Immunohistochemical demonstration of serotonin nerve fibers in the olfactory bulb of the rat,cat and monkey

Summary The distribution of serotonin (5-HT) positive fibers in the olfactory bulb of the rat, cat and monkey was studied using the peroxidase-anti-peroxidase (PAP) immunohistochemical method and highly specific antibodies to 5-HT. In general, 5-HT fibers were present throughout all layers in the olfactory bulb of these species except for the olfactory nerve layer and different as well as restricted laminar patterns of 5-HT distribution were observed. There were also species-related differences in the pattern of 5-HT distribution, in each layer. The most notable species difference was apparent in the glomerular layer of the main olfactory bulb. In case of the rat and cat, a very dense plexus of 5-HT fibers was observed to be diffuse in the glomerulus, while in the monkey, the distribution of 5-HT fibers was scanty and partial, as was seen in the accessory olfactory bulb of the rat.This work was supported by grant (No. 56440022) from the Ministry of Education, Science and Culture, Japan     

Immunohistochemical demonstration of serotonin-containing nerve fibers in the cerebellum

Summary The localization of serotonin (5-HT)-immunoreactive nerve fibers in the cerebellum of the rat and cat was investigated by means of the peroxidase-anti-peroxidase (PAP) method using highly specific antibodies to 5-HT.Serotonin-containing nerve fibers were distributed throughout the entire cerebellum including the deep cerebellar nuclei, while 5-HT-positive neuronal somata were not detected in the cerebellum of either species. A different pattern of 5-HT innervation was found among the three layers of the cerebellar cortex. There were also interspecific differences in the pattern of distribution of 5-HT. In the rat, the pool of 5-HT nerve fibers mainly consisted of tangential elements, which were predominant in the molecular layer, while in the cat only a few 5-HT fibers were found in the molecular layer of the cerebellar cortex; dense networks of 5-HT nerve fibers were present in the granular layer. Some differences are evident in the pattern of distribution of 5-HT fibers in cerebellar regions classified on an anatomical and functional basis.This work was supported by a grant (No. 56440022) from the Ministry of Education, Science and Culture, Japan     

Regional Distribution of Extracellular 5-Hydroxytryptamine and 5-Hydroxyindoleacetic Acid in the Brain of Freely Moving Rats

The extracellular concentrations of 5-hydroxytryptamine (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) have been determined in six brain areas of awake rats (frontal cortex, striatum, hypothalamus, hippocampus, inferior colliculus, and raphe nuclei) using intracerebral microdialysis. The extracellular levels of 5-HT showed no significant differences among the brain regions studied. The tissue levels of 5-HT and 5-HIAA as well as the extracellular concentration of 5-HIAA were significantly higher in raphe nuclei. The regional distribution of tissue and extracellular 5-HIAA were very similar, suggesting that extracellular 5-HIAA depends mainly on the output from the intracellular compartment. On the other hand, extracellular 5-HT and tissue 5-HT showed a different distribution pattern. The tissue/extracellular ratio for 5-HT ranged from 739 in frontal cortex to 2,882 in raphe, whereas it only amounted to 1.8-3.6 for 5-HIAA. The relationship between the present results and the density of 5-HT uptake sites in these areas is discussed.     

活细胞内5-羟色胺可见荧光的多光子激发成像

应用多光子激发激光扫描显微镜对5-羟色胺（5-hydroxytryptamine, 5-HT）孵育的大鼠粘膜型肥大细胞进行自发荧光成像,首次观察到了活细胞内5-HT相关的可见荧光,并对其产生机理进行了初步探讨.实现了对活细胞内5-HT空间分布的高分辨成像,为研究活组织或细胞内5-HT的空间分布和含量与细胞功能状态的关系提供了新的实验方法.     

Distribution of serotonin immunoreactive neurons in the brainstem of the hamster, guinea pig, rabbit, and rat

Immunohistochemical techniques were employed to study the distribution of serotonin (5-HT) immunoreactive neurons in the brainstem of the hamster, guinea pig, rabbit and rat. 5-HT neurons were principally found to be concentrated in the midline raphe nuclei, particularly, the raphe pallidus, raphe obscurus, raphe magnus, raphe median, raphe pontis and raphe dorsalis nuclei. Characteristically, these cell bodies are displayed in bands or wing-like patterns which extend laterally from the raphe into reticular formations. The formations often appear to blend with the catecholamine system. They are particularly evident in the brainstems of the rabbit and hamster which contain wider and more lateral extensions of the serotonergic (5-HT) neurons than those observed in the brainstems of the rat and guinea pig. The widespread distribution of 5-HT immunoreacted cell bodies in the brainstem shows that there are significant prospects of 5-HT in neuronal activities.     

Functional Mapping of Dorsal and Median Raphe 5-Hydroxytryptamine Pathways in Forebrain of the Rat Using Microdialysis

Abstract: Recent neurochemical studies of the properties of 5-hydroxytryptamine (5-HT) pathways arising from the dorsal raphe nucleus (DRN) and median raphe nucleus (MRN) have measured extracellular 5-HT in brain regions with reported preferential DRN or MRN 5-HT inputs. Here, we have tested whether electrical stimulation of the DRN and MRN releases 5-HT in rat forebrain regions in a pattern that fits the reported distribution of DRN/MRN pathways. The effect on extracellular 5-HT of electrical stimulation (5 Hz, 300 µA, 20 min) of the DRN, and then MRN, was determined in six regions of the anaesthetised rat. Stimulation of the DRN evoked a short-lasting but clear-cut release of 5-HT (+70–100%) in regions (frontal cortex, dorsal striatum, globus pallidus, and ventral hippocampus) reported to receive a 5-HT projection from the DRN. Regions receiving an MRN innervation (dorsal hippocampus, medial septum, and ventral hippocampus) released 5-HT (+70–100%) in response to MRN stimulation. Regions reported to receive a preferential DRN innervation (frontal cortex, dorsal striatum, and globus pallidus) did not respond to MRN stimulation. Of two regions (dorsal hippocampus and medial septum) reported to receive a preferential MRN innervation, one did not respond to DRN stimulation (dorsal hippocampus) although the other (medial septum) did. In summary, electrical stimulation of the DRN and MRN released 5-HT in a regionally specific pattern. With the exception of one region (medial septum), this pattern of release bears a strong relationship to the distribution of 5-HT projections from the DRN and MRN reported by anatomical studies. The combination of raphe stimulation with microdialysis may be a useful way to study the in vivo neurochemistry of DRN/MRN 5-HT pathways.     

Cloning, functional expression, and mRNA tissue distribution of the rat 5-hydroxytryptamine1A receptor gene

G protein-coupled receptors comprise a family of genes that share significant sequence similarity. We have screened a rat genomic library under low stringency hybridization conditions with the coding portion of the hamster beta 2-adrenergic receptor gene to isolate new members of this gene family. We show that one of these clones, clone D, codes for a 5-hydroxytryptamine1A (5-HT1A) binding site since: 1) it possesses an intronless open reading frame encoding a protein with seven putative transmembrane domains and 89% amino acid identity with the human 5-HT1A receptor (G21); 2) when transfected into Ltk- cells, it expresses a ligand-binding site with the pharmacology of the 5-HT1A receptor subtype, including 5-HT- and spiroxatrine-displaceable binding of 8-hydroxy-(2-(N,N-di[2,3-3H]propylamino)-1,2,3,4-tetrahydronaphthalene (KH = 0.8 nM). We further show that clone D encodes a functional receptor because its binding site interacts with G proteins and because it mediates agonist-induced inhibition of basal and stimulated cAMP accumulation in transfected GH4C1 pituitary cells. Finally, we have analyzed the tissue distribution of 5-HT1A receptor mRNA in rat brain and have found that 5-HT1A mRNA is present with the expected distribution of the 5-HT1A receptor (highest in septum and hippocampus) but is present as three RNA species (3.9, 3.6, and 3.3 kilobases). These studies represent the first characterization of receptor function and brain distribution of the cloned rat 5-HT1A receptor.     

Distribution of 5-hydroxytryptamine and indolealkylamine metabolites in the American cockroach, Periplaneta americana L

A procedure is described for simultaneous estimation of tryptophan (TP), 5-hydroxytryptophan (5-OHTP), 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), N-acetyl 5-hydroxytryptamine (NA5-HT) and N-acetyldopamine (NADA) using high performance liquid chromatography with coulometric electrochemical detection. The procedure has been used to determine the distribution of these compounds in the central nervous system of the American cockroach, Periplaneta americana. The ratio of TP:5-HT is greatest in the cerebral ganglia (6.5) with lesser ratios evident in the thoracic ganglia (15.5-18.9) and abdominal ganglia (9.6-11.2). Relatively low concentrations of 5-OHTP and NA5-HT were observed in the cerebral ganglia whereas 5-HIAA was not detected. Incubation of ganglia resulted in increased concentrations of NA5-HT. Reserpine reduced levels of 5-HT and NADA whereas probenecid caused a marked reduction in TP and slight elevation of NADA levels. No MAO activity was detected in the central nervous system.     

Regional distribution of dopamine, 5-hydroxytryptamine,and noradrenaline in the rat vas deferens

The concentrations of dopamine (DA), 5-hydroxytryptamine (5-HT) and noradrenaline (NA) in the rat vas deferens divided in eight or four sections were determined by high performance liquid chromatography with electrochemical detection. Dopamine and NA had the same regional distribution; their concentrations were maximal near the prostatic end and decreased towards the epididymis. The concentration of 5-HT also decreased from the prostatic to the epididimal end, but 5-HT did not follow the same regional distribution as DA and NA. Reserpine (0.02 or 0.2 mg/kg, i.p., 24 hr) and 6-hydroxydopamine (2×80 mg/kg, i.v., 6 days) decreased the contents of DA and NA; the concentrations of both amines were modified to a similar extent. Reserpine also diminished the content of 5-HT. Pargyline (200 mg/kg, i.p., 2 hr) increased the concentration of 5-HT whilep-chlorophenylalanine (300 mg/kg, oral, 3 days) decreased the contents of the amine in some sections of the vas deferens. This study suggests that DA and NA co-exist in the same sympathetic neurons. Some of the 5-HT could be stored in mast cells as previously proposed, but the finding that tissue content of 5-HT changes after inhibiting the deamination or synthesis of the amine suggests that other source(s) of 5-HT distinct from mast cells exist in the rat vas deferens.     

蝘蜓消化道5-羟色胺细胞的免疫组织化学定位

应用5-羟色胺(5-Hydroxytryptamine 5-HT)特异性抗血清对蝘蜓(Sphenomorphus iudieus)消化道5-HT细胞的分布及形态进行了免疫组织化学研究。结果表明,5-HT阳性细胞在堰蜓消化道各段均有分布,其中以胃幽门部位分布密度最高,食道与直肠部位其次,空肠部位分布密度最低。消化道各段5-HT细胞形态多样,有圆形、椭圆形、梭形、楔形、不规则形,其中梭形细胞多具有胞突。文中对堰蜓消化道5-HT细胞的分布、形态与功能相适应的特点进行了讨论。     

Amines of the mucosal mast cell of the gut in normal and nematode infected rats

Infection with the nematode N. brasiliensis is accompanied by a marked increase of the number of mucosal mast cells (MMC) and the mucosal content of histamine and 5-hydroxytryptamine (5-HT). We compared amine levels, determined by ion exchange and high performance liquid chromatography (HPLC) with numbers of MMC and enterochromaffin cells (ECC). Furthermore, we measured 5-HT cytofluorometrically in individual MMC and ECC. The cellular distribution of 5-HT was studied immunohistochemically. Our results corroborate previous findings that histamine is stored in MMC. Quotients between histamine content and numbers of MMC decreased throughout the period of worm expulsion, followed by a recovery, suggesting a histamine release during this defense reaction. The HPLC analysis gave no evidence for a storage of dopamine in MMC. ECC and MMC of normal and infected rats showed a formaldehyde induced fluorescence and 5-HT immunoreactivity. The formaldehyde induced fluorescence of MMC from normal rats was about 10% that of ECC, but MMC exceeded ECC three times by numbers. These findings suggest that a considerable proportion of the intestinal 5-HT in the normal rat is stored in MMC. ECC numbers did not change during the infection and their content of 5-HT was unchanged, as judged by cytofluorometry. The cytofluorometric measurements showed that the intensity of the monoamine fluorescence from the MMC of infected animals was about three times as high as that of controls. It was concluded that the increased tissue levels of 5-HT was due to both an increase in MMC numbers and an increase in the 5-HT content of individual MMC. The results suggest a different role for histamine and 5-HT in the defense reaction towards the nematode infection.     

The novel cellular mechanism of human 5-HT6 receptor through an interaction with Fyn

The human 5-HT(6) receptor (5-HT(6)R) is one of the latest cloned receptors among the known 5-HT receptors. Its abundant distribution in the limbic region, which participates in the control of mood and emotion and is involved in nervous system diseases such as depression and Alzheimer disease, has caused it to generate much interest. However, the cellular mechanisms of 5-HT(6)R are poorly understood. In the present study we found, using a yeast two-hybrid assay, that the carboxyl-terminal region of 5-HT(6)R interacts with the Fyn-tyrosine kinase. We also determined using a glutathione S-transferase pulldown assay that this interaction was mediated through the SH3 domain of Fyn and confirmed this by co-immunoprecipitation assays in two different transfected cell lines as well as in adult rat brains. Immunocyto(histo)chemistry also showed prominent co-localization between 5-HT(6)R and Fyn in transfected cells and a similar distribution between 5-HT(6)R and Fyn in the rat brain. Based on this interaction, we further examined the modulation of 5-HT(6)R by Fyn and vice versa. In addition, we demonstrated that the activation of 5-HT(6)R activated the extracellular signal-regulated kinase1/2 via an Fyn-dependent pathway. These findings suggest that Fyn may play an important role in 5-HT(6)R- mediated signaling pathways in the central nervous system.     

Distribution of serotonin uptake and binding sites in the cnidarian Renilla koellikeri: an autoradiographic study

Using [(3)H]-serotonin ([(3)H]-5-HT) as radiolabel and autoradiography, we have mapped the distribution of 5-HT uptake and binding sites in the sea pansy Renilla koellikeri in order to identify potential cellular pathways of 5-HT inactivation and to identify cellular substrates for the previously characterized 5-HT receptor involved in the modulation of peristaltic behavior. Uptake measured in fresh polyp tissues occurred via two processes: a high affinity (uptake(1)), clomipramine-sensitive process with a K(m) of 0.45 muM, and another of lower affinity (uptake(2)) with a Km of 11.6 muM. Autoradiograms of high affinity uptake sites revealed a diffuse distribution of label with higher density in the ectoderm and endoderm, and lower density in the mesoglea. No subsets of cells, including serotonergic neurons, appeared to retain label preferentially, thus suggesting that removal of 5-HT and its chemical conversion is a general property of sea pansy tissues. Under incubation conditions identical to those used in a previous radiobinding analysis, autoradiograms of binding sites were generated on sections from lightly fixed and cryosectioned polyps. In contrast to uptake sites, binding sites appeared as aggregations of label around neurons of the subectodermal, mesogleal and endodermal nerve nets. In the endoderm where the myoepithelia subtend peristalsis, myoepithelial cells appeared unlabeled, suggesting that 5-HT exerts its modulatory effect on peristalsis principally via neurons. Taken together, these results indicate that 5-HT is released as a neurohormone in the sea pansy and that it may act as a broad-range neuromodulator.     

Serotonin 4 receptor (5-HT4R) internalization is isoform-specific: Effects of 5-HT and RS67333 on isoforms A and B

Serotonin 4 receptors (5-HT4Rs) are particularly abundant within the limbic system, where they constitute potential targets for the development of novel, rapid acting antidepressants. However, the population of limbic 5-HT4Rs is not homogenous, comprising various isoforms of which 5-HT4(a) and 5-HT4(b) are among the most abundant variants. Sequence divergence at their C-termini is predictive of specificity in isoform signalling and regulation, but the differences, if any, remain ill-defined. The present study compared isoforms 5-HT4(a) and 5-HT4(b) in their ability to undergo endocytic regulation following exposure to 5-HT and to the putatively fast acting antidepressant RS67333. Both ligands differed in their ability to induce internalization of either isoform, 5-HT being more effective than RS67333 in HEK293 cells and in neurons. In contrast, trafficking induced by 5-HT was isoform-specific. In particular, while PKC, GRK2 and βarrestin were necessary for 5-HT4(a)R internalization, sequestration of 5-HT4(b)Rs required PKC but not GRK2 and relied significantly less on βarrestin. After endocytosis, isoform (b) appeared scattered throughout the intracellular compartment and efficiently recycled to the membrane upon agonist removal. Isoform (a) accumulated in the perinuclear compartment and displayed little recycling. Isoform-specific subcellular distribution was present in HEK293 cells and in neurons. In neurons, where internalization by RS67333 was more pronounced than in HEK293 cells, receptors internalized by this ligand followed the same distribution pattern as observed with 5-HT. These results point to isoform-related differences in the way that 5-HTRs respond to different ligands. Such diversity should be taken into account when developing therapeutic agents that target 5-HT4Rs.     

Synthesis and pharmacological study of radioiodinated serotonin derivative specific of 5-HT1B and 5-HT1D binding sites of the central nervous system

We describe here the synthesis of a new serotonin conjugate, S-CM-GTNH2, and its radioiodinated derivative. Quantitative autoradiographic studies on rat and guinea pig brain sections incubated with 2 nM [3H]5-HT showed a preferential affinity of S-CM-GTNH2 for 5-HT1B and 5-HT1D sites. Autoradiograms from brain sections incubated with 0.02 nM S-CM-G[125I]TNH2 showed a heterogeneous anatomical distribution of the labelling with high densities in regions rich in 5-HT1B or 5-HT1D binding sites, and with no labelling of those rich in 5-HT1A or 5-HT1C sites. The pharmacological profiles of the binding sites corresponded to those of 5-HT1B and 5-HT1D receptor subtypes. The radioligand S-CM-G[125I]TNH2 is a good probe for the study of these sites and will be used for their subcellular localization in electron microscopy.     

The subcellular distribution of endogenous and exogenous serotonin in brain tissue: comparison of synaptosomes storing serotonin, norepinephrine, and gamma-aminobutyric acid

Abstract— We have studied the subcellular distribution of exogenous and endogenous serotinin in slices from the hypothalamus and midbrain of several species. In a procedure which appears to label the endogenous pools, tissue slices were incubated with low concentrations of [³H]5-HT (5 × 10^-8 M), for 45 min, when there was apparent equilibrium between [³H]5-HT of tissue and medium. After the tissue slices were homogenized in 0-32 M-sucrose and subjected to differential centrifugation, the distribution of exogenous and endogenous 5-HT in pellets and supernatant fluid was similar. In some experiments, the crude mitochondrial pellets were resuspended in 0-32 M-sucrose, layered on linear, continuous density gradients of sucrose (1 -5-0-32 M), and centrifuged for short times (incomplete equilibrium centrifugation). The subcellular distribution of particulate tritium, total tritium, and particulate endogenous 5-HT was the same in portions of the gradients containing synaptosomes. The peak distribution of [³H]5-HT in sucrose gradients was separable from the peak for [¹⁴C]GABA by four to five fractions; potassium (a marker for cytoplasm occluded within synaptosomes) occurred in the regions of the gradients containing most of the labelled compounds. The distribution of monoamine oxidase activity (a mitochondrial marker) overlapped the distribution of [³H]5-HT after a 15 min centrifugation but appeared in denser regions of the gradient after centrifuging for 2 h. Particles containing [3H]5-HT and [I4C]NE were slightly but consistently separable in synaptosomal fractions isolated from the hypothalamus or midbrain of rat, guinea pig and hamster.