Analysis of the neurohypophyseal components accumulating in the supraoptic nucleus of the rat after injection of colchicine

[35S]Cysteine has been injected into the supraoptic nuclei of normal rats and of animals given 7 micrograms colchicine into the cerebrospinal fluid to inhibit transport of neurosecretory granules. Analysis of extracts of the supraoptic nuclei 20 min or 6 h after isotope injections showed that (i) colchicine does not affect biosynthesis, i.e., incorporation of tracer into the common precursors of neurohypophyseal hormones and their associated neurophysins, and (ii) processing of precursors inside the arrested granules proceeds normally.     

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.     

Biosynthesis and axonal transport of rat neurohypophysial proteins and peptides

35S-cysteine injected adjacent to the supraoptic nucleus (SON) of the rat is rapidly incorporated into proteins. These 35S-cysteine-labeled proteins in the SON (1-24 h after injection) were separated by polyacrylamide gel electrophoresis, and the distribution of radioactive proteins on the gels was analyzed. 1 h after injection, about 73% of the radioactivity appeared in two peaks (both about 20,000 mol wt). With time, these peaks (putative precursors of neurophysin) decreased, as a 12,000 mol wt peak (containing two distinct neurophysins) increased in radioactivity. Both the 20,000- and 12,000-mol wt proteins are transported into the axonal (median eminence) and nerve terminal (posterior pituitary) regions of the rat hypothalamo-neurohypophysial system. Conversion of the larger precursor protein to the smaller neurophysin appears to occur, in large part, intra-axonally during axonal transport. Six distinct 35S-cysteine-labeled peptides (less than 2500 mol wt), in addition to arginine vasopressin and oxytocin, are also synthesized in the SON and transported to the posterior pituitary where they are released together with labeled neurophysin by potassium depolarization in the presence of extracellular calcium. These data provide support for the hypothesis that the neurohypophysial peptides (vasopressin and oxytocin) and neurophysins are derived from the post- translational clevage of protein precursors synthesized in the SON, and that the conversion process can occur in the neurosecretory granule during axonal transport.     

Swelling of neurosecretory fibers (Herring bodies) in the reorganized hypophyseal stalk following hypophysectomy]

Herring bodies were revealed in the reorganized hypophyseal stalk of rats two months after hypophysectomy both in normal laboratory condition and under salt load. We observed Herring bodies with a storage of neurosecretory granules and neurohormones, as well as with massive destruction and disappearance of neurosecretory granules due to release of hormones in neuroplasm. Many Herring bodies were characterized by degenerative changes probably associated with aging of neurosecretory cells or their lesion due to the operation. Occasionally we revealed Herring bodies which contained a network of dilated neurotubules, that probably reflected either the reparative phase of secretory cycle of corresponding neurosecretory cells or restoration of their functions after hypophysectomy. Herring bodies were often situated around capillaries, but they were separated from perivascular space with narrow sprouts of pituicytes.     

The ultrastructure of the herring bodies in rats subjected to different experimental conditions

Summary A study of the morphology of Herring bodies of the posterior pituitary lobe of rats treated with colchicine and/or exposed to low temperatures has been performed. After treatment with colchicine (20 g in distilled water injected intracisternally) a predominance of Herring bodies with a large number of small synaptic-like vesicles surrounded by neurosecretory granules is found. Exposures to low temperature (4–6° C) result in an increase in the neurosecretory material and the Herring bodies show many neurosecretory granules of different densities. After treatment with colchicine and subsequent exposure to low temperatures, the Herring bodies are characterized by having a great number of autophagic bodies which become more numerous as the length of the exposure is increased; later autophagic vacuoles and lamellar bodies become evident.     

Immunocytochemical observations of corticotropin-releasing-factor-containing neurons in the rat hypothalamus with special reference to neuronal communication

Synapses between neurons with corticotropin-releasing-factor-(CRF)-like immunoreactivities and other immunonegative neurons in the hypothalamus of colchicine-treated rats, especially in the paraventricular nucleus (PVN) and the supraoptic nucleus (SON) were observed by immunocytochemistry using CRF antiserum. The immunoreactive nerve cell bodies and fibers were numerous in both the PVN and the SON. The CRF-containing neurons had synaptic contacts with immunonegative axon terminals containing a large number of clear synaptic vesicles alone or combined with a few dense-cored vesicles. We also found CRF-like immunoreactive axon terminals making synaptic contacts with other immunonegative neuronal cell bodies and fibers. And since some postsynaptic immunonegative neurons contained many large neurosecretory granules, they are considered to be magnocellular neurosecretory cells. These findings suggest that CRF functions as a neurotransmitter and/or modulator in addition to its function as a hormone.     

FINE STRUCTURE OF THE NEUROHYPOPHYSIS OF THE OPOSSUM (DIDELPHIS VIRGINIANA)

The neurohypophysis of the opossum (Didelphis virginiana) was studied by electron microscopy in order to amplify Bodian''s classic light microscopic observations in which he demonstrated a definite lobular pattern. The lobule of the opossum neurohypophysis is divided into three regions: a hilar, a palisade, and a septal zone. The hilar portion contains bundles of nerve fibers, the extensions of the hypothalamo-hypophyseal tract containing neurofilaments but few neurosecretory granules. In the opossum, pituicytes have a densely fibrillar cytoplasm. Herring bodies are prominent in the hilar region. They are large bodies packed with neurosecretory granules that have been described as end bulb formations of axons. From the hilar region, axons fan out into a palisade zone where the nerve terminals packed with neurosecretory granules, mitochondria, and microvesicles abut upon basement membranes. The neurosecretory granules are similar to those present in the neurohypophysis of other mammals, except for an occasional huge granule of distinctive type. Material morphologically and histochemically resembling glycogen occurs as scattered particles and as aggregates within nerve fibers. The septal zone, containing collagen, fibroblasts, and numerous small capillaries, is separated from the adjacent glandular tissue by a basement membrane.     

Ultrastructural observation on the rat supraoptic neurons following acute operative stress

The response of the SON to various forms of stress is well documented. However, the effect of operative stress, which is a common and important clinical event requiring the mediation of vasopressin, has largely escaped attention. The present report describes the ultrastructural changes in the neurons of the caudal (retrochiasmatic) part of the SON following a deepseated linear incision on the dorsum of the rat. The observations were confirmed to the first forty-eight hours after trauma. At 24-hours post-operatively, a marked depletion of the neurosecretory granules was observed. This was associated with a proliferation of the granular and smooth endoplasmic reticulum, Golgi cisternae and ribosomes. A few of the neurosecretory granules were seen to lie in the close vicinity of the Golgi complexes. At 48-hours after trauma, these features persisted. In addition, an accumulation of neurosecretory granules was conspicuous in some axon pre-terminals. From the above findings, it is suggested that an increased demand for vasopressin during the early postoperative period is met by the supraoptic neurons by a liberation of their neurosecretory contents. An attempt at replenishment of the latter is evidenced by a proliferation in the membrane components and ribosomes. The pooling of neurosecretory granules in occasional axon pre-terminals may indicate an imbalance in the synthesis-secretion coupling of vasopressin.     

Ultrastructure of the peptidergic and monoaminergic neurons in the hypothalamic neurosecretory system of Anuran Batracians

Summary In the toad Bufo arenarum Hensel the following regions of the hypothalamic — neurohypophyseal system were studied under the electronmicroscope: preoptic and paraventricular nuclei, median eminence and infundibular process of the neurohypophysis.Neuronal perikarya of the preoptic nucleus are loaded with typical neurosecretory granules of peptidergic nature having a mean diameter of 1660 Å. While most neurons of the winter toad are in a storage stage a few show signs of a more active synthetic activity. A distinctive feature of preoptic neurons is the presence of large lipid droplets. The paraventricular nucleus contains small neurons containing granulated vesicles with a mean diameter of 800-1000 Å. In the region extending between these two nuclei and the median eminence axons containing either neurosecretory elementary granules or granulated vesicles are observed.The inner zone of the median eminence is occupied by axons of the preoptic neurohypophyseal tract; two types of axons, according to the size and density of the neurosecretory granules, may be recognized. The outer zone of the median eminence contains mainly axons and nerve terminals containing granulated vesicles of probable monoaminergic nature and only a few with granules of peptidergic type.The neurohypophysis contains two kinds of axons: one with more dense granules of 1800 Å and the other with granules of lesser electron density and 2100 Å. At the ending proper small clear vesicles of synaptic type are found.A progressive increase in volume of the peptidergic granules along the axon is demonstrated. This is of the order of 218% from the preoptic perikarya down to the infundibular process. The physiological significance of the two neurosecretory systems — i.e. the monoaminergic and the peptidergic — and the probable nature of the two types of peptidergic axons is discussed.Supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas and by the Air Force Office of Scientific Research (AF-AFOSR 963-67).The authors want to express their gratitude to Mrs. Defilippi-Novoa and Mr. Alberto Sáenz for their skillful assistance.     

Ultrastructural localization of neurohypophyseal hormones and their carrier proteins by the "triple bridge" immunoenzymatic technique]

High specific antibodies have permitted the ultrastructural localization of vasopressin, ocytocin and neurophysins 1 and 2. Hormones and carrier proteins are detected in neurosecretory granules of the hypothalamic and neurohypophyseal fibres and so at the granular and extra-granular level of supra-optic pericaryons.     

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.     

Size and degeneration increase in herring bodies during aging in hamsters.

The hypothalamo-neurohypophysial tract of young, adult and aged male hamsters was studied at lateral and ventral regions of hypothalamus by means of electron microscopy. Neurosecretory swelling axons (Herring bodies) were usually found as classically described containing abundant neurosecretory granules, mitochondria, few microtubules and profiles of smooth endoplasmic reticulum in all groups of age. However, in aged hamsters, starting at 18-month-old subjects, we observed that the size of some neurosecretory axons was highly increased. Autophagic and degenerative features were seen in the larger ones. These data could suggest abnormal axonal storage or axonal transport blocked during aging. The implications in the role of hypothalamus-neurohypophysial system during aging are discussed.     

The effect of castration upon the ultrastructure of the rat hypothalamus

Summary The neurosecretory hypothalamic nuclei and the inner zone of the median eminence of castrated rats were studied under the electron microscope. After one month of castration all the neurosecretory neurons of both nuclei show signs of hyperactivity characterized by dilated cisternae of the endoplasmic reticulum containing a macromolecular filamentous material and an increase in the number of ribosomes. After six months of castration, some neurosecretory neurons show an increased number of neurotubules and larger lysosomes than in the controls. Other neurons show a very significant hypertrophy of the endoplasmic reticulum, with large amounts of intracisternal filamentous material. These cells have few neurosecretory granules and in the adjacent synapses the number of granulated vesicles is increased. In the supraoptic nucleus there are two kinds of neurosecretory axons: the clear ones, which are similar to those that appear in control animals and the dark ones, which have smaller elementary granules. In the inner zone of the median eminence the axons show an increase in the number of neurosecretory granules with respect to the controls. After supplementary administration of sexual hormones, all the modifications produced by castration disappear. The ultrastructural changes observed in the neurosecretory nuclei after castration are discussed in relation to those previously described in the neurohypophysis under the same experimental conditions. A feedback regulatory action of sex hormones on hypothalamic neurosecretory neurons is postulated.Supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas and by the Air Force Office of Scientific Research (AF-AFOSR 963-67).We are deeply indebted to Mrs. Defilippi-Novoa and Mr. Alberto Saenz for their skillful assistence.     

Immunocytochemistry and ultrastructure of the neuropil located ventral to the rat supraoptic nucleus

Summary The neuropil located ventral to the SON was investigated by the use of immunoperoxidase staining for neurophysins, oxytocin and vasopressin, and electron miroscopy. The study was performed in six groups of rats: 1) control; 2) infusion of isotonic saline into the CSF; 3) infusion of hypertonic saline into the CSF; 4) drinking hypertonic saline for 4 days; 5) same as group 4 but injection of colchicine into the CSF on second day of dehydration; 6) salt loading for 3 months. In the control rats the ventral neuropil contained a few immunoreactive processes, the general morphology of which was completely different from that of the neurosecretory axons emerging from the SON at its dorsal aspect. In rats of groups 3 to 6 the ventral processes (VP) became loaded with neurosecretory granules, whereas the perikarya and axons were depleted. Based on their general morphology and reactivity pattern it is suggested that the VP are dendrites. Most of these dendrites were embedded in a glial cushion formed by the processes of a particular type of marginal glia. Some of these dendrites enveloped an arteriole penetrating the optic tract. All VP were rich in synaptic contacts. The possibility that the VP of neurosecretory cells may be functionally related to the subarachnoid CSF and the arteriolar blood flow is discussed.Supported by Grant RS-82-18 from Direccíon de Investigaciones, Universidad Austral de Chile     

Ultrastructural studies on the secretory cycle of the neurosecretory cells and the formation of herring bodies in the paraventricular nucleus of the rat

Summary The ultrastructure of the neurosecretory cells in the paraventricular nucleus of the normal male rat was studied by electron microscopy during various functional states. Four morphologically distinct types of neurosecretory cells were observed. It appears that they do not represent different classes of cells but different phases of secretory activity of a single cell type. The perikarya of the neurosecretory cells show a definite cycle of formation and transportation of secretory granules. We have designated the phases of this cycle as: (1) phase of synthesis, (2) phase of granule production, (3) phase of granule storage and (4) phase of granule transport. The neurosecretory granules appear to be moved in bulk into the axons, forming a large axonal swelling filled with granules as a result of one cycle in the neurosecretory process. Thus it may be postulated that a secretory cycle in the perikaryon of the neurosecretory cell seems to result in the formation of a Herring body in its axon, and that its content is then conveyed to the posterior pituitary.     

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.     

Effect of colchicine on the intracellular transport of secretory proteins in rat liver parenchymal cells. Immunocytochemical observations

We studied the effects of colchicine on the intracellular transport of secretory proteins in rat liver parenchymal cells using the direct immunoenzyme technique. Livers were perfusion-fixed 0.5, 1, and 2 h after injection of colchicine. Vibratome sections of the fixed liver were stained using peroxidase-conjugated Fab' of anti-albumin or anti-fibrinogen. By light microscopy, reaction deposits showing albumin and fibrinogen were observed in the cytoplasmic granules of hepatocytes. Such stained granules decreased 30 min after injection, but later increased gradually and crowded in the cytoplasm. The Golgi complex stained for the proteins decreased after 30 min but increased in the juxtanuclear region after 60 min. The analysis of serial sections showed that colchicine severely disturbed the spatial relationship between the Golgi apparatus and the bile canaliculus. We obtained similar results by electron microscopy; a positive reaction for albumin and fibrinogen was observed in a small number of the cytoplasmic granules after 30 min. After 1 h of treatment, most of the Golgi complexes were fragmented and lost their stacked cisternae. However, they reappeared accompanied with vacuolated cisternae and secretory granules, which were partially stained for albumin and fibrinogen. After 2 h, the secretory granules positive for both proteins accumulated further. Some of them lined a long the plasma membrane, and others made a cluster in the cytoplasm. The profiles showing exocytosis were very rarely seen. These results showed that in the first 30 min, colchicine primarily disturbs partially the Golgi assembly but does not affect the post Golgi secretory pathway much. Later, the drug affects both the post Golgi pathway and the Golgi assembly, and it causes a marked accumulation of secretory granules.     

Occurrence of large hyaline bodies in the preoptic nerve cells of Zoarces Viviparus L.

Summary Specially in stress conditoin, some hyaline droplets occur in the perikarya of the neurosecretory hypothalamic cell of Zoarces viviparus. They first appear as filamentous macromolecular material in the intracisternal space, then tend to accumulate in the form of irregular electron dense rods or reticulum. The neurosecretory cells containing such hyaline bodies have usually few elementary neurosecretory granules and dilated endoplasmic reticulum.Aided by a Grant from NATO and the Deutsche Forschungsgemeinschaft.My thanks are due to Prof. Dr. E. Lindner and Doz. Dr. K. H. Andres for their kind help in the explanation of electronmicrographs.     

The hypothalamo-hypophysial system of the frog Rana temporaria

Summary The median eminence (ME) of the adult frog, Rana temporaria, was studied by means of electron microscopy including quantitative electron-microscopic autoradiography. In frogs captured in May and June numerous peptidergic neurosecretory fibres extending via the internal zone to the pars nervosa display large swellings containing few granules, mitochondria, neurotubules and cisternae of the smooth endoplasmic reticulum. In addition, few secretory globules up to 1.5 m in diameter occur in these varicosities. In animals collected during the autumn period many of these neurosecretory swellings filled with neurosecretory granules and polymorphic inclusions resemble Herring bodies. Three types of granule-containing neurosecretory fibres were observed in the external zone (EZ) of the ME of adult R. temporaria. Peptidergic A₁- and A₂-type fibres are characterized by granules 150–220 nm and 100–160 nm in diameter, respectively. Monoaminergic fibres of type B with granules approximately 100 nm in diameter represent 50% of all neurosecretory elements in the EZ of the frog ME; 12% of the total number of granule-bearing axons in the EZ actively taking up radiolabelled 5-hydroxytryptophan are thought to be serotoninergic terminals. Neurosecretory terminals of all types and glial vascular endfeet establish direct contacts with the perivascular space of the primary portal capillaries. Some neurosecretory terminals are separated from the lumen of the third ventricle by a thin cytoplasmic lamella of tanycytes. The possible physiological significance of this structural pattern is discussed.     

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).