Rapid refilling of neurosecretory terminals with secretory granules after an acute stimulus to the isolated neural lobe of the rat

Summary Quantitative ultrastructural techniques were used to study changes in the distribution of intracellular organelles in neurosecretory terminals of the rat neurohypophysis during recovery from a depolarising stimulus in vitro. The volumetric density of neurosecretory granules which, in profiles of nerve terminals sectioned through the area of contact with the basal lamina, was decreased as a result of stimulation, returned to control values within 2 h after cessation of stimulation. We conclude that, even in the absence of the cell body and action potentials propagated from it, granules can migrate quickly within the terminal region of the neurosecretory axons to refill a depleted compartment.     

Microtubules in the neurosecretory neurones of the posterior pituitary of the rat

Summary The microtubules in the neurosecretory neurones of the posterior pituitary were studied using different electron microscopical techniques. Tannic acid staining indicated that the microtubules had a 13 protofilament substructure similar to that described for microtubules from other tissues and organisms; the dimensions of the microtubules were also similar to that previously reported. Albumen pretreatment clearly showed the microtubules running across axonal swellings, but not continuing across the nerve endings. The only organelles showing possible association with the microtubules were small vesicles and smooth endoplasmic reticulum, no association between hormone granules and microtubules could be seen.Acknowledgement: The author is grateful to P. Wilks for skilled technical assistance.     

Morphometric classification of the neurosecretory granules in the rat pars nervosa

Summary The distribution of the diameters of the neurosecretory granules in the rat pars nervosa (measured from electron micrographs taken at 40 000 × ) was compared among axons by nonparametric statistical methods and the axons were classified into five groups with median granule diameters of 143, 155, 167, 180 and 193 nm. We suggested that these five axon types carried different secretory substances contained in the pars nervosa. This investigation is supported by a grant from the Population Council, New York and grant from the Ministry of Education. Authors are grateful to Japan Electron Optics Laboratory Company for their technical assistance with the electron microscopy and to Miss Kazue Yamamoto for her help in preparing the figures.     

Ultrastructure of the rat posterior pituitary gland and evidence of hormone release by exocytosis as revealed by freeze-fracturing

Summary Posterior pituitary glands from normal rats, and rats which had been deprived of water for varying periods, were examined by the freeze-fracture method. This technique reveals large areas of the nerve cell membrane. Images consistent with exocytosis as the mechanism of release of the neurohypophysial hormones were observed. These modifications were most numerous after the rat had been starved of water for 2 days.In normal rats, the large number of neurosecretory granules within the nerve fibres caused a bulging of the nerve cell membrane. The bulges disappeared 2 days after removal of drinking water. Regions of the membrane displaying bulges were characterised by the absence of the typical membrane-associated particles.It is postulated that the close proximity of the neurosecretory granules to the nerve cell membrane may result in rapid fusion of the neurosecretory granules on stimulation of the gland. The change in properties of the nerve cell membrane overlying the neurosecretory granules, as suggested by the loss of membrane-associated particles, may represent a change in the structure of the membrane to a form which is more favourable for fusion.This work was supported in part by a grant from the New Zealand Medical Research Council.     

Pseudopodia formation by neurosecretory granules

Summary Ultrastructural studies of the mouse neurohypophysis, under various experimental conditions, revealed a number of neurosecretory granules (NSG) bearing single pseudopodia-like protrusions. Some NSG adhered to the axolemma via pseudopodia; other NSG, distant from the axolemma, budded electron lucent microvesicles from the tip of the pseudopod.Pseudopodia counts were made on electron micrographs, and calculated as a percentage of the NSG population. In neural lobes from intact mice, small numbers of pseudopodia were observed (0.3%); the count increased significantly after injections of large doses of horseradish peroxidase (HRP) (9.4–14.5%); hypertonic saline augmented the count, as did histamine.In vitro incubation experiments with isolated neural lobes in Krebs Ringer revealed concomitant pseudopodia formation and elevated vasopressin release (measured by antidiuretic bioassay) in the presence of HRP and di-butyryl cyclic AMP respectively. Histamine and excess potassium also increased hormone secretion, but did not induce pseudopodia formation in vitro; pseudopodia were observed neither in controls, nor in the presence of ineffective secretagogues.It is suggested that the pseudopod may represent the active site on the granule membrane. Different ultrastructural images of granule release suggest that several modes of hormone release may be operative in the neurohypophysis. The role of HRP in pseudopodia formation and vasopressin release is enigmatic.This study is dedicated to Prof. Berta Scharrer, esteemed mentor and beloved friendProf. Zwi Selinger, Department of Biological Chemistry of the Hebrew University, kindly collaborated in the in vitro experiments. Thanks are due to Mrs. Ilana Sabnai and Mrs. Sara Eimerl for excellent technical assistance. Research supported by the Binational Israel-United States Science Foundation (BNSF), grant 200     

Effects of colchicine on release of neurosecretory material from the posterior pituitary gland of the rat

Summary The effect of colchicine on the release of neurosecretory material from the posterior pituitary gland was investigated in the rat in vivo and in vitro. Colchicine was administered subarachnoidally when neurophysin, radiolabelled by injection of (³⁵S) cysteine into the supraoptic nucleus, had accumulated in the neural lobe. Dehydration for 3 days of non-colchicine-treated rats was followed by a 100% reduction of neurophysin-bound radioactivity. When colchicine was given prior to dehydration, the reduction of radioactive neurophysin was less marked. Colchicine treatment alone was likewise followed by a lowering of protein-bound radioactivity in the neural lobe, which may indicate that colchicine, in addition to blocking the rapid axonal transport of neurosecretory material, also impedes the slow transport.The release of radioactive neurophysin in response to depolarizing concentration of potassium in vitro was diminished in the presence of colchicine, the reduction being most pronounced after colchicine treatment in vivo. The biochemical data prove the view that colchicine inhibits the release of neurosecretory material from the neural lobe. The ultrastructural findings support the biochemical data. Thus, colchicine treatment alone or followed by dehydration induced a marked increase in the number of organelles, especially of mitochondria and dense bodies. There was a marked increase in the number of enlarged axons filled with dammed organelles in the infundibulum and neurohypophysis. There was an accumulation of dense core vesicles and microvesicles in the axonal terminals in the neurohypophysis after treatment with either colchicine or colchicine followed by dehydration, which indicates an impediment of the release process. Dehydration alone induced a depletion of the dense core vesicles in the terminals. Out from the combined biochemical and ultrastructural findings possible mechanisms for the action of colchicine are discussed.The present study was supported by grants from Svenska livförsäkringsbolags fond för medicinsk forskning, The Swedish Medical Research Council (No. B73-12X-2543-05B), Magnus Bergvalls stiftelse and from the Medical Faculty, University of Göteborg.Miss Gull Grönstedt is thanked for careful secretarial work and the technical assistance by Mrs. Wally Holmberg, Mrs. Elisabeth Norström and Mrs. Ulla Svedin is gratefully acknowledged. Abbreviations used: NSG = neurosecretory granules; NSN = neurosecretory material; SON = supraoptic nucleus.     

Cytochemical duality of neurosecretory material in the hypothalamo-posthypophysial system of the rat as related to hormonal content

Summary Cytochemical methods using silver proteinate, silver methenamine and potassium ferrocyanide + OsO₄ for ultrastructural detection of glycoproteins allow, in the posthypophysis and the magnocellular nuclei of the rat, differentiation of two types of fibres and neurons: one type containing negative granules with a homogeneous content of low electron density, the second type containing granules which demonstrate a ring-shaped deposit either of silver or of potassium ferrocyanide-osmium complex, likely to be related to a glycoproteic component. The difference between these two types is increased by prestaining en bloc with uranyl acetate before the silver proteinate reaction. A similar investigation was carried out on the vasopressin deficient Brattleboro rat; the neurosecretory material, present in some endings and neurones only, is of the nonreactive type, so that it appears justified to correlate the reactivity of granules with vasopressin, and consequently to distinguish neurones and fibres containing vasopressin from those in which oxytocin is quantitatively the main hormonal peptide. This conclusion is strongly supported by the fact that percentages of reactive and negative endings, as determined on this basis in the posthypophysis of normal rats from two different strains, are in good agreement with biochemical data reported in the literature.     

Depletion of neurosecretory granules and membrane retrieval in the sinus gland of the crab

Summary Ultrastructural aspects of hormone release from the sinus gland of the crab Carcinus maenas, have been studied by incubation of glands in vitro (i) in high potassium-containing media to induce hormone release; (ii) in a high potassium-containing calcium-free medium in which depolarisation but no hormone release would be expected; and (iii) in control saline. Uptake of horseradish peroxidase into subcellular organelles was also studied.Many neurosecretory granules could be found in the nerve terminals but, in contrast to mammalian neurosecretory systems, structures resembling microvesicles were extremely scarce. High potassium stimulation in the presence of calcium caused an 18 % loss of granules from the nerve terminals associated with images of single and multiple exocytosis. It further caused an increase in vacuoles which could have accounted for 33 % of the membrane of the granules exocytosed. After incubation in high potassium-containing, calcium-free media there was no evidence either of exocytosis of granules or of an increase in the vacuole population. The population of sparse microvesicle-like structures was not significantly altered by incubation in either high potassium medium. Horseradish peroxidase reaction product could be found only in vacuoles of tissues stimulated by high potassium concentrations in the presence of calcium. It is concluded that this depolarising stimulus produces, in the presence of calcium, the release by exocytosis of about one sixth of all the granules in the sinus gland, and that vacuoles are the organelle responsible for the recapture of membrane after the exocytosis.     

Morphometric classification of neurosecretory granules in the neurohypophysis of the hagfish,Eptatretus burgeri

Summary Neurosecretory axons in the neurohypophysis of the hagfish, Eptatretus burgeri, were statistically classified into six types according to the size of secretory granules. These types are comparable with those in higher vertebrates. The concentration of each axon type is different in three regions: anterior dorsal wall, posterior dorsal wall, and ventral wall. The regional differences of the hagfish neurohypophysis are discussed in relation to the regional differentiation of the tetrapod neurohypophysis into the median eminence and the pars nervosa.We wish to express our appreciation to Professor Hideshi Kobayashi for the guidance of this study. It was aided by grants from the Ford Foundation and the Ministry of Education to Professor H. Kobayashi, and also by grants from the Population Council, New York and the Ministry of Education to Professor S. Ishii.     

Thiamine pyrophosphatase activity in the axonal smooth endoplasmic reticulum of neurosecretory neurons

Summary Neurosecretory cells of the supraoptic-neurohypophysial system of normal mice were investigated with the use of the cytochemical reaction for thiamine pyrophosphatase (TPPase) at the ultrastructural level. In the hypothalamic perikarya dense lead precipitates occur within the cisterns of the mature face of the Golgi apparatus, these being the cisterns that give rise to neurosecretory granules (NSG). Smooth endoplasmic reticulum is occasionally confluent with TPPase-positive Golgi cisterns. Along axons, within swellings, and within terminals distinct profiles of TPPase-positive tubules and cisterns are revealed, apparently part of a network of axonal smooth endoplasmic reticulum (AER). Some NSG appear to be confluent with AER. NSG with TPPase-positive tubular protrusions (likely vestiges of AER) are seen. Apart from reaction product (lead precipitate), the AER often contains an electron dense substance optically similar to that of NSG. TPPase-containing AER is often associated with mitochondria. Profiles of electron-lucent, precipitate-free tubules and cisterns are occasionally seen alongside reactive AER. Optimal TPPase activity in the AER occurs at pH 7.0–7.4, whereas in the Golgi complex intense marking is in the range of pH 6.0–8.5. A faint peppering of precipitate occasionally appears in the AER in controls (incubation medium without substrate), but neither in density nor in extent is this comparable to the reaction product seen after incubation in the presence of TPP. Preliminary comparison has been made between the AER revealed by the TPPase reaction, and that visualized after heavy metal impregnation according to the method of Alonso and Assenmacher (1978a). The nature of the close association between NSG and AER, and the possible roles of this membrane system in neurosecretory cells is discussed.Abbreviations AER axonal smooth endoplasmic reticulum - NSG neurosecretory granules - TPPase thiamine pyrophosphatase - SON supraoptic nucleus Research supported in part by a grant from the Israel Academy of Sciences to M.C.We thank Mrs. Ilana Sabnay for excellent technical assistance     

On the fine structure of the perivascular cells in the neural lobe of rats

Summary The purpose of this study is to investigate the ultrastructural features of perivascular cells as found in the neural lobe of the hypophysis. In particular, an inquiry was made into the nature of the relationship of such cells to neurosecretory fibers and endings. The latter, in fact, are often invaginated within the perivascular cells and enveloped by their processes; furthermore, they often reveal a certain number of empty granules as well as characteristics of degenerative nature. In the course of this study the localization of the perivascular cells has been investigated as well as that of their processes within the extensive interlobular network typical of the hypophysial neural lobe of rodents. Based on the data gathered, the hypothesis is put forward that the perivascular cells play an important role in the turnover of neurosecretory endings, both under physiological and experimental conditions, contributing thereby to the release of post-hypophysial hormones.With the technical assistance of Vincenzo Panetta.     

Elementary granules,small vesicles and exocytosis in the rat neurohypophysis after acute haemorrhage

Summary Neural lobes of rats subjected to severe acute haemorrhage under sodium pentobarbitone anaesthesia were examined electron microscopically and the ultrastructure compared with that in anaesthetised and unanaesthetised controls. Changes in the localisation and numerical distribution of elementary granules and small vesicles in the neurohypophysial nerve endings of bled rats were consistent with the occurrence of exocytosis. The occurrence of exocytotic profiles was observed more frequently in freeze-etched tissue samples as compared with the material fixed for conventional electron microscopy. The ratio of small vesicles: elementary granules was shown to be significantly increased (P<0.005) in the nerve endings of neural lobes from bled rats. Equally, the numbers of exocytotic profiles related to 1000 m² of neurohypophysial tissue area were significantly greater (P<0.005) in bled rats.The study was supported by Medical Research Council of Canada. The authors are grateful to Dr. W. Costerton, Biology Department, The University of Calgary, for use of facilities for freeze-etching, and to Miss Y. Carter for technical assistance.Research Associate, Consejo Nacional de Investigationes Cientificas y Tecnicas, Argentina.Associate, Medical Research Council of Canada.     

Daily fluctuations of nucleoli in neurosecretory cells of the rat supraoptic nucleus

Summary Daily fluctuations of nucleoli and nucleolar fibrillar centres in neurosecretory cells from the supraoptic nucleus (SON) were investigated in rats artificially synchronized for 3 weeks to a set 12 h light/12 h dark cycle with free access to food and water. Groups of 3 animals were sacrificed by intracardiac perfusion every 4 h for a 24-h period and every 2 h between 22.00 h and 07.00 h. The SON of each animal was removed, and the mean nucleolar volume and the mean volume of the nucleolar fibrillar centres were estimated by a stereological analysis. The quantitative data showed that the fluctuations in the nucleolar volume of SON neurons depend on the time of sacrifice. A peak value was found in animals sacrificed at 03.00 h which was 1.5 times the value found in animals sacrificed at 19.00 h. The volume of fibrillar centres underwent small, but not significant changes over the 24-h period. None of the large fibrillary centres that can be observed in the superior cervical ganglion were found in the SON. Our results demonstrate that in these neurons the size of the nucleolus undergoes daily fluctuations. These results are discussed in the light of previous studies conducted at our laboratory on the circadian rhythm of nucleolar volume and of nucleolar components in neurons of the superior cervical ganglion.     

Freeze-fracture study of the rat parathyroid gland under hypo- and hypercalcemic conditions,with special reference to secretory granules

Summary Freeze-fracture images of the parenchymal cells in the parathyroid gland of rats were observed after vitamin D₂ plus calcium chloride-suppression and EGTA-activation of secretion. In cells of the suppressed glands, large bulges protruded from the Golgi cisternae, and large granules with a stalk, which are identified as storage granules, suggest that, during maturation, some storage granules may be connected by long tubules with the Golgi cisternae and supplied with secretory products from the Golgi cisternae via these tubules.In the activated glands, presumptive exocytotic and endocytotic specializations of intramembranous particles of the parenchymal cell plasma membrane were frequently observed. In addition, elevations and complementary shallow depressions of various shape and extent were occasionally encountered in the intercellular space. From their morphological characteristics it was concluded that these originated from secretory granule cores, which are discharged from the parenchymal cells into the intercellular space by exocytosis, and it was suggested that discharged granule cores may retain their spherical shape until they fuse to form a flat conglomerate.     

Isolation and compositional analysis of secretion granules and their membrane subfraction from the rat parotid gland

Summary A secretory granule fraction has been isolated from rat parotid by discontinuous gradient centrifugation using hyperosmotic sucrose-Ficoll solutions of low ionic strength. The secretion granule fraction comprises 25% of the total tissue -amylase activity and is judged to be of high purity, both morphologically and by its low level of contamination by enzyme activities associated with other organelles.Secretion granules were lysed by capitalizing on their lability in KCl-containing media, and the low density granule membranes were separated from residual organelle and soluble contaminants by flotation in a sucrose gradient. Residual, poorly extractable secretory contaminants of the granule membrane subfraction were selectively removed by a saponin- (10 g/ml) Na₂SO₄ (0.3m) wash, apparently with negligible disruption of granule membrane structure. Based on detailed consideration of the extent of contamination by residual mitochondria and incompletely removed secretory polypeptides, it is possible to estimate that 95% of the protein associated with the purified secretion granule membrane is bona fide granule membrane protein. Further analyses indicate that -glutamyltransferase constitutes a marker enzymatic activity shared by granule membranes and the apical domain of the plasma membrane.Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoretograms of radio-iodinated granule membrane polypeptides are characterized by 20–25 radioactive bands of which 5–6 are suggested to be glycoproteins by virtue of their binding of concanavalin A. The limited polypeptide composition of the secretion granule membrane (in comparison to membranes of other cellular compartments) and the high phospholipid-protein ratio (4.4 mg/mg) may reflect the functional specialization of this storage container for secretory proteins.     

Fluorescent granules in the guinea-pig hypothalamus and their possible relation to neurosecretory substance

Summary Sections of guinea-pig hypothalamus stained by various methods have been studied under bright-field and fluorescence microscopy to determine the characteristics of refractile granules originally observed in fixed tissue stained for cholinesterases but also seen in unfixed, unstained material.When sections of fresh tissue or tissue fixed in formaldehyde: Na₂SO₄ were examined by fluorescence microscopy, the granules emitted with a wave-length between 600 and 620 m. The density and distribution of the granules closely paralleled that of one of the components that stained with the aldehyde-fuchsin and chrome-alum techniques for neurosecretory substance; granules were absent, however, from the area characterized by Herring substance, and from the stained perikarya and beaded axons of the supraoptic and paraventricular nuclei.The granules were most numerous in the median eminence and infundibular nuclei. Their concentration was about average in mature males and in lactating and hypophysectomized females; they were more abundant in late pregnancy and after ovariectomy and were particularly plentiful some weeks after hysterectomy. They were virtually absent from immature males and females.The possible relation of the granules to releasing factors or to vasotocin-like neurosecretory material is discussed.The authors are most grateful to Dr. J. S. Perry who carried out the surgery involved in these experiments. They also wish to express their thanks to Dr. R. B. Heap who, with Dr. Perry, gave invaluable help in planning the experiments and assessing the data; to Mr. S.P. Mann and Dr. D. F. Sharman for advice on the fluorescence technique and to Miss M. Hamon for skilled technical assistance.Publication of the colour pictures was made possible by a generous grant from the Wellcome Foundation; this support is gratefully acknowledged.Thanks are also due to the University of Alberta for a contribution towards the cost of reprints.     

Ultrastructural study of the neurosecretory granules in the sinus gland of the blue crab,Callinectes sapidus

Summary Seven morphologically different types of neurosecretory granules have been found in the axon terminals of the sinus gland of the blue crab, Callinectes sapidus. They differ from each other in size, shape, staining characteristics, solubility characteristics, core matrix characteristics, axon terminal matrix characteristics, presence or absence of space between the granule membrane and granule core matrix, and frequency of occurrence. Five of the types are segregated in different axon terminals and are believed to represent different hormone-protein complexes. Two of the types, which have lost part or all of their granular contents, are thought to be variants of the other five types. The differences in granular morphology are better revealed by some fixation procedures than others. Palade's acetate-veronal buffered osmium tetroxide, in particular, reveals striking differences. The following observations suggest that different hormone-protein complexes are segregated in different axon terminals and that these complexes may be morphologically distinguished at the level of the electron microscope.Supported by USPHS-NIH Training Grant GM-00669 and Grant GB-7595X from the National Science Foundation.     

Ultrastructural localization of acid phosphatase in the posterior pituitary of the dehydrated rat

Summary Acid phosphatase (AcPase) was localized by means of electron-microscopic histochemistry and estimated biochemically in the posterior pituitary of rats deprived of water, given 2% NaCl ad libitum, or given tap water ad libitum over 6 days. Autophagic vacuoles, some of which gave a positive AcPase reaction, often contained neurosecretory granules (NSG) in nerve endings of control animals on tap water. Nerve endings of water-deprived or salt-treated rats were depleted of NSG, but frequently contained dense membranous residual bodies, some of which appeared to enclose microvesicles. Smooth endoplasmic reticulum located in axons and terminals appears to be a source of hydrolytic enzymes for neurohypophysial lysosomes. The total amount of AcPase per posterior lobe increased progressively to 40% above control levels after 6 days of water deprivation or salt administration, and this increase may reflect accelerated production of neuronal components in neurohypophysial cells whose secretory rate has been stimulated by elevated body osmolarity.Supported by the Medical Research Council of Canada.Medical Research Associate of the Medical Research Council of Canada.     

Ultrastructural appearance of neurosecretory granules in the sinus gland of the crab after different fixation procedures

Summary The appearance of neurosecretory granules in the crab sinus gland was studied after fixation at different pHs. Whereas at pH 7.0 the neurosecretory granules were pleomorphic with respect to electron density, at pH 5.0 or 6.0 all the granules remained electron dense. The possible role of maturation as an explanation of this observation is discussed.ERA 493 CNRS