Immunoelectron microscopic study of tyrosine hydroxylase immunoreactive nerve fibers and ganglion cells in the rat adrenal gland.

The present immunocytochemical study used an antiserum to tyrosine hydroxylase (TH), the first enzyme in the biosynthetic pathway of catecholamines, and revealed TH immunoreactivity in the ganglion cells and in the varicose nerve fibers of the cortex and medulla in the rat adrenal gland. TH immunoreactive nerve fibers in the cortex and medulla contained large and small granular vesicles, and also small clear vesicles. The immunoreactive nerve fibers were in close apposition to cortical cells in the cortex and in apposition to smooth muscle cells of blood vessels in both the cortex and medulla. Furthermore, TH immunoreactive nerve fibers were sometimes in close apposition to pericytes of blood vessels in the cortex and chromaffin cells in the medulla. The present results suggest that the catecholaminergic nerve fibers in the rat adrenal gland may be both intrinsic and extrinsic in origin.     

GABA receptor modulation of tyrosine hydroxylase gene expression in the rat adrenal gland

Chromaffin cell gamma-aminobutyric acid (GABA) receptors play a role in modulating catecholamine secretion. The present experiments examined the role of GABA receptors in modulation of tyrosine hydroxylase (TH) induction in rat adrenal gland. Administration of bicuculline, a GABA antagonist, had no effect on TH activity or TH mRNA. However, bicuculline potentiated reserpine's effect on TH activity and TH mRNA induction. These data suggest that GABA receptors modulate induction of TH and TH mRNA in the adrenal gland.     

Fine structural survey of tyrosine hydroxylase immunoreactive terminals in the myenteric ganglion of the rat duodenum

We have examined whether the noradrenergic neurons have direct synaptic projections to the myenteric ganglion neurons of the duodenum and the ultrastructure of their terminals by using immunogold–silver labeling for tyrosine hydroxylase. In the neuropil of the myenteric ganglia, about half of the axon terminals contained round clear vesicles and the rest of them contained pleomorphic clear vesicles. The sizes of axon terminals contacting the dendrites as a whole were 1.62 ± 0.07 μm. All axon terminals formed asymmetric synaptic contacts with dendrites or somata. Immunohistochemical study revealed that the tyrosine hydroxylase-immunoreactive nerve terminals were distributed throughout the ganglia and contained exclusively pleomorphic clear synaptic vesicles (about 20–80 nm long). The tyrosine hydroxylase-immunoreactive terminals were generally large (1.99 ± 0.07 μm). A considerable number of the tyrosine hydroxylase-immunoreactive terminals made asymmetric synaptic contacts with small dendrites, spines or somata of the myenteric ganglion neurons. Serial ultrathin sections through the myenteric neurons revealed that about 16% of the total number of axosomatic terminals showed tyrosine hydroxylase immunoreactivity. These results indicated that the myenteric ganglion neurons of the duodenum receive direct synaptic projection of sympathetic noradrenergic neurons and that their terminals contain pleomorphic vesicles and form asymmetric synaptic contacts.     

Immunohistochemical features of substance P-immunoreactive chromaffin cells and nerve fibers in the rat adrenal gland

The distribution of substance P (SP) immunoreactivity and the colocalization of SP with other bioactive substances in chromaffin cells and nerve fibers were investigated in the rat adrenal gland at the light microscopic level. In the capsule and cortex, SP immunoreactivity was seen in some nerve fibers around blood vessels and in thick nerve bundles passing through the cortex directly into the medulla. In the medulla, the SP immunoreactivity was observed in a small number of chromaffin cells; these SP-immunoreactive chromaffin cells were either phenylethanolamine N-methyltransferase (PNMT) immunoreactive or immunonegative, indicating that they were either adrenaline cells or noradrenaline (NA) cells. SP-immunoreactive varicose nerve fibers were also found in the medulla and were in contact with a cluster of the NA cells showing catecholamine fluorescence, which suggests that SP from medullary nerve fibers may regulate the secretory activity of the NA cells. Because no SP-immunoreactive ganglion cell was present in the rat adrenal gland, the intra-adrenal nerve fibers were considered to be extrinsic in origin. The double-immunostaining method further revealed that the SP-immunoreactive chromaffin cells also exhibit immunoreactivities for calcitonin gene-related peptide (CGRP), and neuropeptide tyrosine (NPY), suggesting that these peptides can also be released from the chromaffin cells by certain stimuli. The intra-adrenal nerve fibers in the medulla were composed of SP-single immunoreactive, and SP/CGRP-, SP/choline acetyltransferase (ChAT)-, SP/nitric oxide synthase (NOS)-, SP/pituitary adenylate cyclase activating polypeptide (PACAP)-, ChAT/NOS-, and ChAT/PACAP-immunoreactive nerve fibers, which may affect the secretory activity of the NA cells. In the adrenal capsule, the nerve fibers were present around blood vessels and showed immunoreactivities for SP/ CGRP, SP/NPY, SP/NOS, and SP/vasoactive intestinal polypeptide, suggesting that the origin of nerve fibers in the capsule may differ from those in the medulla.     

Fine structural survey of tyrosine hydroxylase immunoreactive terminals in the myenteric ganglion of the rat duodenum

We have examined whether the noradrenergic neurons have direct synaptic projections to the myenteric ganglion neurons of the duodenum and the ultrastructure of their terminals by using immunogold–silver labeling for tyrosine hydroxylase. In the neuropil of the myenteric ganglia, about half of the axon terminals contained round clear vesicles and the rest of them contained pleomorphic clear vesicles. The sizes of axon terminals contacting the dendrites as a whole were 1.62 ± 0.07 μm. All axon terminals formed asymmetric synaptic contacts with dendrites or somata. Immunohistochemical study revealed that the tyrosine hydroxylase-immunoreactive nerve terminals were distributed throughout the ganglia and contained exclusively pleomorphic clear synaptic vesicles (about 20–80 nm long). The tyrosine hydroxylase-immunoreactive terminals were generally large (1.99 ± 0.07 μm). A considerable number of the tyrosine hydroxylase-immunoreactive terminals made asymmetric synaptic contacts with small dendrites, spines or somata of the myenteric ganglion neurons. Serial ultrathin sections through the myenteric neurons revealed that about 16% of the total number of axosomatic terminals showed tyrosine hydroxylase immunoreactivity. These results indicated that the myenteric ganglion neurons of the duodenum receive direct synaptic projection of sympathetic noradrenergic neurons and that their terminals contain pleomorphic vesicles and form asymmetric synaptic contacts.     

Orexin-A immunoreactive cells and fibers in the central nervous system of the axolotl brain and their association with tyrosine hydroxylase and serotonin immunoreactive somata

Orexin-A-like immunoreactivity in the axolotl brain was investigated by immunohistochemistry. Immunoreactive somata formed a single group in the hypothalamus, but were distributed beyond several nuclei, namely, the ventral aspect of the nucleus preopticus posterior, dorsal aspect of the nucleus suprachiasmaticus and anterior aspect of the pars ventralis hypothalami. Immunoreactive fibers were distributed throughout the brain from the olfactory bulb to the spinal cord except the cerebellum. The densest immunoreactive fibers were seen in the medial forebrain bundle and caudal lateral forebrain bundle. The largest number of immunoreactive puncta were seen in the mesencephalic tectum in addition to the hypothalamus. Immunoelectron microscopic analysis revealed the presence of synaptoid connections of immunoreactive fibers on neuronal somata in the tectum. The function of the mesencephalic system in the urodele seems to be sensory integration, suggesting that the orexin-A nervous system is associated with the modulation of sensory inputs. Orexin-A immunoreactive puncta were also observed on catecholaminergic and serotonergic somata. In view of the restricted somatic distribution in the hypothalamus, wide distribution of fibers throughout the central nervous system (CNS), and intimate association with monoaminergic somata, the orexin nervous system in the axolotl CNS is similar to those of other vertebrates, suggesting that this system is essential for brain functions throughout vertebrates.     

Localization of 5-hydroxytryptamine-like immunoreactive cells and nerve fibers in the rat female reproductive system

The presence of 5-hydroxytryptamine (5-HT)-like immunoreactivity (IR) was studied in the rat female reproductive system using polyclonal antibodies directed against 5-HT. Moreover, 5-HT levels in the ovary, Oviduct, uterus, and cervix were measured by high-pressure liquid chromatography with electrochemical detection. The highest 5-HT concentrations were found in the oviduct, followed in descending order by the cervix, the ovary, and the uterus. Most 5-HT-like IR was observed in the cytoplasm of mast cells. These cells were found in the connective tissue around the fimbria, in the oviduct, in the uterus, and in the ovary. Mast cells are clustered in the proximity of the parenchymal blood vessels. Moreover, a few 5-HT-like nerve fibers were found distributed mainly perivascularily in the uterine cervix and in the uterine horns as well as in the oviduct. IR nerve fibers were rarely seen within the ovary. The present data provide direct evidence that 5-HT in the female reproductive system not only is associated with mast cells but is located in nerve fibre-like structures as well. The functional significance of this probable 5-HT-ergic innervation of the female reproductive tract discovered in the present study should be clarified in future investigations. © 1992 Wiley-Liss, Inc.     

Localization of 5-hydroxytryptamine-like immunoreactive cells and nerve fibers in the rat female reproductive system.

The presence of 5-hydroxytryptamine (5-HT)-like immunoreactivity (IR) was studied in the rat female reproductive system using polyclonal antibodies directed against 5-HT. Moreover, 5-HT levels in the ovary, oviduct, uterus, and cervix were measured by high-pressure liquid chromatography with electrochemical detection. The highest 5-HT concentrations were found in the oviduct, followed in descending order by the cervix, the ovary, and the uterus. Most 5-HT-like IR was observed in the cytoplasm of mast cells. These cells were found in the connective tissue around the fimbria, in the oviduct, in the uterus, and in the ovary. Mast cells are clustered in the proximity of the parenchymal blood vessels. Moreover, a few 5-HT-like nerve fibers were found distributed mainly perivascularily in the uterine cervix and in the uterine horns as well as in the oviduct. IR nerve fibers were rarely seen within the ovary. The present data provide direct evidence that 5-HT in the female reproductive system not only is associated with mast cells but is located in nerve fibre-like structures as well. The functional significance of this probable 5-HT-ergic innervation of the female reproductive tract discovered in the present study should be clarified in future investigations.     

Distribution of calcitonin gene-related peptide immunoreactive nerve fibers in the human submandibular gland

The indirect immunofluorescence technique was used to study the distribution of calcitonin gene-related peptide (CGRP) in human submandibular gland. A relatively low number of thin varicose fibers with intense immunofluorescence for CGRP was seen in samples from seven glands. These CGRP-immunoreactive (CGRP-IR) nerve fibers were mainly seen around or in close contact with intra- and interlobular blood vessels. Some CGRP-IR nerve fibers were also found in association with intra- and interlobular salivary ducts and a few around the submandibular acini. By visual estimation there was no difference in the density of CGRP-IR nerve fibers between specimens of recurrent duct obstruction and laryngeal carcinoma. The present results show that the distribution of CGRP-IR nerve fibers in the stroma and in the glandular secretory elements of the human submandibular gland is quite similar to that seen in the rat and the ferret, which have been reported earlier. Furthermore, the regional distribution of CGRP-IR fibers in the human submandibular gland suggests that CGRP has a physiological role in the regulation of salivary gland function in human salivary glands, e.g. blood flow and secretion.     

Enkephalin-like immunoreactive principal ganglion cells and nerve fibres in the inferior mesenteric ganglion of the cat

The occurrence and distribution of methionine-enkephalin (ME), leucine-enkephalin (LE) and methionine-enkephalin-Arg6-Gly7-Leu8 (MERGL)-like (LI) immunoreactive material in the inferior mesenteric ganglion (IMG) of the cat were studied by immunohistochemical techniques using the peroxidase-antiperoxidase method. Numerous ME-Li, LE-Li and MERGL-Li immunoreactive fibres with the same distribution pattern were observed. They were varicose and often surrounded closely neighbouring unlabelled ganglion cell bodies. Sometimes they ran in strands between ganglion cells. ME-Li immunoreactive material was detected in a number of cell bodies, the diameter of which was similar to that of unlabelled principal ganglion cell bodies, and which were probably Enk-Li-containing principal ganglion cells. These immunoreactive cells were often surrounded by ME-Li immunoreactive fibres. No LE-Li or MERGL-Li immunoreactive ganglion cell bodies were observed. The presence of ME-Li immunoreactive principal ganglion cells raises the possibility that the Enk-Li immunoreactive fibres present in the IMG may have a prevertebral ganglionic source. The possibility that the Enk-Li material present in nerve fibres might be derived from preproenkephalin-A was suggested by the occurrence of MERGL-Li immunoreactivity.     

Localization of tyrosine hydroxylase and phenylethanolamine N-methyltransferase immunoreactive cells in the medulla of the dog

The tyrosine hydroxylase (TH)- and phenylethanolamine N-methyltransferase (PNMT)-immunoreactive cells of the medulla are closely associated with cardiovascular control in both the cat and rat. Although it is often the species of choice for cardiovascular studies, no previous study had characterized these cell groups in the dog. The TH- and PNMT-immunoreactive cells of the dog were distributed much as they are in both cat and rat but with some species variations, which may be indicative of their functional role.     

Co-localization of vasoactive intestinal polypeptide and neuropeptide Y immunoreactivities in the nerve fibers of the rat adrenal gland

The co-localization of Vasoactive Intestinal Polypeptide (VIP) with Neuropeptide Y (NPY) or its C-flanking peptide (C-PON) was investigated with immunocytochemistry methods in the adrenal gland of the rat. Most of the VIP immunoreactive (+) nerve fibers found in the capsule/glomerular zone also exhibited NPY or C-PON immunoreactivity (IR). We found that at least two populations of VIP varicose nerve fibers can be observed, the most prevalent exhibited both VIP/NPY or VIP/C-PON IR and the other which was rather scarce lacked NPY or C-PON IR. In the superficial cortex VIP/NPY or VIP/C-PON IR nerve fibers were often associated with capsular or subcapsular vascularization and extended into the zona glomerulosa. In the deeper layers of the adrenal cortex radial fibers were closely associated with the inner vascularization of the zona fasciculata and reticularis. In the adrenal medulla NPY or C-PON immunoreactivity was associated with ganglion neurons as well as chromaffin cells; these last cells were always VIP (-). VIP and NPY/C-PON IR could be co-localized in catecholaminergic nerve terminals of the adrenal cortex but not in the adrenal medulla.     

Immunoelectron microscopic localization of relaxin in endometrial gland cells of the pregnant guinea pig

Endometrial gland cells in uteri from late-pregnant guinea pigs (day 60 to parturition) resembled typical protein-secreting cells. Extensive rough endoplasmic reticulum and well-developed Golgi complexes were evident. The most striking features of endometrial gland cells were accumulations of large (≈ 0.5 μm in diameter), dense, membrane-bounded granules. The granules were located in the supranuclear region of the cell and frequently occurred in close proximity to the plasma membrane adjacent to the lumen of the gland. Thin sections of endometrial gland cells treated with relaxin antiserum and either colloidal gold-protein A or colloidal gold-goat antirabbit IgG demonstrated that the granules contained relaxin. These studies provide additional evidence that the uterus of the guinea pig produces relaxin and support the hypothesis that uterin relaxin may play an important role in pregnancy and parturition in the guinea pig.     

Immunoelectron microscopic localization of thyroglobulin in the human thyroid gland

The microidentification of the organelles containing thyroglobulin (TG) in the follicular cells of human thyroid glands were studied by the immunoelectron microscopic method. Fresh human thyroid glands were used for the purification of TG. TG was purified by differential salt fractionation and DEAE-cellulose chromatography. Anti-human TG rabbit IgG antibody was obtained by immunization of this purified TG. F(ab')2 fragments of anti-TG rabbit IgG antibody were prepared by pepsin digestion. The specificity of the antibody was tested on an Ouchterlony immunodiffusion plate. Conjugation of the purified F(ab')2 fragments of anti-TG rabbit IgG antibody to horse radish peroxidase was performed in order to use the direct peroxidase labelled antibody method. Under immunohistochemical light microscopy, the luminal colloid and the follicular cells appeared heavily stained. Under immunoelectron microscopy, positive reactions were observed in the rough endoplasmic reticulum, the Golgi apparatus, and the apical cell border. To date, no clear direct evidence of the presence of TG in the rough endoplasmic reticulum and the Golgi apparatus of human thyroid cells has ever been reported in immunoelectron microscopic studies. This study indicates that the use of F(ab')2 fragments of anti-TG rabbit IgG antibody in immunoelectron microscopic study is useful for the identification of TG in organelles of human thyroid follicular cells.     

Serotonin immunoreactive nerve fibers and terminals in the rat pituitary--light- and electron-microscopic studies

Serotonin-immunoreactive nerve fibers were demonstrated to form a plexus in the intermediate lobe of rat pituitary. Such fibers were sporadic in the posterior lobe where they occurred exclusively in the zone adjacent to the intermediate lobe, and in the anterior lobe. With the aid of electron-microscopic immunocytochemistry, labeled fibers were shown to constitute the majority of neuronal processes found in the intercellular region of the intermediate lobe. They were frequently seen to establish synaptoid contacts with endocrine cells.     

Neuropeptide tyrosine (NPY)-like immunoreactivity in adrenal chromaffin cells and intraadrenal nerve fibers of rats

The present peroxidase-antiperoxidase immunohistochemical study demonstrated that approximately 50% of the total chromaffin cells of the rat adrenal medulla exhibited NPY-like immunoreactivity. The immunoreactive material was localized in the core of the chromaffin granules as well as diffusely in the cytoplasm. By combination of immunohistochemistry with noradrenaline-fluorescence microscopy, all NPY-immunoreactive chromaffin cells are nonfluorescent, indicating that all NPY-chromaffin cells co-store adrenaline. A comparison of two consecutive sections, each of which was processed for the immunostaining with anti-NPY and anti-Met-Enk-Arg-Gly-Leu antisera, respectively, indicated that NPY and preproenkephalin A and its derivatives coexist in approximately one-fifth of the total NPY-immunoreactive cells. In addition to the NPY-immunoreactive cells, a plexus of NPY-immunoreactive nerve fibers with varicosities was found in the subcapsular regions of the adrenal gland. The nerve fibers were often associated with small blood vessels and extended into the zona glomerulosa. Single NPY-immunoreactive fibers were sparsely distributed in the deeper regions of the cortex and in the medulla. Ganglion cells in the adrenal gland were not seen exhibiting intensely positive NPY-like immunoreactivity. The NPY-immunoreactive nerve fibers contained abundant small clear vesicles mixed with a few small and large granular vesicles. The immunoreactive material appeared on the granular cores as well as in the axoplasm. The NPY fibers were closely apposed to smooth muscle cells and pericytes of small blood vessels in the cortex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decreased tyrosine hydroxylase activity in the adrenal gland of adult rats that were handled in infancy

Rats that have been handled for the first 20 days of life have less adrenal tyrosine hydroxylase activity than animals that have not had this stimulation. This decrease in activity is observed at 35, 100, and 230 days of age.     

Early expression of chromogranin A and tyrosine hydroxylase during prenatal development of the bovine adrenal gland

The present study was undertaken to define the temporal pattern and distribution of cells positive for chromogranin A (CgA) and tyrosine hydroxylase (TH) in various developmental stages of fetal bovine adrenal gland. CgA is an acidic protein, co-stored and co-released with amines and a variety of peptide hormones and neurotransmitters in dense core vesicles of neural and endocrine cells and can be used as a marker for these cells and their malignant counterparts. TH is the rate-limiting enzyme in catecholamine biosynthesis and reflects noradrenergic differentiation. The expression of CgA and TH was examined by immunohistochemistry. CgA immunoreactivity appears first in 35-day-old bovine fetuses. By the end of the second month, CgA-labelled cells are scattered throughout the entire primordium of the adrenal gland, and at a fetal age of 85–91 days most of these cells concentrate in the developing adrenal medulla. From this stage onwards, immunoreactive cells of the marginal zone of the medulla exhibit significantly stronger CgA immunoreaction than the central area. TH immunoreactivity appeared in the adrenal primordium for the first time at the end of the second month of gestation. The distribution pattern of TH-positive cells was similar to that described for CgA, and no significant differences in topographical arrangement between TH- and CgA-positive cells can be detected. The results show that bovine adrenal chromaffin cells express CgA already during their earliest stages of development and prior to TH. The stronger immunoreaction of marginal adrenal medullary cells suggests an adrenalcortical effect of glucocorticoids on the expression of CgA.     

An electron microscopic study on substance P-like immunoreactive nerve fibers in the pars distalis of the adenohypophysis in the dog.

The pars distalis of the adenohypophysis has recently been proved to be innervated by substance P-like immunoreactive nerve fibers in the monkey and the dog. The present study investigated immuno-electron microscopically the morphology of these nerve fibers and their relationship to anterior pituitary gland cells in the dog. The nerve fibers were unmyelinated and varicose. Direct contacts between them and every cell type of the gland could be ascertained, including folliculostellate cells. Typical synapses were identified on somatotropes and corticotropes. Most of them were of asymmetrical type with round to oval small clear vesicles and scattered large dense-cored vesicles. Occasional symmetrical type synapses were also seen. The results imply that substance P-like immunoreactive nerve fibers have an effector role in the pars distalis of the dog.