SERT (-56% versus -30%) and TPH (-59% versus -32%). Striatal serotonin concentration was similar in the subset of patients reported to have had dyskinesias versus those not reported to have had this drug complication. Previous findings of decreased SERT binding are likely explained by loss of SERT protein. Reduced striatal levels of all of the key serotonergic markers (neurotransmitter and metabolite, transporter protein, synthesizing enzyme protein) provide strong evidence for a serotonergic disturbance in PD, but with some patients affected much more than others. The more marked caudate reduction suggests that raphe neurons innervating this area are more susceptible to 'damage' than those innervating putamen and that any functional impairment caused by striatal serotonin loss might primarily involve the caudate. Questions related to the, as yet undetermined, clinical consequences in PD of a striatal serotonin deficiency (caudate: cognitive impairment?) and preservation (putamen: levodopa-induced dyskinesias?) should be addressed in prospective brain imaging and pharmacological studies.

Expression of serotonin transporters by peripheral blood mononuclear cells of rhesus monkeys ( Macaca mulatta)

It has been well established that serotonin (5-hydroxytryptamine, 5-HT) plays a key role in neuro-endocrine-immune networks, mostly through its receptors and/or transporters. Although the presence of 5-HT receptor mRNAs in peripheral blood mononuclear cells (PBMCs) of rhesus monkeys has been reported, there is little information about serotonin transporter (SERT) expression by these cells. To examine SERT expression at the transcription and translation level, one-step RT-PCR, confocal microscopy and flow cytometry were used to detect SERT mRNA and protein expression by rhesus monkey PBMCs. It was found that SERT mRNA could be detected by RT-PCR from all of the rhesus macaque PBMC RNA samples and the nucleotide sequence of the amplicons was identical to the published SERT mRNA sequence. Low level SERT immunoreactivity was also demonstrated on the surface of rhesus PBMCs by confocal microscopy. Almost all lymphocytes and most monocytes were positive for SERT by flow cytometry. In the 2 rhesus macaques examined by multicolor flow cytometry, SERT bright cells were more than 84%, 94%, and 96% among CD20+, CD3+, and CD3+CD4+ lymphocytes respectively. These data demonstrate expression of SERT by rhesus macaque PBMCs, and indicate that rhesus macaques would be suitable models to test the in vivo immune regulatory effects of 5-HT or drugs targeting SERT.

High Mucosal Serotonin Availability in Neonatal Guinea Pig Ileum Is Associated With Low Serotonin Transporter Expression

5-Hydroxytryptamine (5-HT) is a neurotransmitter and paracrine signaling molecule in the gut. Paracrine signaling by enterochromaffin cells (EC), which release 5-HT, has not been studied in neonates. Our aim was to compare 5-HT disposition in the intestinal mucosa of neonatal and adult guinea pigs. 5-HT was locally measured in vitro from intestinal segments using a diamond microelectrode and continuous amperometry. The serotonin transporter (SERT) was measured using immunohistochemical and Western blot techniques. 5-HT intestinal content was measured using immunohistochemistry and high-performance liquid chromatography with electrochemical detection. An oxidation current, reflective of local 5-HT release, was recorded with the microelectrode near the mucosal surface, and this current was larger in neonatal than in adult tissues. Mechanically stimulating the mucosa with a fine glass probe evoked an additional current in adult but not neonatal tissues. Oxidation currents were reduced by tetrodotoxin and were blocked in calcium-free solutions. Fluoxetine (1 microM) potentiated oxidation currents in adult but not neonatal tissues. SERT levels were lower in neonatal vs adult tissues. There was no difference in 5-HT content between neonates and adults but 5-hydroxyindole acetic acid/5-HT ratios were higher in adults. EC cell counts showed no difference in cell number, but EC cells were found in the crypts in neonatal and along the villi in adult tissues. SERT expression is low in neonates, and this is associated with high levels of free mucosal 5-HT and reduced metabolism. Postnatal maturation of 5-HT signaling may be important for development of neurohumoral control of intestinal motor reflexes.

Persistent alterations to enteric neural signaling in the guinea pig colon following the resolution of colitis

Functional changes induced by inflammation persist following recovery from the inflammatory response, but the mechanisms underlying these changes are not well understood. Our aim was to investigate whether the excitability and synaptic properties of submucosal neurons remained altered 8 wk post-trinitrobenzene sulfonic acid (TNBS) treatment and to determine whether these changes were accompanied by alterations in secretory function in submucosal preparations voltage clamped in Ussing chambers. Mucosal serotonin (5-HT) release measurements and 5-HT reuptake transporter (SERT) immunohistochemistry were also performed. Eight weeks after TNBS treatment, colonic inflammation resolved, as assessed macroscopically and by myeloperoxidase assay. However, fast excitatory postsynaptic potential (fEPSP) amplitude was significantly increased in submucosal S neurons from previously inflamed colons relative to those in control tissue. In addition, fEPSPs from previously inflamed colons had a hexamethonium-insensitive component that was not evident in age-matched controls. AH neurons were hyperexcitable, had shorter action potential durations, and decreased afterhyperpolarization 8 wk following TNBS adminstration. Neuronally mediated colonic secretory function was significantly reduced after TNBS treatment, although epithelial cell signaling, as measured by responsiveness to both forskolin and bethanecol in the presence of tetrodotoxin, was comparable with control tissue. 5-HT levels and SERT immunoreactivity were comparable to controls 8 wk after the induction of inflammation, but there was an increase in glucagon-like peptide 2-immunoreactive L cells. In conclusion, sustained alterations in enteric neural signaling occur following the resolution of colitis, which are accompanied by functional changes in the absence of active inflammation.

Subcellular Redistribution of the Serotonin Transporter by Secretory Carrier Membrane Protein 2

The serotonin transporter (SERT) belongs to the SLC6 family of sodium- and chloride-dependent neurotransmitter transporters responsible for uptake of amino acids and biogenic amines from extracellular spaces. Their activities and subcellular distributions are regulated by various cellular mechanisms, including interactions with other proteins. Using the yeast two-hybrid approach we screened a human brain cDNA library and identified secretory carrier membrane protein 2 (SCAMP2) as a novel SERT-interacting protein. GST-pulldown assays confirmed the physical interaction between SCAMP2 and the N-terminal domain of SERT. In addition, SERT was found to form a complex with SCAMP2 as demonstrated by co-immunoprecipitation from a heterologous expression system and from rat brain homogenate. Co-expression of SERT and SCAMP2 in mammalian cells results in the subcellular redistribution of SERT with a decrease in cell surface SERT and a concomitant reduction in 5-HT uptake activity. Using confocal microscopy we show that in neuronal cells endogenous SERT co-localizes with SCAMP2 in discrete structures also containing the lipid raft marker flotillin-1 and the SNARE protein syntaxin 1A. In contrast, SERT immunoreactivity is clearly segregated from transferrin receptor-containing endosomes. A single amino acid mutation, cysteine-201 to alanine, within the conserved cytoplasmic E peptide of SCAMP2, abolished SCAMP2-mediated down-regulation of SERT, although this mutation had no effect on the physical interaction between SERT and SCAMP2. Taken together, our results suggest that SCAMP2 plays an important role in the regulation of the subcellular distribution of SERT.

Differences in chemo‐ and cytoarchitectural features within pars principalis of the rat anterior olfactory nucleus suggest functional specialization

The anterior olfactory nucleus (AON) lies between the olfactory bulb and piriform cortex and is the first bilaterally innervated structure in the olfactory system. It is typically divided into two subregions: pars externa and pars principalis. We examined the cytoarchitecture of pars principalis, the largest cellular area of the region, to determine whether it is homogeneously organized. Quantitative Nissl studies indicated that large cells (cell body area >2 standard deviations (SD) larger than the mean cell size) are densest in lateral and dorsolateral regions, while small cells (>1 SD smaller than the mean) are more numerous in medial and ventral areas. Further evidence for regional differences in the organization of the AON were obtained with immunohistochemistry for calbindin (CALB), parvalbumin (PARV), glutamic acid decarboxylase (GAD), and choline transporter (CHT). Cells immunopositive for CALB (CALB+) were denser in the deep portion of Layer II, although homogeneously dispersed throughout the circumference of the AON. PARV+ cells were located in the superficial half of Layer II and were sparse in ventral and medial regions. CHT+ and GAD+ fibers were denser in lateral versus medial regions. No regional differences were found in GAD+ somata, or in norepinephrine transporter or serotonin transporter immunoreactivity. The observed regional differences in cyto- and chemoarchitectural features may reflect functional heterogeneity within the AON.

Serotonin transporter function and expression are reduced in mice with TNBS‐induced colitis

Regulated release of serotonin (5-HT) from enterochromaffin (EC) cells activates neural reflexes that are involved in gut motility, secretion, vascular perfusion and sensation. The 5-HT-selective reuptake transporter (SERT) terminates serotonergic signalling in the intestinal mucosa. The aim of this investigation was to determine whether mucosal 5-HT content, release, and/or reuptake are altered in a murine model of immune cell-mediated colitis. Experiments were conducted 6 days after colitis was induced by 2,4,6-trinitrobenzene sulfonic acid, a time point when macroscopic and histological damage scores indicated significant inflammation. During inflammation, SERT transcript levels and immunoreactivity were reduced, and the uptake of [3H] 5-HT was impaired. Increases in mucosal 5-HT content and the number of 5-HT-immunoreactive mast cells in the lamina propria were also detected in the inflamed region, whereas EC cell numbers did not change. Mucosal 5-HT released under basal and stimulated conditions was unchanged in animals with colitis. These data suggest that murine colitis alters 5-HT signalling by increasing 5-HT availability through decreased 5-HT uptake by mucosal epithelial cells. These findings support the concept that altered 5-HT signalling could be a contributing factor in altered gut function and sensitivity in inflammatory bowel disease.

Enteroendocrine cells and 5-HT availability are altered in mucosa of guinea pigs with TNBS ileitis.

Enteroendocrine cells act as sensory transducers, releasing 5-HT and numerous peptides that are involved in regulating motility, secretion, and gut sensation. The action of mucosal 5-HT is terminated by a 5-HT reuptake transporter (SERT). In this study, we examined the hypothesis that ileitis leads to changes in enteroendocrine cell populations and mucosal 5-HT availability. Ileitis was induced in guinea pigs by intraluminal injection of 2,4,6-trinitrobenzenesulfonic acid and experiments were conducted 3, 7, and 14 days after treatment. The number of somatostatin, neurotensin, and 5-HT-immunoreactive cells increased at 3 and 7 days of ileitis, respectively, whereas no significant changes in the numbers of cholecystokinin, glucagon-like peptide-2, glucose-dependent insulinotropic peptide, and peptide YY-immunoreactive cells were observed. Chemical stimulation of the inflamed mucosa with sodium deoxycholic acid significantly increased 5-HT release compared with basal release. Mechanical stimulation of the mucosa potentiated the effect of the chemical stimuli at day 7. Epithelial SERT immunoreactivity was significantly reduced during the time course of inflammation. Thus changes in enteroendocrine cell populations and 5-HT availability could contribute to the altered motility and secretion associated with intestinal inflammation by disrupting mucosal signaling to enteric nerves involved in peristaltic and secretory reflexes.

Molecular defects in mucosal serotonin content and decreased serotonin reuptake transporter in ulcerative colitis and irritable bowel syndrome

Background & Aims: Serotonin (5-HT) is a critical signaling molecule in the gut. 5-HT released from enterochromaffin cells initiates peristaltic, secretory, vasodilatory, vagal, and nociceptive reflexes. Despite being pathophysiologically divergent, ulcerative colitis (UC) and irritable bowel syndrome (IBS) are both associated with clinical symptoms that include alterations in the normal patterns of motility, secretion, and sensation. Our aim was to test whether enteric 5-HT signaling is defective in these disorders. Methods: Rectal biopsy specimens were obtained from healthy controls and patients with UC, IBS with diarrhea (IBS-D), and IBS with constipation (IBS-C). Key elements of 5-HT signaling, including measures of 5-HT content, release, and reuptake, were analyzed with these samples. Results: Mucosal 5-HT, tryptophan hydroxylase 1 messenger RNA, serotonin transporter messenger RNA, and serotonin transporter immunoreactivity were all significantly reduced in UC, IBS-C, and IBS-D. The enterochromaffin cell population was decreased in severe UC samples but was unchanged in IBS-C and IBS-D. When 5-HT release was investigated under basal and mechanical stimulation conditions, no changes were detected in any of the groups relative to controls. Conclusions: These data show that UC and IBS are associated with similar molecular changes in serotonergic signaling mechanisms. While UC and IBS have distinct pathophysiologic properties, these data suggest that shared defects in 5-HT signaling may underlie the altered motility, secretion, and sensation. These findings represent the first demonstration of significant molecular alterations specific to the gut in patients with IBS and support the assertion that disordered gastrointestinal function in IBS involves changes intrinsic to the bowel.

This month in Gastroenterology

This month in Gastroenterology

The distribution of serotonin transporter immunoreactivity in hippocampal formation in monkeys and rats

To elucidate the anatomical distribution of the serotonergic neurotransmitter system, we identified serotonin transporter (5-HTT) in the hippocampus of rats and monkeys by immunohistochemistry. A widespread and heterogeneous distribution of 5-HTT-immunoreactive fine fibers was noted in the rat brain. However, in monkeys, punctuate 5-HTT-immunoreactive deposits and fewer fibers were observed. The species difference in 5-HTT immunohistochemical staining pattern may be caused by differences in localization of 5-HTT between species.

Age-associated changes in the serotonergic system in rat superior colliculus and pretectum

The purpose of this study was to investigate whether aging alters serotonergic innervation of the superior colliculus and pretectum in rats. The superior colliculus has one of the highest concentrations of serotonin in the rat central nervous system. Young and old male F344 rats (<6 months, and >18 months, albino and pigmented) were used in all experiments. Coronal sections through the superior colliculus and pretectum were incubated with antibodies to serotonin, the serotonin 2A receptor, and the serotonin transporter. Immunocytochemical staining was analyzed semi-quantitatively. The results indicate that with age there is an increase in serotonin immunoreactivity throughout the entire superior colliculus and pretectum, a decrease in levels of serotonin 2A receptor staining in select layers of superior colliculus, and no change in serotonin transporter immunoreactivity. Albino rats differ from pigmented rats in that they have enhanced serotonergic immunoreactivity in the superficial layers of superior colliculus, a region that receives direct retinal input. These data suggest that the age-related changes in the serotonergic system in the superior colliculus and pretectum may account for some of the alterations in light-mediated behaviors with aging.

Immunocytochemical localization of serotonin and serotonin transporter (SET) in taste buds of rat

We used an immunocytochemical approach to study the localization of serotonin and its termination system, serotonin transporter (SET), in the taste buds of rats using specific antibodies against serotonin and SET. Under confocal laser scanning microscopy, both serotonin and SET immunoreactivity were detected in the taste buds of rat vallate papillae. Serotonin immunoreactivity was seen in the spindle-shaped cells with apical processes that seemed to be light (Type II) taste cells. SET-immunoreactivity was mainly localized in the periphery or interfaces between the taste cells. Double staining studies revealed that all serotonin-containing taste cells were immunoreactive for SET, while a subclass of SET-positive cells showed serotonin immunoreactivity. These data support the hypothesis that serotonin plays a transmitter role in taste receptor cells and suggest that the serotonin-induced sensation of taste is terminated by serotonin uptake through serotonin transporter.

Expression and development of a functional plasmalemmal 5-hydroxytryptamine transporter by thyroid follicular cells.

5-Hydroxytryptamine (5-HT) is synthesized and secreted by thyroid parafollicular (PF) cells. As all PF granules contain 5-HT, it is released whenever PF cells secrete. Because 5-HT stimulates follicular (F) cells and can modulate their response to TSH, 5-HT has been proposed to be a paracrine PF to F cell transmitter. This role would require a thyroid mechanism to rapidly inactivate 5-HT. A 5-HT transporter (SERT) in the plasma membrane of serotonergic neurons inactivates neuronal 5-HT. We thus tested the hypothesis that this molecule is expressed in the thyroid. Messenger RNA encoding SERT was demonstrated in both the human thyroid and a rat F cell line (FRTL-5). SERT immunoreactivity was detected in rat F, but not PF, cells. Transporter-mediated uptake of [3H]5-HT by F cells arose early in development (E13 in mice) and was maintained in adult life in mice, guinea pigs, bats, and rats (FRTL-5 cells). These observations indicate that a functional SERT is expressed in the thyroid, not by the 5-HT-secreting PF cells, but by their putative F cell targets.