Biochemical and immunohistochemical determination of 5-hydroxytryptamine located in mast cells in the trigeminal ganglion of the rat and guinea pig

The presence of 5-hydroxytryptamine (5-HT), as well as its precursor (5-HTP) and metabolite (5-HIAA), were biochemically determinated in the trigeminal ganglion of the guinea pig and rat. The distribution of 5-HT in the ganglion and in its posterior root was studied using both indirect immunofluorescence and the peroxidase-antiperoxidase method. In order to increase the possible 5-HT content of primary sensory neurons for subsequent immunohistochemical visualization, animals were first treated with nialamide, an inhibitor of monoamine oxidase, and then loaded with L-tryptophan. Another group of animals received colchicine to inhibit intra-axonal transport of transmitter substances. However, even combined use of loading and colchicine treatment did not reveal 5-HT immunoreactivity in ganglion cells. The only source of 5-HT immunoreactivity in the trigeminal ganglion and its posterior root was mast cells. These cells were located around the ganglion in adjacent leptomeningeal and connective tissues, as well as between the ganglion cells and nerve fibers. Only occasionally were mast cells found in the posterior root of the ganglion.     

Substance P-like immunoreactivity in the trigeminal ganglion. A fluorescence, light and electron microscope study

The trigeminal ganglion of rat and guinea pig was studied for the presence of immunoreactive substance-P using fluorescence, light and electronmicroscopy. In untreated animals substance P containing cells, with a diameter of 15 to 50 micron, were distributed throughout the ganglion and comprised 10-30% of all ganglion cells. Colchicine, injected intraventricularly to inhibit intra-axonal transport, had no effect on the number of substance P cells; but when the drug was injected directly into the posterior root of the ganglion, the proportion of these cells increased to as much as 50%. In the electron microscope, immunoreactive substance-P was confined to ganglion cells classified as B type according to the arrangement of subcellular organelles, and to unmyelinated nerve fibers. Subcellularly the immunoreactivity appeared in cytoplasmic vesicles, as well as dispersed in the nerve fibers and the perikarya of neurons. The great number of substance P immunoreactive ganglion cells suggests that they do not comprise a well defined subpopulation of the B-cells. However, the immunoreactivity was restricted to a distinct ultrastructural type of neurons with unmyelinated nerve fibers, suggesting that they also may share some distinct functions.     

Substance P-like immunoreactivity in the trigeminal ganglion

Summary The trigeminal ganglion of rat and guinea pig was studied for the presence of immunoreactive substance-P using fluorescence, light and electronmicroscopy. In untreated animals substance P containing cells, with a diameter of 15 to 50 m, were distributed throughout the ganglion and comprised 10–30% of all ganglion cells. Colchicine, injected intraventricularily to inhibit intra-axonal transport, had no effect on the number of substance P cells; but when the drug was injected directly into the posterior root of the ganglion, the proprotion of these cells increased to as much as 50%. In the electron microscope, immunoreactive substance-P was confined to ganglion cells classified as B type according to the arrangement of subcellular organelles, and to unmyelinated nerve fibers. Subcellularily the immunoreactivity appeared in cytoplasmic vesicles, as well as dispersed in the nerve fibers and the perikarya of neurons. The great number of substance P immunoreactive ganglion cells suggests that they do not comprise a well defined subpopulation of the B-cells.However, the immunoreactivity was restricted to a distinct ultrastructural type of neurons with unmyelinated nerve fibers, suggesting that they also may share some distinct functions.     

Localization of serotonin-like immunoreactivity in the eyestalk of the prawn Palaemon serratus (Crustacea, Decapoda, Natantia)

A serotonin-like substance in the organ of Bellonci in the eyestalks of embryos, larvae, and adults of the prawn Palaemon serratus was visualized by the use of two specific antisera against serotonin (5-hydroxytryptamine; 5-HT) in combination with peroxidase-antiperoxidase (PAP). The organ of Bellonci, characterized by compact onion bodies distally and degenerating onion bodies proximally, was the only site of the serotonin-like substance in adults, as well as during development in embryos and larvae. Variations in the content of the 5-HT analogue in the adult were detected during the molting cycle. There was more immunoreactivity in specimens fixed at night than in those fixed in daytime. Likewise, colchicine and nialamide injections enhanced the immunoreactivity of the serotonin-like substance. Extirpations of the medulla externa X organ (MEX), a neurosecretory cell group of the optic ganglion medulla externa, produced the same effect.     

Calcitonin gene-related peptide coexists with substance P in capsaicin sensitive neurons and sensory ganglia of the rat

Immunohistochemical and radioimmunoassay studies revealed that both CGRP- and SP-like immunoreactivity in the caudal spinal trigeminal nucleus and tract, the substantia gelatinosa and the dorsal cervical spinal cord as well as in cell bodies of the trigeminal ganglion and the spinal dorsal root ganglion is markedly depleted by capsaicin which is known to cause degeneration of a certain number of primary sensory neurons. Higher brain areas and the ventral spinal cord were not affected by capsaicin treatment. Furthermore CGRP and substance P-like immunoreactivity were shown to be colocalized in the above areas and to coexist in cell bodies of the trigeminal ganglion and the spinal dorsal root ganglia. It is suggested that CGRP, like substance P, may have a neuromodulatory role on nociception and peripheral cardiovascular reflexes.     

Fluorescence microscopy of the 5-HTP turnover in the exocrine pancreas of mice and rats

Summary The turnover ofl-5-HTP,d-5-HTP and 5-HT in the exocrine pancreas have been studied by means of the fluorescence method ofFalck andHillarp. l- andd-5-HTP are easily taken up by the acinar cells, whereas 5-HT seems to pass into the cells only to a minor extent. After the administration ofl-5-HTP (and in some cases after 5-HT administration), specific fluorescence is seen in the form of apically located granules (probably identical with the zymogen granules) for a short period, which is prolonged, if the animals are pretreated with a MAO inhibitor. Decarboxylase inhibition prevents the appearance of these fluorescent granules. Administration ofd-5-HTP does not give rise to this granular fluorescence but to a diffuse fluorescence throughout the cells. Thus, there are reasons to assume that the granular fluorescence derives from 5-HT. The results obtained in this work correspond well with those from a similar study withl-DOPA and some of its analogues.abbreviations DOPA 3,4-dihydroxyphenylalanine - DA dopamine - NA noradrenaline - A adrenaline - 5-HTP 5-hydroxytryptophan - 5-HT 5-hydroxytryptamine - MAO monoamine oxidase This work was supported by grants from the Swedish Medical Research Council (B68-12X-712-03B and B68-14X-56-04B), the United States Public Health Service (06701-02) and the Faculty of Medicine, University of Lund, Lund, Sweden.     

5-Hydroxytryptamine immunoreactivity is detectable in sympathetic nerve fibres in rat oral tissues

Summary The aim of this investigation was to examine if 5-hydroxytryptamine (5-HT) is detectable not only in mast cells but also in sympathetic nerve fibres in oral sites of the rat, including the periodontal ligament, pulp, palatal mucosa, and vestibular sulcus. Antibodies against 5-HT and tyrosine hydroxylase were used. Maxillae from rats were dissected free, fixed, decalcified, cut transversally, and processed for immunohistochemistry. Nerve fibres showing 5-HT-like immunoreactivity were regularly observed in the walls of the arteries and arterioles in the vestibular sulcus and the periodontal ligament. However, 5-HT-like immunoreactivity was not seen in the walls of the vessels of the palatal mucosa. Interestingly, 5-HT-like immunoreactivity coexisted with tyrosine hydroxylase-like immunoreactivity in the innervation of the periodontal ligament and the vestibular sulcus. Thus, the present study gives morphological correlate for the occurrence of effects of 5-HT derived not only from mast cells but also from sympathetic nerve fibres in oral tissues. The source of 5-HT in the nerve fibres as well as the functional implications of the observations remain to be determined.     

Localization of substance P-like immunoreactivity in the palate and trigeminal ganglion of Rana pipiens

This study was undertaken to localize substance P-like immunoreactivity (SP) in the nerve fibers innervating the palate, identify the ganglion of the palatine nerve and determine whether it contains SP cell bodies, in the frog Rana pipiens. The palatine nerve which is a branch of the maxillo-mandibular subdivision of the trigeminal nerve was traced to the trigeminal ganglion that connects to the medulla by the trigeminal nerve root. Using an immunocytochemical method, SP containing fibers with varicosities were found in the connective tissue layer of the palate. Some of these fibers were observed adjacent to blood vessels to the epithelial layer of the palate in apparent innervation of the ciliated epithelial and mucus cells. SP-labeling was also observed in small to medium cells of the trigeminal ganglion. These results appear to support the pharmacological studies of SP on the regulation of mucociliary activity in the frog R. pipiens.     

Localization of l-glutamate decarboxylase immunoreactivity in the major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex of the rat

Summary The localization of l-glutamate decarboxylase (GAD), the GABA-synthesizing enzyme, was studied in the rat major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex by indirect immunofluorescence technique with a specific antiserum raised in rabbits. GAD immunoreactivity was demonstrated in small cells of these ganglia. The GAD-immunoreactive small cells were 10–20 m in diameter and formed clusters or occured as solitary cells. The principal neurons were non-reactive but they were surrounded by immunoreactive processes. Studies on colocalization of GAD with tyrosine hydroxylase (TH), the rate-limiting enzyme of the catecholamine synthesis, in the major pelvic ganglion and in the coeliac-superior mesenteric ganglion complex indicated that all GAD-immunoreactive small cells were also labelled with TH. In the major pelvic ganglion all TH-immunoreactive SIF cells were also immunoreactive for GAD. However, in the coeliac-superior mesenteric ganglion complex there occured TH-immunoreactive small cells which showed no immunoreactivity to GAD. It is suggested that the small GAD-immunoreactive cells represent small intensely fluorescent (SIF) cells.     

Galanin-immunoreactive nerves in the rat iris: alterations induced by denervations

Summary The iris and choroid membrane of the adult rat contain nerve fibers expressing immunoreactivity to the neuropeptide galanin. The density and distribution of galanin-positive nerve fibers varied from iris to iris and, particularly, among animals. Smooth, non-terminal axons were seen running in nerve bundles consisting of otherwise negative fibers. From the choroid membrane these bundles reached the iris via the ciliary body. Axons were frequently seen to branch giving rise to a sparse system of varicose, single fibers in the dilator plate and sphincter area. Galanin-positive fibers were sometimes also seen outlining blood vessels.Capsaicin, in a dose that causes permanent depletion of substance P- and cholecystokinin-immunoreactive fibers in the iris, caused no change in amount of galanin-positive fibers. Removal of the superior cervical ganglion caused a rapid and pronounced increase in the number of galanin-immunoreactive nerve fibers. Similarly, removal of the ciliary ganglion appeared to increase galanin immunoreactivity, while removal of the pterygopalatine ganglion was less effective. Lesioning of the trigeminal ganglion caused a disappearance of galanin immunoreactivity. The sympathetectomy-induced increase was counteracted by capsaicin.Galanin-positive nerve cell bodies were present in both the superior cervical and the trigeminal ganglia. In the superior cervical ganglion, immunoreactive galanin did not seem to coexist with neuropeptide Y-positive cells; in the trigeminal ganglion, some galanin-positive cells also contained calcitonin gene-related peptide (CGRP) immunoreactivity, while most cells did not. In the iris, double-staining suggested that CGRP and galanin immunoreactivities were contained in different fiber populations.We conclude that the rat iris and choroid membrane contain a sparse plexus of nerve fibers expressing galanin-like immunoreactivity. It is suggested that these fibers are derived from the trigeminal ganglion. The iris is able to respond with a pronounced increase in number of galanin-immunoreactive nerve fibers to certain denervation procedures.     

A quantitative cytochemical study of the growth of individual mast cells

Summary For individual mast cells, relationships between their dry mass and their content of heparin and 5-hydroxytryptamine (5-HT) were studied. This was achieved by measuring these parameters successively on identical cells, by means of quantitative cytochemical techniques. The peritoneal mast cells have a very long life span and a slow turnover of granule components. Increase of the dry weight of the cells may therefore be taken as an expression of cellular growth. Mast cell populations from younger and older animals were analysed in an attempt to evaluate the influence of cell-aging and animal-aging on the growth of the mast cells. The analysis was based on allometric (log-log) plots and linear regressions. Within the cell populations there were strong mutual correlations between the cell parameters studied, without any obvious deviations from linearity. However, the slopes of the allometric lines indicated a somewhat different mode of growth for mast cells from younger and older animals. The capacity of the mast cells to accumulate 5-HT after a single injection of its precursor, 5-hydroxytryptophan, was used as a functional test. In relation to the cell weight, the induced increase of 5-HT was greater for lighter than for heavier mast cells. This difference between light and heavy mast cells was greater for cells from younger than from older animals. These differences in growth and functional properties between mast cells from younger and older animals were interpreted as an effect of aging of the animals rather than of aging of the cells.Supported by grants from the Swedish Medical Research Council, Project no 2235I wish to express my sincere thanks to Professor Lennart Enerbäck for valuable and constructive criticism of this paper. I also wish to thank Lecturer Erik Leander for valuable statistical advice     

Distribution of serotonin-immunoreactive nerve cells and fibers in the rat gastrointestinal tract

 The distribution of serotonin-immunoreactive (5HT-IR) nerve cells and fibers was thoroughly investigated immunohistochemically in the rat stomach, duodenum, jejunum, ileum, and colon. The immunoreactivity of the 5HT neurons was compared between non-treated controls and animals treated with colchicine, colchicine plus 5-hydroxytryptophan (5HTP), colchicine plus pargyline, and reserpine. The intensity of immunoreactivity in nerve fibers as well as nerve cell bodies was enhanced mostly in colchicine plus pargyline treated animals, therefore these animals were used for an observation of precise localization of 5HT in the rat gastrointestinal (GI) tract. Immunoreactivity in the nerve cell bodies and fibers was completely abolished in the GI tract of reserpine treated animals. The pattern of localization and projection of 5HT-IR neurons was similar in all segments of the rat GI tract. 5HT-IR nerve cell bodies were located in the myenteric plexus and showed the distinctive features of Dogiel type I neurons. Prominent bundles of varicose fibers traversed the myenteric ganglia and some of them surrounded the cell bodies of immunopositive and immunonegative neurons. 5HT-IR nerve fibers were located in the submucous plexus, densely entwined about the submucosal blood vessels. Most characteristically, 5HT-IR nerve fibers invaded the lamina propria of mucosa where they underlay the crypt epithelium. In conclusion, the present study showed that 5HT-IR neurons located in the myenteric plexus projected fibers widely in the rat GI tract. The localization of fibers in the lamina propria of mucosa implies that this neuron may exert an important role in the epithelial function of the GI tract. Accepted: 8 October 1996     

Combined retrograde tracing and immunohistochemistry of trigeminal ganglion neurons projecting to gingiva or tooth pulps in the lower jaw of the cichlid Tilapia mariae

Rat trigeminal ganglion neurons projecting to the oral mucosa or to tooth pulps have different cell diameters and contain different chemical markers. In the present paper we examine whether trigeminal ganglion neurons sending axons to gingiva or tooth pulps in the lower jaw of the cichlid Tilapia mariae differ in a similar way. Retrograde tracing with fluorescent latex microspheres revealed labelled gingival and pulpal neurons in the caudal part of the trigeminal ganglion. The gingival neurons had a unimodal size distribution (peak 11 μm; range 8–14 μm) and the pulpal neurons exhibited a bimodal size distribution (peaks 12 and 25 μm; range 10–40 μm). Immunohistochemistry revealed a calcitonin gene-related peptide-like immunoreactivity in some 40% of the gingival neurons and a substance P-like immunoreactivity in 30%. Of the small pulpal neurons about 60% exhibited a calcitonin gene-related peptide-like immunoreactivity and 15% showed a substance P-like immunoreactivity. Of the large pulpal neurons some 70% exhibited a calcitonin gene-related peptide-like immunoreactivity. These neurons did not show a substance P-like immunoreactivity. In some animals a few trigeminal ganglion neurons showed a neuropeptide Y- or a vasoactive intestinal polypeptide-like immunoreactivity. Perikarya with a tyrosine hydroxylase- or a choline acetyl transferase-like immunoreactivity were not observed. We conclude that gingiva and tooth pulps in the lower jaw of T. mariae are innervated by trigeminal ganglion neurons, the cell diameters and neuropeptide contents of which differ in a pattern similar to that in the rat. Hence, this seems to represent a conserved evolutionary pattern.     

Growth hormone-releasing factor (GRF)-like immunoreactivity in sensory ganglia of the rat

Summary Growth hormone-releasing factor (GRF)-like immunoreactivity has been demonstrated in the trigeminal and spinal ganglia of fetal, young and adult rats by use of peroxidase-antiperoxidase immunohistochemistry. GRF-like-immunoreactive cells first appear during the second half of embryonic life, as early as day 17. In untreated animals the GRF-immunoreactive elements form approximately 1% of all ganglion cells in the trigeminal and spinal ganglia; their numbers do not change significantly during development. The granular immunoreaction product is confined to perikarya, especially to the perinuclear region. Nerve fibers displaying GRF-like immunoreactivity were found neither in the ganglia, nor in the corresponding central and peripheral areas of termination. The possible role of GRF in sensory ganglia is discussed.     

Gastro-intestinal and neurohormonal peptides in the alimentary tract and cerebral complex ofCiona intestinalis (Ascidiaceae)

Summary Polypeptide-hormone producing cells were localized in the alimentary tract and cerebral ganglion ofCiona intestinalis using cytochemical, immunocytochemical and electron-microscopical methods.Antisera to the following peptides of vertebrate type were employed: bombesin, human prolactin (hPRL), bovine pancreatic polypeptide (PP), porcine secretin, motilin, vasoactive intestinal polypeptide (VIP),-endorphin, leu-enkephalin, met-enkephalin, neurotensin, 5-hydroxytryptamin (5-HT), cholecystokinin (CCK), human growth hormone (GH), ACTH, corticotropin-like intermediate lobe peptide (CLIP) and gastric inhibitory peptide (GIP).Immunoreactive cells were found both in the alimentary tract epithelium and in the cerebral ganglion for bombesin, PP, substance P, somatostatin, secretin and neurotensin. Additionally, in the cerebral ganglion only, there were cells immunoreactive for-endorphin, VIP, motilin and human prolactin. 5-HT positive cells, however, were restricted to the alimentary tract.No immunoreactivity was obtained either in the cerebral ganglion or in the alimentary tract with antibodies to leu-enkephalin, met-enkephalin, CCK, growth hormone, ACTH, CLIP and GIP. Prolactin-immunoreactive and pancreatic polypeptide-immunoreactive cells were argyrophilic with the Grimelius' stain and were found in neighbouring positions in the cerebral ganglion.At the ultrastructural level five differently granulated cell types were distinguished in the cerebral ganglion. Granules were present in the perikarya as well as in axons. The possible functions of the peptides as neurohormones, neuroregulators and neuromodulators are discussed.     

Protein content,dry mass and chemical composition of individual mast cells related to body growth

Summary Recently developed quantitative microscopical techniques were used to study relations between body growth and protein content as well as dry mass of individual mast cells. Since previous studies had shown an age-related increase of mast cell content of 5-hydroxytryptamine (5-HT) and heparin, these mast cell components were also included in the present study. The cells were obtained from the peritoneal cavity of rats aged 44–269 days (body weights 189–610 g). All studied mast cell parameters showed an increase that was related to the growth of the animals. The dry mass increased 60%, protein 50%, heparin 50% but 5-HT increased as much as 260% during the studied growth period. There was a mutual and linear correlation between all studied mast cell parameters. Population studies, based on large scale measurements of individual mast cells from young and adult rats, were made. These studies showed that histograms of 5-HT content, protein content and dry mass of individual mast cells were skewed with a tail towards higher values and approximately lognormal. On the other hand, the frequency distribution of heparin content of individual mast cells was approximately normal.Supported by grants from the Swedish Medical Research Council, Project no 2235     

Intracellular localization of serotonin in mast cells of the colon in normal and colitis rats

Intracellular localization of serotonin (5-HT) in the mast cells of two phenotypes in normal rat colon and dextran sodium sulphate-induced colitis was studied by immunoelectron microscopy with a quantitative analysis of the distribution of immunogold labelling. Mucosal mast cells in normal rats contained round shape secretory granules with varying electron density. Immunogold labelling for 5-HT was concentrated over the secretory granules. In mucosal mast cells from colitis rats, vacuolated granules without 5-HT labelling were frequently observed and immunogold labelling over the secretory granules was significantly increased compared to controls. On the other hand, connective tissue mast cells in normal rats contained oval shape secretory granules with homogeneous electron density. Their immunogold labelling was diffusely scattered over the secretory granules as well as over the cytoplasm. In connective tissue mast cells from colitis rats, secretory granules with high electron density were increased and the immunogold labelling over the secretory granules was much higher than that in controls. The present results suggest that intracellular localization of 5-HT is different in two phenotypes of mast cells and they may release 5-HT in a different manner. Mucosal mast cells may release 5-HT by a degranulation or exocytosis, while connective tissue mast cells may release 5-HT by a diacrine manner of secretion.     

Trigeminal ganglion cells cocultured with gut express vasoactive intestinal peptide

The plasticity of neural crest cells for the expression of adrenergic and cholinergic transmitter phenotypes has been well studied. The object of this study was to determine if cells of a sensory ganglion are capable of neuropeptide transmitter plasticity. We studied whether cells of the trigeminal ganglion, which do not express the neuropeptide vasoactive intestinal peptide (VIP) in vivo, would express this peptide when grown with a tissue the gut, that contains large numbers of VIP neurons. Embryonic aneural chick rectum was explanted with the embryonic quail trigeminal ganglion on the chorioallantoic membrane of chick hosts for 7-8 days. The explants were fixed, sectioned, and stained for VIP immunoreactivity (IR), for neurofilament protein immunoreactivity, and for the quail nucleolar marker. In sections of the explants we observed two populations of quail neurons: small (10-13 microns) VIP-IR cells and large (25-32 microns) cells lacking VIP-IR and resembling native trigeminal neurons. Trigeminal ganglia explanted with embryonic heart or trigeminal ganglia explanted alone lacked small VIP-IR cells but contained large VIP-negative neurons. These results show that cells of the trigeminal ganglion grown with the gut can express a neuropeptide they do not express in the absence of the gut or in vivo. Thus the embryonic trigeminal ganglion contains cells that are plastic with respect to neuropeptide expression.     

Localization of serotonin/tryptophan-hydroxylase-immunoreactive cells in the brain and suboesophageal ganglion of Drosophila melanogaster

We previously demonstrated that tryptophan hydroxylase (TPH), the rate-limiting enzyme of serotonin (5-HT) synthesis, was commonly present in the brains of some insects. The current study was aimed at determining the number of serotonergic neurons in the brain and suboesophageal ganglion of adult Drosophila melanogaster and to investigate further the differences in immunoreactivity between 5-HT and TPH. Brain sections of Drosophila were immunostaind with sheep anti-TPH polyclonal antibody and rabbit anti-5-HT antiserum. The 5-HT-like immunoreactive neurons were also immunoreactive for TPH and bilaterally symmetrical; 83 neurons were found in each hemisphere of the brain and suboesophageal ganglion of adult Drosophila. This technique of colocalizing 5-HT and TPH revealed a larger number of serotonergic neurons in the brain and suboesophageal ganglion than that previous reported, thus updating our knowledge of the 5-HT neuronal system of Drosophila.     

Rhombomere formation and hind-brain crest cell migration from prorhombomeric origins in mouse embryos

Prior to rhombomere development, structures called prorhombomeres appear in the mammalian hindbrain. This study clarifies the developmental relationship between prorhombomeres and their descendent rhombomeres and hindbrain crest cells in mouse embryos by focal dye injections at various levels of prorhombomere A (proRhA), proRhB, and proRhC, as well as at their boundaries. ProRhA gives rise to two rhombomeres, rhombomeres 1 and 2 (r1 and r2), as well as to crest cells that migrate into the first pharyngeal arch, including the trigeminal ganglion. ProRhB develops into r3 and r4 and produces crest cells populating the second arch and acousticofacial ganglion. The anterior portion of proRhC gives rise to r5 and r6 and to crest cells migrating into the third pharyngeal arch and the IXth ganglion; its posterior portion develops into r7 and releases crest cells into the fourth pharyngeal arch region as well as the Xth ganglion. These results suggest that the boundaries between prorhombomeres serve as lineage restrictions for both hind-brain neuroepithelial cells and for segmental origins of crest cell populations in mouse embryos. The Hox code of the mouse head can be schematized in a much simpler way based on this prorhombomeric organization of the hind-brain, suggesting that prorhombomeres primarily underlie mammalian hind-brain segmentation.