Urinary bladder innervation in human foetuses of 13 to 25 weeks has been studied by electron microscopy and glyoxylic acid and acetylcholinesterase histochemical methods. Cholinergic fibers appear as a faintly stained dense plexus at 13 weeks. An increase in thickness and intensity of staining of the cholinergic nerve bundles occurs over the foetal age period under study. At 25 weeks, the finer cholinergic plexuses are demonstrable on the muscle fibers. Adrenergic innervation, on the other hand, becomes apparent at 16 weeks as faintly stained plexuses along the blood vessels in the trigone and is also sparsely seen in the wall of the fundus. By 25 weeks the intensity of staining increases. plexuses appear around the ganglionic clusters in the adventitia and in the wall of the urinary bladder; intensely fluorescent neurons and individual adrenergic fibers in the muscle layers are also seen. Ultrastructurally the muscle fibers undergo maturation by 19 weeks with the development of myofibrils and increasing number of intercellular contacts. The axonal profiles in all the layers of the bladder wall of foetuses from 13 to 19 weeks contain large numbers of small granulated vesicles and some have large vesicles with a halo around them. Neuromuscular synaptic zones seen by 16 weeks do not show the typical structure and generally contain clear vesicles in the presynaptic varicosities.
One of the histochemical characteristics of ceroid pigment is the existence of ethylenic double bonds in protein-bound unsaturated lipids. For electronmicroscopic demonstration of these double bonds, the authors modified the bromine silver method, which was developed by Norton et al. originally for light microscopic demonstration of unsaturated lipids. Using this method, we observed a precipitation of the fine silver grains in highly or moderately electron-dense areas of ceroid-like pigment bodies, evidently showing the presence of double bonds at these sites.
Calmodulin is a small, acidic calcium-binding protein concentrated in the nervous system. The immunohistochemical localization of calmodulin in the developing mouse brain was studied light-microscopically using the specific anti-calmodulin IgG which was elicited in the rabbit with native calmodulin of bovine brain. The immunoreactivity of the cell somata and their processes found in the adult brain did not appear simultaneously but in some sequential order with the regions of the brain. On the other hand, immunoreactivity disappeared during the development. This reaction of disappearance was also abolished by the neutralization of the antibody with calmodulin. From the results of the appearance as well as the disappearance of the immunoreactivity, the pattern of the immunostaining reached that of the adult by 17 days of age.
The activities of Δ5-3β-hydroxysteroid dehydrogenase (3β-HSD; EC 220.127.116.11) and glucose-6-phosphate dehydrogenase (G-6-PDH; EC 18.104.22.168) in the adrenal cortex of virgin rats during the estrous cycle were measured by microspectro-photometry. 3β-HSD activity was found throughout the cortex, and was especially high in the outer part of the zona fasciculata and the juxtamedullar cell layer during estrus. The HSD-positive cells had a high lipid content, a well developed smooth endoplasmic reticulum (SER) and globular mitochondria with many vesicular cristae. Reaction products of 3β-HSD were found on the outer surface membrane of the SER, and in the cristae of the mitochondria, particularly in the swollen cristae. The distribution of G-6-PDH was similar to that of 3β-HSD. These results suggest that steroidogenesis in the adrenal cortex increases in estrus, activating both the SER and mitochondria in cells of the zona fasciculata and the juxtamedullar cell layer.
Immunocytochemical localization of glutathione-peroxidase (GSH-PO) in the rat adrenocortical cells was observed not only in cytosol (CYTOSOL GSH-PO) but in mitochondria (MITOCHONDRIAL GSH-PO) by the direct peroxidase labeled antibody method using anti-GSH-PO IgG Fab' fragment. In the untreated rat adrenocortical cells, CYTOSOL GSH-PO was observed mainly in the outer part of the zona fasciculata and zona reticularis. MITOCHONDRIAL GSH-PO was observed mainly in the inner part of zona fasciculata. Biochemical GSH-PO activity in the cytosol and mitochondrial fractions obtained from whole adrenal gland was comparable to the immunocytochemical results. In the hypophysectomized rat adrenocortical cells, markedly atrophic inner part of zona fasciculata showed only CYTOSOL GSH-PO, and biochemical activity of the cytosol GSH-PO was markedly increased. It has been reported by us that this atrophic zone contains accumulated cholesterol esters. By ACTH administration to the hypophysectomized rats, markedly, hypertrophic outer part of zona fasciculata showed increased immunocytochemical localization of MITOCHONDRIAL GSH-PO. And biochemically, mitochondrial GSH-PO activity in this group was higher than that of the hypophysectomized group. These results suggest that the CYTOSOL GSH-PO has its role in the reduction of cholesterol ester hydroperoxides and that the MITOCHONDRIAL GSH-PO in the reduction of sterol hydroperoxides produced during steroidogenesis.
Immunohisto-cytochemical localization of glutathione-peroxidase (GSH-PO) in adrenocortical cells was studied on rats treated with aminoglutethimide (Elipten), which blocks the conversion of cholesterol into pregnenolone, and 4-aminopyrazolopyrimidine (4-APP), which increases the adrenocortical cholesterol synthesis. In a normal untreated group, GSH-PO was observed immunohistochemically in zonae fasciculata and reticularis and intracellular localization of GSH-PO was observed not only in cytosol but in mitochondria. In Elipten treated group, immunohistochemically, GSH-PO was mainly observed in lipid accumulated zona fasciculata. And intracellular localization of GSH-PO was mainly observed in cytosol near the electron dense lipid droplets. Intensity of immunocytochemical staining of GSH-PO was stronger than that in the normal untreated group. However, number of GSH-PO positive mitochondria was less than that in the normal untreated group. In 4-APP treated group, immunohistochemical localization of GSH-PO was mainly observed in inner fasciculata-reticularis and intracellular localization of GSH-PO was mostly observed in cytosol near the smooth endoplasmic reticulum. These findings may suggest that GSH-PO plays an important role in prevention of damage to mitochondrial and microsomal components with lipid peroxidation.
In the goldfish brain, eleven groups of tyrosine hydroxylase (TH) -positive (catecholaminergic neurons and four groups of serotonin (5-HT) -positive (serotonergic) neurons are described as follows by immunofluorescence and light microscopic level of PAP technique: 1) The olfactory bulb (TH); 2) The area ventralis telencephali (TH); 3) The nucleus preopticus (TH); 4) The nucleus dorsolateralis thalami (TH); a) The pretectal area (5-HT); 5) The nucleus ventromedialis thalami (TH); 6) The nucleus posterioris periventricularis (PVO-accompanying cells) (TH); b) The nucleus recessus lateralis (NRL) (5-HT); 7) The rostro-medial part of the nucleus recessus lateralis (NRL) or the paraventricular organ (PVO) pars anterior (TH); b) PVO pars anterior (5-HT); 8) The nucleus posterior tuberis (NPT) (TH); c) The nucleus recessus posterioris (NRP) (5-HT); 9) The lateral isthmal region (TH); d) The raphe nuclei of the brainstem (5-HT); 10) The reticular formation of the medulla oblongata (TH); and 11) The post-obecular region (TH). By immunoelectron microscopy, two kinds of cerebrospinal fluid (CSF) -contacting neurons were confirmed in the PVO pars anterior. One contained TH-positive granules (80-120 nm) and the other 5-HT-immunoreactive granules (100-200 nm in diameter). In the telencephalon, preoptic nucleus (PON) and nucleus posterior tuberis (NPT), TH-positive non-CSF-contacting neurons showed similar ultrastructure such as well developed rough surfaced endoplasmic reticulum, and immunoreactive granules (106-110 nm).
Vasopressin-, oxytocin-, somatostatin-, neurotensin-, and TRH-like immunoreactive neurons in respect to their distribution in the paraventricular nucleus of the rat were examined by light microscopic immunocytochemistry using the peroxidase-anti-peroxidase (PAP) method. Distinct and characteristic patterns were observed for each peptide neuron. The coexistence of different peptide-containing neurons and the effect of the ascending catecholamine neuron system upon peptide neurons in the paraventricular nucleus are briefly discussed.
Morphological study at the light and electron microscopic levels was carried out on the posterior group of the magnocellular neurons in the hypothalamus of the rat as revealed by monoamine oxidase (MAO) histochemistry. A conspicuously high activity of type B MAO was found in the perikarya and dendritic processes of these neurons. The dendritic processes ramified into fine varicose fibers which ran in all directions throughout the hypothalamus. Axodendritic synapses were frequently observed on the MAO-positive dendritic trunks and varicosities, some of which were filled with the small round vesicles. The ventrally directed dendrites richly branched into a dense network at the basal surface of the posterior hypothalamus and penetrated the limiting astrocytes where they appeared to have a close association with cerebrospinal fluid. In addition, these neurons showed an ability to synthesize serotoin after administration of the immediate precursor, 5-hydroxytryptophan. The possible neuronal and humoral functions of the magnocellular neurons in the posterior hypothalamus is discussed.