Kazuyuki Ono

Immunohistochemical and histochemical evidence for the presence of noradrenaline, serotonin and gamma-aminobutyric acid in chief cells of the mouse carotid body

The immunohistochemical study revealed tyrosine hydroxylase (TH), dopamine β-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), serotonin, glutamate decarboxylase (GAD) and γ-aminobutyric acid (GABA) immunoreactivities in the mouse carotid body. TH and DBH immunoreactivities were found in almost all chief cells and a few ganglion cells, and in relatively numerous varicose nerve fibers of the carotid body. The histofluorescence microscopy showed catecholamine fluorescence in almost all chief cells. However, no PNMT immunoreactivity was observed in the carotid body. Serotonin, GAD and GABA immunoreactivities were also seen in almost all chief cells of the carotid body. From combined immunohistochemistry and fluorescence histochemistry, catecholamine and serotonin or catecholamine and GABA were colocalized in almost all chief cells. Thus, these findings suggest that noradrenaline, serotonin and GABA may be synthesized and co-exist in almost all chief cells of the mouse carotid body and may play roles in chemoreceptive functions.

Ganglion cells immunoreactive for catecholamine-synthesizing enzymes, neuropeptide Y and vasoactive intestinal polypeptide in the rat adrenal gland

Immunohistochemistry has been used to demonstrate tyrosine hydroxylase (TH), dopamine-β-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT), neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) immunoreactivities, and acetylcholinesterase (AChE) activity was demonstrated in rat adrenal glands. The TH, DBH, NPY and VIP immunoreactivities and AChE activity were observed in both the large ganglion cells and the small chromaffin cells whereas PNMT immunoreactivity was found only in chromaffin cells, and not in ganglion cells. Most intraadrenal ganglion cells showed NPY immunoreactivity and a few were VIP immunoreactive. Numerous NPY-immunoreactive ganglion cells were also immunoreactive for TH and DBH; these cells were localized as single cells or groups of several cells in the adrenal cortex and medulla. Use of serial sections, or double and triple staining techniques, showed that all TH- and DBH-immunoreactive ganglion cells also showed NPY immunoreactivity, whereas some NPY-immunoreactive ganglion cells were TH and DBH immunonegative. NPY-immunoreactive ganglion cells showed no VIP immunoreactivity. AChE activity was seen in VIP-immunopositive and VIP-immunonegative ganglion cells. These results suggest that ganglion cells containing noradrenaline and NPY, or NPY only, or VIP and acetylcholine occur in the rat adrenal gland; they may project within the adrenal gland or to other target organs. TH, DBH, NPY, and VIP were colocalized in numerous immunoreactive nerve fibres, which were distributed in the superficial adrenal cortex, while TH-, DBH- and NPY-immunoreactive ganglion cells and nerve fibres were different from VIP-immunoreactive ganglion cells and nerve fibres in the medulla. This suggests that the immunoreactive nerve fibres in the superficial cortex may be mainly extrinsic in origin and may be different from those in the medulla.

Quantitative Light Microscopic Observations on Paneth Cells of Germ-Free and Ex-Germ-Free Wistar Rats

Germ-free rats were inoculated with bacteria from feces of SPF rats, and the Paneth cells in the ileal crypts were observed at different time intervals after inoculation. 12 h after inoculation, the Paneth cells showed a striking degranulation and the occurrence of supranuclear vacuoles. The Paneth cell area was significantly reduced. Four days to 3 weeks after inoculation, the secretory granules of Paneth cells were abundant, and both number and area of Paneth cells showed a progressive increase coming close to the data in SPF rats. The present study demonstrates interrelationships between secretory activity of Paneth cells and the microbial milieu in the small intestine and that the presently used experimental model is well suited for examining the histophysiology of Paneth cells. Moreover, the results suggest that Paneth cell numbers may also be closely related to crypt length.

Configuration of myoepithelial cells in various exocrine glands of guinea pigs

To study the configuration of myoepithelial cells, we isolated glandular endpieces of various guinea pig glands by collagenase, and visualized the myoepithelial cells by immunohistochemistry for actin, or by Bodipy-phallacidin, under both a light microscope and laser scanning confocal microscopes. In parotid and mandibular glands, the glandular acini were small (about 20–30 μm diameter) and spherical, and each acinus had one or two myoepithelial cells attached that were stellate in shape (central cell body and four to six thin processes). Most of the basal surface of the glandular cells was not covered by myoepithelial cells, and processes often extended to the neighboring acinus. The tubular glandular endpieces of the major sublingual gland, which secretes a mucous substance, were almost fully encircled by bandlike myoepithelial cells (about 3–6 μm wide). Although there were many differences between the lacrimal gland and the Harderian gland (e.g., the secretory product of the lacrimal gland was mucous, and glandular lumina were narrow; the Harderian gland secreted lipids and showed wide lumina), the outer contours of both glandular endpieces were the same (about 50–100 μm diameter, ellipsoid or spherical in shape). In both glands, 5–20 stellate myoepithelial cells were attached onto a glandular endpiece, and their arrangement had a lacy appearance. Actin filaments in myoepithelial cells aggregated and formed bundles in the broad processes and cell bodies. The bundles ran across the cell body, but there was no point where the bundles converged. In the arborization, some distal processes reversed their direction. We conclude that the configuration of myoepithelial cells depends on the outer contour of the glandular endpieces rather than on the secretory material or luminal width. The variety of myoepithelial cell configurations in the different exocrine glands we examined suggests that it is quite difficult to assign to myoepithelial cells the general role of expelling secretory products from glandular lumina. These cells seem to maintain the contour of the glandular endpieces, serving as the exoskeleton of the endpieces.

Immunocytochemical study of tyrosine hydroxylase and dopamine β-hydroxylase immunoreactivities in the rat pancreas

An immunohistochemical and immunoelectron microscopic study was used to demonstrate tyrosine hydroxylase (TH) and dopamine β-hydroxylase (DBH) immunoreactivities in the rat pancreas. Small TH immunoreactive cells were found in close contact with large TH immunonegative ganglion cells among the exocrine glands and were occasionally found in some islets. Some of these TH immunoreactive cells were also DBH immunopositive. The immunoreaction product was seen diffusely in the cytoplasm and in the granule cores of TH immunoreactive cells. All intra-pancreatic ganglion cells were immunoreactive for DBH, but not for TH. The TH immunoreactive cells were identified as small intensely fluorescent (SIF) cells due to their localization and morphological characteristics and showed no insulin, glucagon, somatostatin or pancreatic polypeptide immunoreactivities. These results indicate that SIF cells may release dopamine or noradrenaline to adequate stimuli while the intra-pancreatic ganglion cells with only DBH may not synthesize catecholamines in a normal biosynthetic pathway. TH immunoreactive nerve bundles without varicosities and fibers with varicosities, associated or unassociated with blood vessels, were found in both the exocrine and endocrine pancreas. Close apposition of TH immunoreactive nerve fibers to the smooth muscle and endothelial cells of the blood vessels was observed. A close apposition between TH immunoreactive nerve fibers and exocrine acinar cells and islet endocrine cells was sometimes found in the pancreas. The immunoreaction product was seen diffusely in the axoplasm and in the granular vesicles of the immunoreactive nerve fibers. Since no TH immunoreactive ganglion cells were present in the rat pancreas, the present study suggests that noradrenergic nerve fibers in the pancreas may be extrinsic in origin, and may exert an effect on the regulation of blood flow and on the secretory acitivity of the acinar cells, duct cells and endocrine cells.

Secretion Mode of the Harderian Gland of Rats after Stimulation by Cholinergic Secretagogues

We studied the morphological changes in rat Harderian glands 30 min after injection of cholinergic secretagogues. In controls, the glands exhibited a tubuloalveolar structure with relatively wide lumina, in which some osmiophilic dense droplets exocytosed from glandular cells were observed. Also two types of glandular cells (type A cells and type B cells sometimes showing exocytotic figures of lipid-secretory vacuoles) and myoepithelial cells were recognized. After injection of carbamylcholine chloride (subcutaneously, 0.1 mg/kg body weight), which has both nicotinic and muscarinic actions, many of the alveolar lumina dilated and contained a small number of osmiophilic droplets. Exocytotic figures in both types of cells and a pronounced decrease in the number of vacuoles in the glandular cells were observed. However, there was no evidence of apocrine or holocrine secretion. The injection of the higher dose of carbamylcholine (1.0 mg/kg) caused fusion of secretory vacuoles in the apical cytoplasm and contraction of myoepithelial cells. Most alveoli showed no clear lumina; their centers were jammed with cytoplasmic fragments and accumulated secretory products. Massive discharge of cytoplasmic fragments containing some secretory vacuoles was often observed. This may be classified as apocrine secretion. Bethanechol chloride (subcutaneous injection, 1.0 mg/kg), a muscarinic agonist, stimulated the Harderian-gland secretion, and enhanced exocytosis was observed. The discharge from the glandular cells, following injection of various doses of carbamylcholine, were almost inhibited by atropine sulfate, a muscarinic antagonist. The present results suggested that the cholinergic systems regulate the secretion of rat Harderian-gland cells which have muscarinic receptors.

Immunohistochemical observations of immunoglobulin A in the Paneth cells of germ-free and formerly-germ-free rats.

SummaryThe localization of secretory immunoglobulin A (IgA) in Paneth cells was immunohistochemically studied in germ-free (Gf) and ex-Gf rats that had been injected with feces obtained from specific-pathogen-free (SPF) rats. In Gf as well as SPF rats, the secretory granules of Paneth cells and the brush borders of crypt cells exhibited IgA immunoreactivity. At 12 and 24 h after inoculation, it was found that, concomitant with the occurrence of considerable degranulation, the IgA immunoreactivity in Paneth cells disappeared, except of the margin of supranuclear vacuoles. In contrast, the IgA immunoreactivity of the crypt-cell brush borders was unchanged. Four days after inoculation, secretory granules exhibiting IgA immunoreactivity reaccumulated in Paneth cells. The present study suggests that Paneth cells regulate the bacterial milieu in the intestine by releasing secretory granules containing IgA into the crypt lumen.

Gamma-aminobutyric acid (GABA) immunoreactivity in the mouse adrenal gland

Gamma-aminobutyric acid (GABA) immunoreactivity was revealed by immunocytochemistry in the mouse adrenal gland at the light and electron microscopic levels. Groups of weakly or faintly GABA immunoreactive chromaffin cells were often seen in the adrenal medulla. By means of immunohistochemistry combined with fluorescent microscopy, these GABA immunoreactive chromaffin cells showed noradrenaline fluorescence. The immunoreaction product was seen mainly in the granular cores of these noradrenaline cells. These results suggest the co-existence of GABA and noradrenaline within the chromaffin granules. Sometimes thick or thin bundles of GABA immunoreactive nerve fibers with or without varicosities were found running through the cortex directly into the medulla. In the medulla, GABA immunoreactive varicose nerve fibers were numerous and were often in close contact with small adrenaline cells and large ganglion cells; a few, however, surrounded clusters of the noradrenaline cells, where membrane specializations were formed. Single GABA immunoreactive nerve fibers, and thin or thick bundles of the immunoreactive varicose nerve fibers ran along the blood vessels in the medulla. The immunoreaction deposits were observed diffusely in the axoplasm and in small agranular vesicles of the GABA immunoreactive nerve fibers. Since no ganglion cells with GABA immunoreactivity were found in the adrenal gland, the GABA immunoreactive nerve fibers are regarded as extrinsic in origin.

Immunohistochemical observations of lysozyme in the Paneth cells of specific-pathogen-free and germ-free mice.

The localization of lysozyme, which may function as an antibacterial agent, was immunohistochemically studied on the mouse Paneth cell secretory granules showing bipartite substructures (central core and peripheral halo). The lysozyme immunoreactivity was observed in the core, but not in the halo. Even in germ-free mice, Paneth cells have many secretory granules and their cores show lysozyme immunoreactivity. It seems likely that mice Paneth cells possess the ability to produce secretory granules containing lysozyme in disregard of the intestinal bacterial milieu.

Postnatal development of neuropeptide Y- and calcitonin gene-related peptide-immunoreactive nerves in the rat urinary bladder

The postnatal development of neuropeptide Y- and calcitonin gene-related peptide-immunoreactive (NPY-IR and CGRP-IR) nerve fibers in the rat urinary bladder was investigated using whole-mount preparations and cryostat sections. In newborn and 3-day-old rats, many NPY-IR nerve fibers were observed in the subserous and muscle layers. Many NPY-IR nerve cell bodies clustered at branching points of the subserous nerve bundles. Within 4 weeks after birth, these cell bodies drastically decreased in number and spread along the bundles, although the number of NPY-IR nerve fibers increased moderately. In contrast, CGRP-IR nerve fibers in newborn and 3-day-old rats were less developed, and no CGRP-IR nerve cell body was observed in any rat. However, CGRP-IR nerve fiber distribution in the urinary tissues conspicuously increased within 4 weeks after birth. Especially, an increase of the infraepithelial fibers showing a meshwork appearance was prominent in the fundus and corpus of the bladder. The infra- and intraepithelial CGRP-IR nerve meshwork of the ventral wall was more dense than that of the trigone. At 4 weeks, NPY-IR and CGRP-IR nerves were similar to those of the adult rat (8–12 weeks old). The present study suggests a correlation between the development of the peripheral nervous system in the urinary bladder and maturation of micturition behavior in the rat.