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Are adrenergic neurons subject to a serotoninergic influence in the nucleus tractus solitarii? A morphological and biochemical study in the rat.

The possible relationships between adrenaline-synthesizing neurons and serotoninergic afferent fibers in the nucleus tractus solitarii of the rat were investigated both morphologically and biochemically. Adrenergic elements (cell bodies, dendrites and nerve endings) were detected simultaneously with serotoninergic axonal varicosities in the same electron-microscopic sections by means of combined phenylethanolamine-N-methyltransferase immunocytochemistry and [3H]serotonin-uptake radioautography. Among some 500 serotoninergic varicosities scanned in the areas of significant overlap between the 2 types of labeling, only 3 were directly apposed to an adrenergic process, identified as a dendrite in each case. No synaptic membrane differentiations were seen at these occasional sites of contact. Destruction of the serotonin input by 5,7-dihydroxytryptamine had no significant effect on the tyrosine hydroxylase dopamine-beta-hydroxylase and phenylethanolamine-N-methyltransferase enzymatic activities in the C2 adrenergic region, but induced 22% and 38% increases of tyrosine hydroxylase and dopamine-beta-hydroxylase activities, respectively, in the neighboring A2 noradrenergic area. Taken together, these results suggest that serotoninergic and adrenergic neurons do not significantly interact in the nucleus tractus solitarii; this implies that the possible catecholaminergic relays for the action of serotonin in autonomic regulation at this level could consist of noradrenergic neurons rather than of their adrenergic counterparts.

Animals↗

Morphological correlates of serotonin-neuropeptide Y interactions in the rat suprachiasmatic nucleus: combined radioautographic and immunocytochemical data.

The morphological substrate of putative serotonin (5-HT)/neuropeptide Y (NPY) interactions in the suprachiasmatic nucleus (SCN) was investigated by combined radioautography and immunocytochemistry after intraventricular administration of (3H)5-HT in the rat. In the ventral portion of the SCN, the distribution of (3H)5-HT uptake sites overlapped closely the NPY-immunoreactive terminals. Previous investigations have shown that the dense 5-HT and NPY innervations of the SCN originate in different structures, i.e., the midbrain raphe nuclei and the ventral lateral geniculate nucleus, respectively. Accordingly, in the present study, destruction of 5-HT afferents by 5,7-dihydroxytryptamine was not found to induce any modification in NPY staining and, in ultrastructural immuno-radioautographic preparations, two distinct pools of axonal varicosities could be identified. Both 5-HT and NPY terminals established morphologically defined synaptic junctions, sometimes on the same neuronal target. Some cases of direct axo-axonic appositions between the two types of terminals were also encountered. These data constitute additional criteria for characterizing the cytological basis of the multiple transmitter interactions presumably involved in the function of the SCN as a central regulator of circadian biological rhythms.

Animals↗

Ultrastructural identification of catecholaminergic fibers in the goldfish pituitary. A high-resolution radioautographic study after in vitro 3H-dopamine administration.

The monoaminergic innervation of the goldfish pituitary gland was studied by means of light- and electron-microscopic radioautography after in vitro administration of 3H-dopamine. The tracer was specifically incorporated and retained by part of the type-B fibers innervating the different lobes of the pituitary. In the rostral pars distalis labeled fibers were most frequently observed in contact with the basement membrane separating the neurohypophysis and the adenohypophysis. In the proximal pars distalis and the pars intermedia, labeled profiles were detected in the neural tissue and in direct contact with the different types of secretory cells. According to the previous data concerning the uptake and retention of tritiated catecholamines in the central nervous system, it is assumed that the labeled fibers are mainly catecholaminergic (principally dopaminergic). This study provides morphological evidence for a neuroendocrine function of catecholamines in the goldfish.

Animals↗

Rate of protein synthesis in rat salivary gland cells after pilocarpine or feeding. II. Protein synthesis in vivo in cells of the submandibular and parotid gland after secretion.

The effect of pilocarpine and food uptake on the rate of incorporation of [3H]-leucine in vivo was measured by means of quantitative radioautography in three exocrine cells of the rat: the acinar and the granular duct cells of the submandibular and the acinar cells of the parotid gland. The three cell types react differently. The submandibular acinar cells showed a decrease in incorporation rate after pilocarpine administration but not after feeding. The incorporation rate of the granular duct cells of the submandibular gland remains constant after both stimulations. The acinar cells of the parotid gland show an increase in incorporation rate of [3H]-leucine in response to both. The contrast between the submandibular and the parotid gland could also be demonstrated radiobiochemically, the results reflecting the incorporation rates of the acinar cells of both glands, giving no information on the contribution of other cell types. The decrease in incorporation rate of the submandibular gland acinar cells is accompained by a shift of polyribosomes towards monomers.

Animals↗

Dynamic properties of axonal transport of proteins and glycoproteins: a study based on the effects of metaphase blocking drugs in the developing optic pathway of chick embryos.

Some properties of the axonal transport of proteins and glycoproteins along the optic pathway of chick embryos and newly hatched chicks were studied by labelling retinal ganglion cells with 3H-proline or 3H-fucose. A study of the effects of colchicine (COL) and vinblastine (VLB) on embryonic axonal transport was also carried out. Marked changes in the efficiency of axonal transport were found throughout development. In particular, the fraction of retinal ganglion cell proteins which is rapidly exported toward tectal terminals increases during embryonic life but steadily decreases after hatching. Glycoprotein transport behaves similarly except that its efficiency is relatively higher at stages when critical events of synaptic maturation in the tectum are reported to occur. Embryonic axonal transport is blocked by COL and VLB at very low intravitreal concentrations. Retinal protein synthesis and the morphology of ganglion cells are profoundly altered by the drugs: in general, COL and VLB effects were much more marked in embryonic than in mature neurons. An analysis of the time course of rapid transport along embryonic optic axons was carried out by reducing the efflux of labelled proteins from the eye by giving VLB intravitreally 2 h after the pulse. It revealed some peculiar features in the retino-tectal migration of glycoproteins and confirmed their progressive accumulation within terminals as previously described by radioautography. These results suggest that axonal transport of proteins during embryonic life undergoes changes in parallel with synaptic maturation. It may thus be considered as one of the factors controlling the genesis of neuronal networks.

Animals↗

Radioautographic study of glycoprotein biosynthesis and renewal in the ovarian follicles of mice and the origin of the zona pellucida.

L-fucose-3H was injected intravenously into mice which were killed at several time intervals after injection and semi-thin sections of their ovaries were processed for radioautography and analysed quantitatively; At the same time the specific activity of serum glycoproteins was determined. Glycoprotein biosynthesis was demonstrated in the oocytes, granulosa and stromal cells. The silver grain density of the follicular fluid in large follicles reached a peak at 4 h, remained high at 8 h after injection and decreased steadily at the subsequent intervals. It was demonstrated that the labeling pattern of the follicular fluid depends on the secretory activity of the granulosa cells and also on the specific activity of serum glycoproteins. The collapsed zonae pellucidae which represent the highest degree of follicular atresia are able to take up glycoprotein macromolecules. Based on this finding and also on the labeling pattern of the large follicles it was shown that there is very little synthesis of specific glycoproteins for the zona pellucida in large follicles. A more specific labeling of the zona pellucida occurred in the medium follicles. Following the growth of these follicles having a previously labeled zona pellucida, it was demonstrated that this extracellular structure is secreted by the oocyte.

Animals↗

Radioautographic study of the rat brain and pituitary after injection of 3H dexamethasone.

The central nervous system and the pituitary of adrenalectomized male rats injected with 3H-dexamethasone were examined by radioautography. At 1 hr after the injection, radioactivity concentration was high in the medial basal hypothalamus, the pituitary and the pineal gland. In the hypothalamus, radioactive material was found to be selectively concentrated in neurons in the ventral part of nucleus arcuatus and in the infundibular region. In the anterior pituitary, a large proportion of cells showed silver grains both in the cytoplasm and over the cell nuclei. However, in a small number of cells, the radioactive material was associated with the cell nuclei. Less radioactivity was present in the intermediate and posterior lobes. The pineal gland contained more silver grains than did other regions of the brain. The results obtained in the present study suggest essentially an action of dexamethasone in the medial basal hypothalamus and at the level of the pituitary.

Adrenalectomy↗

Localization of vitamin A in the small intestine of mouse. An electron microscope radioautographic study.

Localization of vitamin A in the small intestine of mice was studied with electron microscope radioautography after administration of tritiated vitamin A. The label was concentrated over lipid droplets in cells distributed in the lamina propria and the submucous layer. The cells were similar both to fibroblasts and to fat-storing cells in their morphological features. The name "Vitamin A-Storing Cell" is proposed for these labeled cells, including the fat-storing cell in the liver.

Animals↗

Light microscope radioautographic study of glycoprotein secretion in the hypothalamic-neurohypophysial system of the rat, after L-fucose-3H injection.

Fucose-3H was injected into the cerebral ventricle of rats that were killed at several time intervals after injection. Semi-thin sections of the supraoptic nucleus and neurohypophysis were processed for radioautography and analysed quantitatively. Silver grains indicating the site of fucoselabeled glycoproteins were first located at the perinuclear region of the secretory neurons. The highest silver-grain density in these cells was observed at 2 h after injection , declining afterwards. Silver grains over the neurohypophysis were observed from 2 h on, reached a peak at 1 day after injection and decreased in the subsequent time intervals. The distributions of the silver grains over the neurohypophysis fitted Poissonian distributions and these were shown to be heterogeneous at the several time intervals. Pituicytes were not labeled. The percentage of silver grains over the Herring bodies increased with time. In rats deprived of water after fucose-3H injection there was a great increase in the release of labeled glycoproteins from the neurohypophysis. These results indicate that the glycoproteins synthesized by the secretory neurons of the hypothalamic nuclei are secreted in the neurohypophysis.

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Electron microscopic observations on the castration-induced X zone in the adrenal cortex of male mice.

The secondary X zone induced by castration in the adrenal cortex of adult male mice was examined by electron microscopy and radioautography with 3H-thymidine. 10-15 days after castration a thin layer of small eosinophilic cells is formed in the inner-most cortex. Such eosinophilic cells contain irregulary shaped nuclei and spherical or ellipsoidal mitochondria with tubulolamellar cristae, 20-25 days after castration a prominent zone of small eosinophilic cells was clearly identified as the secondary X zone. The typical secondary X zone cells were characterized by the formation of peculiar mitochondrial complexes and whorled sER. The X zone cells with their characteristic organelles incorporated 3H-thymidine. The ultrastructure and formation of the secondary X zone were very similar to those of the primary X zone which appears during normal postnatal development. We demonstrate here the capacity of reticularis cells of adult male mice to transform into typical X zone cells following castration.

Adrenal Cortex↗

Ultastructural localization of polysaccharides in the vacuolar system of an established cell line.

Polysaccharides in the vacuolar system of an established line of monkey kidney epithelial cells, were investigated by high resolution radioautography following incorporation of 3H-fucose and by the periodic acid-thiosemicarbazide-silver proteinate staining method. Significant amounts of glycogen and glycoproteins were found in different lysosomes and prelysosomes. The radioautographic labeling of these substances correlated with the cytochemical observations. Analysis of the 3H-fucose pattern of labeling suggests that glycoproteins are transported through the different components of the vacuolar system of the cell in a sequential fashion rather than via independent pathways. The possible functional significance of polysaccharides in lysosomes is discussed. It is suggested that glycogen is taken up by lysosomes through autophagic segregation and through infolding and vesiculation of the lysosomal surface, and that the majority of glycoprotein in the lysosomal membranes does not have acid hydrolase activity.

Animals↗

Intracellular transport of sulfated macromolecules in parotid acinar cells.

Intracellular transport of sulfated macromolecules in parotid acinar cells was investigated by electron microscopic radioautography after injection of 35S-sulfate. Ten minutes after injection radiosulfate was concentrated in the Golgi region. By 1 hr, much of the radioactive material had been transported to condensing vacuoles. These vacuoles were subsequently transformed into zymogen granules which contained almost 70% of the radioactivity 4 hrs after injection. These results indicate that, in addition to its packaging function, the Golgi apparatus in parotid acinar cells is capable of utilizing inorganic sulfate for the production of sulfated macromolecules. These molecules, following an intracellular route similar to that taken by digestive enzymes, become an integral component of zymogen granules. The possibility that sulfated macromolecules play a role in exocrine secretion by aiding in the packaging of exportable proteins is discussed.

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Cell death and the regulation of populations of cells in the periodontal ligament.

The contribution of cell death in regulating cellular populations of periodontal ligament was studied in young adult rats. Mandibular first molar periodontium was prepared for light-microscopic radioautography after a pulse of 3H-thymidine in 6 rats and for electron microscopy in 4 rats. The labeling index for 3H-thymidine and the density of fibroblast-like cells were computed from radioautographs. The percentages of dying or dead cells and macrophages were computed from electron micrographs. The labeling index of cells within 20 microns of bone and cementum was significantly lower (p less than 0.01) than the labeling index within the body of the periodontal ligament. The patterns of cellular density and indices of death were the inverse of the labeling indices. Macrophages were plentiful (% macrophages = 3.68% +/- 0.30) and were clustered around blood vessels (mean distance from blood vessel = 2.3 microns). However, only 10% of dying or dead cells were within 10 microns of blood vessels. These data show that death of cells in the periodontal ligament may, in part, balance production of cells by mitosis. The relationships between labeling index, index of death, and cellular density suggest that cells born in the middle of the periodontal ligament may migrate to regions of high cellular density near bone and cementum, and that they may die there. Macrophages do not appear to be associated with dying cells of the periodontal ligament.

Animals↗

Incorporation of L-(3H)-fucose into glycoproteins of the adrenal gland of mice. Light microscope radioautographic study on semi-thin sections.

To study the biosynthesis and intracellular migration of glycoproteins in the adrenal gland, adult mice were injected intravenously with L-(3H)-fucose and killed from 10 min to 14 days after injection. Semi-thin sections of the adrenal glands were then processed for radioautography. Incorporation of labeled fucose occurred in the steroid-secreting cells of the three zones of the cortex as well as in the adrenalin (A) and noradrenalin (NA) cells of the medulla. At short intervals after injection, the main site of incorporation was the paranuclear region of the cells, suggesting uptake by the Golgi apparatus. Subsequently, labeled glycoproteins migrated from the paranuclear region to other cell sites. The labeling pattern observed in the adrenocortical parenchyme strongly suggests that the glycoproteins are transferred to lysosomes, lipofuscin granules and the cell coat (glycocalyx). Counts of silver grains clearly indicate that these glycoproteins undergo renewal. The qualitative and quantitative analysis of the radioautographs also suggest that glycoproteins, acting as intracellular carriers of steroids, may be released to the extracellular environment together with the hormones. Most of the glycoproteins synthesized by the A and NA cells of the adrenal medulla seem to be transferred to secretion granules in which they may play some role in the cytophysiology of these structures. It is likely that glycoproteins are released from the cells during exocytosis of secretory granules.

Adrenal Cortex↗

Quantitative radioautographic light and electron microscopic analysis of the localization of monoamines in the median eminence of the rat. I. Catecholamines.

By means of light and electron microscopic radioautography a quantitative study of the regional distribution of catecholamines in the median eminence of the rat was carried out. One hour after intraventricular injection of 3H-dopamine the highest radioautographic reaction was recorded in the external zone, especially in the lateral palisade zone where many neurosecretory terminals are separated from the basal lamina of the portal pericapillary space by a glial "cuff". This area showed the highest percentage (52%) of labelled catecholamine containing neurosecretory terminals as well as the maximal silver grain density per one terminal. In the medial palisade zone where direct neurovascular contacts with the capillary loops prevail, only 27 per cent of neurosecretory terminals were found to harbour tritiated dopamine. On the average 35 per cent of neurosecretory terminals in the median eminence of the rat contain catecholamines (both dopamine and noradrenaline). Pretreatment of animals with recretory terminals as well as grain density over terminals were decreased in both the medial and lateral palisade zones, although to a lesser degree in the latter. The site of origin of catecholamine fibers as well as the mode of catecholamine action at the level of the median eminence are discussed.

Animals↗

3H-fucose incorporation into bone following exposure to parathyroid hormone, dibutyryl cyclic-AMP and colchicine.

Radioautographic and scintillation counting procedures were used to examine the effect of parathyroid hormone (PTH), dibutyryl cyclic-AMP (DB-cAMP), and colchicine on the incorporation of 3H-fucose into macromolecular material in organ cultures of bone. Radioautography demonstrated 3H-fucose incorporation into bone cells, with the heaviest uptake occurring in osteoclasts. A minimal incorporation occurred in pre-osteoblasts and osteoblasts of the osteogenic periosteum, and in fibroblasts of the fibrous periosteum. PTH appeared to produce a heavier label in association with osteoclasts while decreasing the limited labeling associated with cells of the osteogenic and fibrous periosteum. DB-cAMP and colchicine both markedly reduced the labeling associated with osteoclasts, while the minimal labeling of other bone cells remained. By contrast, scintillation counting results indicated that PTH had little or no effect on 3H-fucose incorporation, while DB-cAMP and colchicine considerably reduced the amount of labeled macromolecular material. The incorporation of 3H-fucose into glycoproteins and the role of glycoproteins are discussed.

Animals↗

Quantative radioautographic light and electron microscopic analysis of the localization of monoamines in the median eminence of the rat.

Serotonin containing structures in the median eminence of the rat have been studied by quantitative light and electron microscopic radioautography following intraventricular infusion of tritiated 5-hydroxytryptophan. One hour after injection of the tracer the highest density of silver grains was recorded in the ependymal and external zones, especially in the lateral palisade zone. The proportion of labelled neurosecretory terminals was also larger in the lateral palisade zone (29%) as compared with the medial palisade zone (13%), although the mean number of developed silver grains per one terminal was higher in the latter. On the average, 16% of neurosecretory terminals sequestered radiolabelled 5-hydroxytryptophan in the external zone of the rat median eminence. It is suggested that serotonin, like catecholamines, is discharged from neurosecretory terminals localized in the external zone and via the portal circulation affects the function of the anterior pituitary. The sites of origin of serotoninergic structures of the median eminence as well as the possible role of monoamine (catecholamine and indolamine) neurohormones in a dual peptidergic and monoaminergic control of anterior pituitary functions are discussed.

Animals↗

Glycoprotein secretion in the hypothalamo-neurohypophyseal system of the rat.

Although the secretory products of the hypothalamo-neurohypophyseal system are not glycoproteins, synthesis and migration of these macromolecules occur within its secretory neurons. After being labeled with 3H-fucose in the Golgi apparatus, newly synthesized glycoproteins migrate to secretion granules, lysosomes and the plasma membrane of the secretory neurons, as demonstrated by quantitative electron-microscopic radioautography. Secretion granules bearing newly synthesized glycoproteins migrate to the pars nervosa, the labeling pattern of which was studied in rats killed from 4 h to 14 days after the isotope injection. Most of the silver grains were observed to overly the secretory axons. Labeling of pituicytes was negligible and the number of silver grains over the perivascular spaces was about 10% of the total at certain postinjection intervals. In the secretory axons, most of the silver grains over the different portions of the secretory axons changed with time. At the longer intervals, the percentage of silver grains increased over the nerve swellings (including Herring bodies) and decreased concomitantly in the undilated portions of the axons and in the nerve endings. This labeling pattern conforms with observations of the secretion products. Water deprivation increased the release of neurosecretion as well as glycoproteins from the pars nervosa. However, glycoproteins inside the Herring bodies were not easily releasible. There was a parallel decrease in the amount of secretion granules and 3H-fucose-labeled glycoproteins indicating that the glycoproteins are predominantly a constituent of the granule content. Some newly synthesized glycoproteins were probably also used in the renewal of the axonal membrane. The labeling of smooth vesicles in nerve endings was discussed. In conclusion, most of the glycoproteins synthesized in the perikarion of the hypothalmic secretory neurons migrate inside secretion graules along the axon to the pars nervosa where they are secreted.

Animals↗