On degeneration of peptidergic neurosecretory fibres in the albino rat

Three types of degenerating peptidergic neurosecretory fibres have been found in the posterior pituitary of chronically dehydrated albino rats. "Dark" neurosecretory fibres and their swellings contain neurosecretory granules, neurotubules, shrunken mitochondria and diffusely distributed fine dense material. Some swellings are filled with synaptic vesicles and/or conglomerations of dense membranes. The transitional forms exist between these fibres and extracellular accumulations of electron dense material. Synaptic vesicles, single neurosecretory granules, lipid-like droplets and lamellar bodies occur in the latter. Some neurosecretory fibres and swellings have numerous polymorphous inclusions arising due to degradation of secretory inclusions and organelles, mitochondria and neurotubules in particular. "Dark" neurosecretory elements and those with numerous polymorphous inclusions are enveloped by pituicyte cytoplasm. Sometimes the plasma membranes both of the pituicytes and neurosecretory fibres are destroyed or transformed into a multi-membrane complex. It is assumed that pituicytes may phagocytize degenerating neurosecretory elements. N urosecretory fibres with a locally dissolved neuroplasm and/or large lucent vacuoles seem to be due to axonal degeneration by the "light" type. These neurosecretory elements, the largest of them in particular, may transform into large cavities bordered by a membrane and containing flake-like material and single-membrane vacuoles. Degeneration of neurosecretory elements seems to occur mainly due to hyperfunction of the hypothalamo-hypophysial neurosecretory system.     

Neurosecretory granule formation in ligated axons: additional arguments for a local differentiation from a Golgi apparatus extension

The sorting domain for the different types of granules and small synaptic vesicles in neurosecretion is still largely a matter of debate. Some authors state that an exocytotic process has to precede granule formation. In previous studies, we favoured the idea that neurosecretory packages in terminals are assembled from axonal reticulum membranes simply by differentiation at the axon ending, the axonal reticulum being an extension of the Golgi apparatus. By ligating bovine splenic nerve, a de novo differentiation can be induced. After ligation, granules and granulo-tubular complexes appear. They were immunoreactive for SV2, VMAT2 and synaptobrevin II, which are all known to be highly enriched in large dense granules. Previously the granulo-tubular structures have already been recognized as precursor stadia of neurosecretory granules.It is concluded that at a de novo differentiation, a sorting out and aggregation is taking place of molecules typical for large dense granules. The small dense granules and tubules can be considered unripe, precursor forms of the large dense granules. All this occurs in the absence of signs of exocytosis. The present findings corroborate the view that granule formation occurs via local differentiation at an axon ending.     

鲫鱼尾部神经分泌系统显微和亚显微结构的季节性变化

鲫鱼尾部神经分泌系统的神经分泌细胞和它的轴突中可观察到各种不同电子密度的颗粒。在性腺各个不同的发育阶段,该系统的分泌物具有累积、充满、释放和恢复这样一种周期性变化,由此说明鲫鱼的尾部神经分泌系统和它的生殖有关。     

The smooth endoplasmic reticulum in neurohypophysial axons of the rat: Possible involvement in transport,storage and release of neurosecretory material

Summary The intra-axonal organization of the smooth endoplasmic reticulum was studied in the neurohypophysis of rats during and after water deprivation. Parallel to conventional electron microscopy, the material was treated with a double impregnation staining technique specifically designed to contrast the intracellular membranous system. In conventionally stained ultrathin sections from severely dehydrated rats most axons appeared to be free of membranous organelles, whereas corresponding axons treated with the double-impregnation technique generally exhibited a highly developed system of smooth endoplasmic reticulum. In axonal endings, both techniques revealed a profusion of microvesicles in intimate relationship with tubular elements of the smooth endoplasmic reticulum. In short-term (12 h) rehydrated rats, a similarly developed system of smooth endoplasmic reticulum was still observed at all axonal levels with both procedures. After 24 to 48 h of rehydration the tubules of the smooth endoplasmic reticulum exhibited, in double impregnated material, numerous dilatations which resembled the adjacent neurosecretory granules. In conventionally stained ultrathin sections, an accumulation of electron dense material occurred within tubules of the smooth endoplasmic reticulum in the more proximal axonal segments, while in the more terminal segments, which contained numerous elongated granules, membrane continuity was frequently observed between newly formed granules and the smooth endoplasmic reticulum. After 7 days of rehydration the general pattern of the axonal smooth endoplasmic reticulum was comparable to that in untreated rats. These results are discussed in the light of a suggested involvement of the axonal smooth endoplasmic reticulum in the non-granular transport of neurosecretory material in connection with (1) storage in distally formed granules, and (2) release via microvesicles. Acknowledgements: The authors wish to express their gratitude to Mrs. M. Balmefrézol for her skillful technical assistance     

Herring bodies; an electron microscopic study of local degeneration and regeneration of neurosecretory axons

Summary The Herring bodies in the posterior lobe of the bovine hypophysis are very large (2–600 ) and can be classified into three types. The type I Herring body contains an accumulation of neurosecretory granules. These Herring bodies are very scarce and should not be confused with the numerous, but small, axonal swellings which also contain neurosecretory granules.The type II Herring body is characterized by the presence of a varying number of normal, moderately electron dense and empty vesicles, autophagic vacuoles, multilamellate bodies and occasional mitochondria. These Herring bodies are frequently observed.The type III Herring body is typified by the presence of dense vesicles connected to tubular formations which contain material of variable electron density, of filaments, and of long slender and very numerous mitochondria.The presence of multilamellate bodies and autophagic vacuoles suggests that the type II Herring body is in a degenerating phase. This concept is further substantiated by the similarity between this type of Herring body and transected neurosecretory axons in which degeneration is occurring.A similar comparison suggests that the type III Herring body is undergoing a regenerative process. Our current concept of the structure and function of Herring bodies is revised in the discussion.This work was supported by grants 5 RO1 NB 06641 NEUA and 5 R0107492 NEUA from the National Institutes of Health and the Space Sciences Research Center of the University of Missouri. The technical assistance of Mrs. G. Clark and Mr. R. Faup, and the clerical assistance of Mrs. S. Schmidt are gratefully acknowledged.Fellow of the Conséjo National de Investigaciones Científicas y Tecnicas de la República Argentína.     

Investigations on the hypothalamo-neurohypophysial neurosecretory system of the grass frog (Rana pipiens) after transection of the proximal neurohypophysis

Summary With the onset of degeneration of the neurosecretory nerve fibers following transection of the proximal neurohypophysis, the pituicytes phagocytize these nerve fibers. Concomitant with a considerable increase in the size of the pituicytes, which reaches a peak between 8 and 10 days after the transection, the following sequence of events can be observed: reduction of the amount of intergranular axoplasm, increase in the size of some granules, partial or total loss of the electron density of the neurosecretory granules, loss of granule membranes, fusion of some granules, polymorphous axonal content in digestion vacuoles, formation of multilamellate bodies, digestion vacuoles with moderately electron dense peripheral material, empty digestion vacuoles. At about 12 days after the transection many vacuoles appear which subsequently disappear as the pituicytes shrink. Free neurosecretory granules resulting from the disappearance of the axolemma remain intact in the intercellular and perivascular connective tissue spaces and are eventually phagocytized by pituicytes and pericytes.Phagocytosis is considered to be a basic function of pituicytes. The problems related to this function as well as the possible implications for the interpretation of Herring bodies are discussed.This investigation was supported by Grant No. NB-06641 of the National Institute of Neurological Diseases and Blindness. The technical assistance of Mrs. Mildred Floyd and Christa Cooper is gratefully acknowledged.Dedicated to Prof. Dr. H. Grau on the occasion of his 70th birthday.     

Retrograde axoplasmic transport of neurosecretory material

The axonal transport of neurosecretory material was studied in neurosecretory axons of the supraoptico-posthypophyseal system after in-situ transection of the median eminence. Two hours, 8 h, and 18 h after the lesion, both vasopressin and oxytocin antibodies revealed progressive accumulations of immunoreactive material not only in the proximal but also in the distal stumps of the transected axons. The electron-microscopic examination of these axonal portions revealed that such intense immunopositive labelings could be correlated, in both stumps, to a conspicuous accumulation of neurosecretory granules. It is concluded that, under normal physiological conditions, a significant amount of axoplasmic neurosecretory material is transported in retrograde direction and that such a retrograde transport mainly involves neurosecretory granules.     

Ultrastructural studies on the afferent synaptic input to oxytocin-containing neurons in the paraventricular nucleus of the hypothalamus

A diverse afferent synaptic input to immunostained oxytocin magnocellular neurons of the paraventricular nucleus of the rat hypothalamus is described. By electron microscopy, immunoreactive material is present within cell bodies and neuronal processes and it is associated primarily with neurosecretory granules and granular endoplasmic reticulum. Afferent axon terminals synapse on perikarya, dendritic processes, and possibly axonal processes of oxytocin-containing neurons. The presynaptic elements of the synaptic complexes contain clear spherical vesicles, a mixture of clear spherical and ellipsoidal vesicles, or a mixture of clear and dense-centered vesicles. The postsynaptic membranes of oxytocinergic cells frequently show a prominent coating of dense material on the cytoplasmic face which gives the synaptic complex a marked asymmetry.     

The ultrastructure of corpus allatum of the earwig Euborellia annulipes lucas (Dermaptera: Labiduridae)

The ultrastructure of corpus allatum of the earwig, Euborellia annulipes has been described. The corpus allatum (CA) is an oval body. The gland is covered by a thin stromal sheath which is wavey and acellular in composition. The gland consists of either parenchymal cells with distinct, double-layered, smooth or slightly undulated plasma-membranes. Golgi bodies and endoplasmic reticulum are poorly developed. The mitochondria are found in abundance and are oval, spherical, elongate, and Y-shaped, with oblique and longitudinal cristae. The gland is innervated by neurosecretory and non-neurosecretory axons. The neurosecretory axons are of two types. The type NS-II, contain electron dense granules of 40...120 nm in diameter and the type NS-I, small less electron dense granules (of mixed nature) 40...90 nm in diameter. Branches of tracheoles also occur that penetrate the gland. The large electron dense granules 100...220 nm in diameter occuring abundantly in aorta, and representing the secretions of the medial A-cells are absent in the CA.     

Autolysis in axon terminals of a new neurohaemal organ in the cockroach Periplaneta americana

An ultrastructural investigation showed that there was a neurohaemal organ in the wall of the ampulla of the antennal pulsatile organ. The neurosecretory axon terminals occurred singly or in small groups rather than closely packed together as in other neurohaemal organs. All axons contained the same type of neurosecretory granule. The granules had varying electron density and a diameter in the range 1000–2500 Å. Some terminals contained small, elliptical, electron-transparent vesicles and the axolemma was apposed to the stroma. Other terminals were large and enveloped by glial tissue and the contents of the terminals exhibited varying degrees of autolytic degeneration. Autolysis was characterized by the occurrence of dense bodies and multilaminate bodies which enclosed mitochondria and neurosecretory granules. It was suggested that the neurosecretory material affects antennal function.     

Histology, histochemistry, and ultrastructure of neurosecretory cells in the optic lobe of the cockroach, Periplaneta americana

In the region of the distal optic chiasma of each optic lobe of Periplaneta americana, there is a group of about 120 monopolar neurosecretory cells. These cells do not stain with paraldehyde fuchsin but remain acidophilic after oxidation. They stain red or sometimes indigo with the azan technique. Histochemically, the neurosecretory material is positive for protein and the amino acids tryptophan and arginine but negative for 1, 2-glycols and strongly acidic groups. At the ultrastructural level, the cytoplasm of the cells contain many elementary neurosecretory granules 100 to 170 nm in dia. The cells also contain well-developed Golgi bodies and endoplasmic retieulum. The axons from these cells run toward the interior of the optic lobe. In this region, axons containing dense granules (mean diameter 70 nm) and synaptic vesicles synapse onto the axons from the neurosecretory cells. The neurosecretory axons then cross over to the anterior side of the optic lobe and run towards the brain. The function of these neurosecretory cells is unknown, but they may be involved with photoperiodically controlled activity rhythms.     

Studies on the transport of secretory granules in the magnocellular hypothalamic neurons

Summary The axonal flow of neurosecretory elementary granules has been studied in the paraventricular neurons of the rat (PVN), with the help of three techniques: light microscopy, radioautography after labelling with ³⁵S-L-cysteine, and electron microscopy.Colchicine treatment does not alter the uptake of ³⁵S cysteine in the PVN but the flow of labelled neurosecretory material towards the neurohypophysis is interrupted. Interruption of the axonal flow is also evidenced by the stagnation of neurosecretory granules at the periphery of the neuronal cytoplasm and by the presence of numerous axonal swellings, heavily loaded with neurosecretory granules and often containing abnormal elongated granules, surrounded by a single membrane, oriented more or less parallely to the long axis of the axons. Other cell organelles and neurotubules are not altered. The present experiments bring further evidence of the arrest by colchicine of the axonal flow of secretory granules without apparent changes of the neurotubules.This work was supported by a grant (1970/1971) from the Belgian National Fund for Scientific Research (J. F.-D), and by grant No 1120 from the Belgian National Fund for Medical Research (P. D).The authors wish to thank Mrs. A.-M. Hunninck-Couck for her devoted and skillful technical assistance, and are endebted to Dr. J. C. Heuson for kindly supplying the rats.     

Sequence of ultrastructural changes induced by activation in the posterior neurosecretory cells in the brain of Rhodnius prolixus with special reference to the role of lysosomes.

The posterior neurosecretory cell (PNC) group in the brain of Rhodnius prolixus is composed of five ultrastructurally identical cells. The PNC were examined in the unfed fifth instar and at seven stages (from 15 min to 14 days) after activation was initiated by feeding. Each stage examined revealed successive changes in morphology which can be related to the synthesis, maturation, storage and transport of neurosecretory material. It is suggested, in particular, that the lysosomal system (dense bodies and multivesicular bodies) may play a role in the maturation of the secretory granules.     

Improved visualization of the immunoreactive hypothalamo-neurohypophysial system by use of immuno-gold techniques

Summary Ultrastructural post-embedding immuno-gold techniques were applied to the supraoptic nucleus and the neurohypophysis of mice and rats. The primary antibodies were three different monoclonal antineurophysins, used in protein A-gold and immunoglobulin-gold procedures. Conventional plastic embedding as well as hydrophilic media (L.R. White) were used; non-osmicated and osmicated tissues were immunolabeled; sodium metaperiodate oxidation was used, but was not essential for immunolabeling.Vasopressinergic and oxytocinergic NSGs were identified by the specific immunoreactivity of their respective neurophysins on adjacent thin sections, and by sequential double labeling on the same thin section using two different antibodies associated with gold probes of different diameters. The immunoidentification indicates that vasopressin NSGs can additionally be differentiated as larger, with more electron-dense matrix, and susceptible to damage by sodium metaperiodate.The only organelles consistently labeled were neurosecretory granules (NSGs), either intact or within lysosomal configurations. Some lysosomal dense bodies were immunoreactive even when discrete NSGs were no longer morphologically recognisable within them. Labeled NSGs were located within neuronal cell bodies, along axonal shafts and within axonal swellings and endings; occasionally immunoreactive NSGs were observed within synaptic boutons. Labeling intensity was semi-quantitatively gauged by counting gold particles in relation to numbers of NSGs per axonal varicosity.The precise localisation achieved with particulate immunogold labeling surpasses that previously obtained with diffuse electron-dense immunoreaction products.     

Morphology of the neural lobe of the hypophysis in rats treated with furosemide. I. Neurosecretory axons

The changes found in the neurosecretory axons of the neural lobe of the hypophysis were studied in rats treated with furosemide for three days: over the whole period of treatment, the animals, according to each group, were deprived or not of water. In the animals with free access to water the axons contained neurosecretory granules with scarce content and low electron density; only some of them had vacuoles and autophagic bodies. In the animals deprived of water axon morphology was variable and axons showing vacuoles and autophagic bodies were abundant, as well as those presenting lamellar and dense bodies and also those in which filaments prevailed. The most outstanding modifications in this last group of animals were related to the dehydration and were not found in those animals which were given access to water again and which were in consequence rehydrated.     

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     

Ultrastructure of the hypothalamic neurosecretory system of the dog

Summary The hypothalamic neurosecretory system of normal dogs was studied by light and electron microscopy after perfusion-fixation. In the supraoptic nucleus most neurons are loaded with elementary neurosecretory granules having a content of low electron density. Neurons with less neurosecretory material and signs of enhanced synthetic activity, as recognized by the changes in the endoplasmic reticulum, were also observed.The vesiculated neurons ofJewell were studied under the electron microscope and various stages of development were described. It was postulated that they originate by a localized process of cytoplasmic cytolysis which ends in the formation of a large aqueous intracellular cavity limited by a plasma membrane. The possible significance of these vesiculated neurones is discussed. Some few myelinated neurosecretory axons are found in the supraoptic nucleus.The neurons of the paraventricular nucleus are smaller and contain less neurosecretory material. This is abundant and very pale in the axons. The median eminence consists of an inner zone, mainly occupied by the neurosecretory axons of the hypothalamic-neurohypophysial tracts, and an outer zone in which some neurosecretory axons end on the capillary of the portal system. This outer zone contains numerous axons with the axoplasm rich in neurofilaments and some containing granulated and non-granulated synaptic vesicles. Some neurons with granulated vesicles were observed in this region. The adrenergic nature of these neurons and axons is postulated.The infundibular process of the neurohypophysis shows small axons with discrete amounts of elementary granules and vesicles of synaptic type at the endings. Some enlarged axons having, in addition, large polymorphic bodies are observed and related to the Herring bodies.The size and morphology of the granules are analyzed along the entire hypothalamic-neurohypophysial system. The changes in diameter and electron density are related to the maturation of the granules and the possible significance of such evolution.Supported by grants from the Consejo Nacional de Investigaciones Cientificas y Técnicas and by the Air Force Office of Scientific Research (AF-AFOSR 963-66).     

Energy dispersive X-ray microanalysis of neurosecretory granules of mouse pituitary on fresh air-dried tissue spreads

Electron microscopy of fresh air dried spreads of unstained posterior lobe tissue from mouse pituitary disclosed neurosecretory granules. Each granule showed a seemingly homogeneous dense core surrounded by a halo and a bounding membrane. The area between granules in the cytoplasm was relatively well preserved. The energy dispersive X-ray microanalysis revealed peaks for sulfur, chlorine and potassium in two granules. The third granule displayed peaks for phosphorus and chlorine. These elements probably contribute to the high electron density of the granules. There was no peak for calcium, in contrast to the dense bodies of human blood platelets.     

Intralamellar neurohemal complexes in the cerebral commissure of the leech Macrobdella decora (Say, 1824): An electron microscope study

Electron microscopy of the cerebral ganglionic commissure of the leech Macrobdella decora (Say, 1824) revealed numerous neurosecretory axons terminating in the neural lamella of both the inner and outer capsules, and in the neural lamella deep within the neuropile. The proximal protions of the terminals, with an investment of glial tissue, contain either numerous large homogeneously electron dense granules, or numerous large granules of varying electron density. The distal portions, often devoid of glia, display numerous infoldings, omega profiles, and electron dense focal sites, and contain numerous neurosecretory granules, small lucent vesicles, and, occasionally, acanthosomes. Statistical analysis of the size distribution and morphology of the neurosecretory granules showed that in many individual terminals the granules are not significantly different from those seen within four groups of neurosecretory cells found in the cerebral ganglion. These terminals, because of their diffuse nature, probably represent a neurohemal complex of a primitive nature. The term “intralamellar complexes” is proposed to describe the form and location of these neurosecretory terminals.     

Granulolysis in neurosecretory neurons of the rat supraoptico-posthypophyseal system

Ultrastructural and cytochemical observations on neurosecretory neurons of the rat supraoptico-posthypophyseal systems were made under experimental conditions which resulted in striking changes in the amount of neurosecretory granules and lysosomes. Attention was focused on granulolysis. At the onset of rehydration following a 4 days water deprivation, very active autophagy took place in neurosecretory axons of the neural lobe involving the marked increase in smooth endoplasmic reticulum, microvesicles and neurosecretory granules, although the latter were still very few due to previous depletion. When axonal transport was inhibited by colchicine at the onset of rehydration, granules accumulated in the perikarya while granule reloading of the neural lobe was delayed. However autophagy, although always active in axons, remained scarce in perikarya. Moreover, in the latter there was only slight evidence of crinophagy. Hypophysectomy also induced granule accumulation in the perikarya, although accompanied by little granulolysis. Images indicative of crinophagy as shown by acid phosphatase localization were few and exclusively restricted to perikarya, while autophagy occurred essentially in axons. Autophagy appeared to be the predominant process for granulolysis and might be considered here as an aspect of the general turnover of cell constituents, related to the sudden regression of hyperactivity-induced hyperthrophy, rather than as an expression of a specific regulation of an excess of secretory material.