Effect of colchicine on vasopressin and melanin-concentrating hormone neurons of rat hypothalamus: hybridocytochemistry and immunocytochemistry studies]

48 hrs. after an intra-cerebroventricular injection of colchicine (100 micrograms), antisera to three putative peptides included in the rat melanin-concentrating hormone (MCH) precursor, strongly stained the secretory granules accumulated in perikarya. In control rats, these antisera stained endoplasmic reticulum, Golgi apparatus, or neurosecretory granules respectively. Colchicine also induced a dramatic decrease in hybridization signal obtained with a probe complementary to the prepro-MCH-mRNA. Similarly, colchicine induced a strong increase in vasopressin immunoreactivity in neurons of the paraventricular and supraoptic nuclei, and a strong decrease of the vasopressin precursor mRNA. These results demonstrated that, in two peptidergic neuron populations of the rat hypothalamus, colchicine lowers mRNAs and impairs neuropeptide protein synthesis, consecutively to the accumulation of neurosecretory granules in perikarya.     

Effects of small doses of colchicine on the components of the hypothalamo-neurohypophysial system of the rat

Summary Small doses (3.5 g and 7 g) of colchicine injected intracisternally caused an interruption of transport of secretory material from the supraoptic and paraventricular nuclei of the hypothalamus to the neural lobe of the pituitary gland. Transport was assessed by direct measurement of the incorporation of [³⁵S] cysteine into neurophysins, by radioimmunoassay of accumulated material in discrete areas of the system and by immunocytochemistry. The larger dose (7 g) switched off transport completely during the first 24 h but the system began to recover within three to four days. Colchicine had little, if any, effect on synthesis; comparison of the relationships of the apparent amounts of immunoreactive neurophysins and immunoreactive hormones in the arrested product led to the conclusion that processing of the hormone precursors continues within the secretory granules which accumulated in the perikarya.Supported by the Medical Research Council and the Science Research Council. E.M.R. was in receipt of an exchange fellowship under the auspices of CONICYT of Chile and The Royal Society. The authors are grateful for the careful technical assistance of Mr. Peter Rees     

Morphometric and electron microscopic studies of the secretory granules of neurosecretory cells of the supraoptic and paraventricular nuclei of white rats and mice]

The morphometrical and electron microscopic analysis of secretory granules in the perikaryons of neurosecretory cells of the supraoptic and paraventricular nuclei in male rats and mice has shown than in the cells of these nuclei in both species of animals there occur secretory granules of the same kind and size. Therefore this method fails to determine which of them contain oxytocin and which of them contain vasorpressin. The neurosecretory granules located in the Golgi apparatus zone are of a less size and have more osmiophilic cnetral material than the granules localized on the periphery which mainly have granular central material and are of a greater size. The distinctions in the size and type of secretory granules are associated with certain stages of their "maturation". Granular particles appear to be "swallen", more active forms of storing neurohormones. The presence of larger granular particles in the supraoptic nucleus of mice allows to suggest greater reactivity of this nucleus than in rats which is likely to be associated with a higher ability of mice, as compared with rats, to adaptate to disturbances in water-salt metabolism.     

Effects of colchicine on axonal transport and ultrastructure of the hypothalamo-neurohypophyseal system of the rat

Summary The effect of colchicine on the transport of proteins in the hypothalamo-neurohypophyseal tract of the rat was studied after injection of (³⁵S) cysteine into the supraoptic nucleus (SON) region. Colchicine, dissolved in distilled water and administered subarachnoidally, inhibited the axonal transport of labelled proteins into the neurohypophysis: the radioactivity that was recovered in neurohypophyseal TCA precipitable material was markedly decreased and hardly any radioactivity was found in the neurohypophyseal proteins which were separated by polyacrylamide gel disc electrophoresis.As revealed by electron microscopy the SON cell bodies showed marked changes after treatment with colchicine: a deeply folded nucleolemma; a pronounced, granular nucleolus; a dispersed chromatin; a zonal distribution of cell organelles with mitochondria and lysosomes accumulated at the periphery, crowded ribosomes, often arranged as polyribosomes and richly branching short profiles of endoplasmic reticulum filled with filamentous material forming an inner perinuclear zone separated by enlarged Golgi complexes.The profiles of elongated Herring bodies in the infundibulum were increased. The axon terminals were filled with heavily osmiophilic neurosecretory granules. The neurofilaments were slightly or moderately increased in number. No apparent changes were observed with regard to the neurotubuli in the SON neurons. The glial cells of the supraopticoneurohypophyseal tract showed reactive changes with a proliferation of filamentous elements. The biochemical and ultrastructural findings are discussed especially with respect to the mechanisms of transport and release of neurosecretory granules.     

Vasopressinergic axon collaterals and axon terminals in the magnocellular neurosecretory nuclei of the rat hypothalamus

Axon collaterals emerging from the vasopressinergic neurons of the supraoptic (SON) and paraventricular (PVN) nuclei and recurving back towards their respective nuclei have been previously reported. Since such axon collaterals can play a role in the neuromodulation of SON and PVN, these nuclei have been further investigated immunohistochemically under the light and electron microscope. The PAP technique, using a commercial antibody, was employed. Vasopressin-positive axon collaterals were seen to recurve towards their nuclei of origin. In the latter, vasopressinergic intrinsic neurons were also observed. Under the electron microscope, axon terminals containing vasopressin-immunoreactive neurosecretory granules were noted. Such terminals presumably arise from the vasopressin-positive recurrent axon collaterals or from the intrinsic neurons for the purpose of neuromodulation within the SON and PVN.     

The fine structure of the hypothalamic secretory neurons of the White-crowned Sparrow,Zonotrichia leucophrys gambelii (Passeriformes: Fringillidae)

Summary The fine structures of the neurons and neuropils of the magnocellular supraoptic nucleus and the parvocellular nuclei of the rostral hypothalamus, including the suprachiasmatic and medial, lateral and periventricular preoptic nuclei, and the neuronal apparatus of the organum vasculosum laminae terminalis, have been examined in the male White-crowned Sparrow, Zonotrichia leucophrys gambelii, by correlated light and electron microscopy.The magnocellular supraoptic nucleus is characterized by large neurosecretory perikarya which contain a well developed Golgi complex and densecored granules 1,500–2,200 Å in diameter. The neuropil displays axons, dendrites and glial fibers. Some axonal profiles contain dense-cored vesicles 800–1,000 Å in diameter and clear vesicles 500 Å in diameter. Axo-somatic and axo-dendritic synapses are conspicuous in this nuclear region.The suprachiasmatic nucleus is characterized by an accumulation of small neurons with moderately developed cellular organelles and some dense-cored granules, approximately 1,000 Å in diameter. The profiles of axons within the neuropil contain dense-cored granules 800–1,000 Å in diameter and clear vesicles 500 Å in diameter.The neurons of the medial preoptic nucleus are relatively large and exhibit well developed cellular organelles and dense-cored granules 1,300 to 1,500 Å in diameter. Granular materials are formed within the Golgi complex. The medial preoptic nucleus is rich in secretory perikarya.Occasionally, neurons with granules 1,500–2,200 Å in diameter are encountered in the lateral preoptic and periventricular preoptic nuclei. They may be considered as scattered elements of the magnocellular (supraoptic and paraventricular) system.The organum vasculosum laminae terminalis consists of three layers, i.e., ependymal, internal and external zones, and exhibits a vascular arrangement similar to that of the median eminence. The perikarya of the parvocellular neurons and their axons in the internal zone contain numerous secretory granules ranging from 1,300 to 1,500 Å in diameter.This investigation was supported by Grant No. 5R040 Japan-U.S. Cooperative Science Program of the Japan Society for the Promotion of Science to Professor H. Kobayashi and Professor S.-I. Mikami, by a Scientific Research Grant No. 56019 from the Ministry of Education of Japan to S.-I. Mikami, by support from the Deutsche Forschungsgemeinschaft (Schwerpunktprogramm Biologie der Zeitmessung) to Prof. A. Oksche and by Grant No. GF 33334, U.S.-Japan Cooperative Science Program of the National Science Foundation to Prof. D.S. Farner.Herrn Professor Dr. Dres h.c. Wolfgang Bargmann zu seinem 70. Geburtstag am 27. Januar 1976 gewidmet.     

Maturation of the human hypothalamo-hypophyseal neurosecretory system in prenatal ontogeny. A light-optical and electron microscopic study

The hypothalamic-hypophysial neurosecretory system (HHNS) was studied in the human foetuses from the age of 8 weeks till birth. The hypothalamus of 8 weeks old foetuses is weakly differentiated, no individual cell groups, so-called nuclei, are identified. The supraoptic (SON) and paraventricular (PVN) nuclei are identified from the age of 12 weeks on. The size of cell nuclei increases with the age. The Homori-positive granules were first found in some SON and PVN cell and in neurohypophysis in the 18 weeks old foetuses. It was shown under the electron microscope that the neurohypophysis of 8 weeks old foetuses consisted mainly of pituicytes with axons among the cytoplasmic processes of the latter. After the age of 10 weeks, the area of parenchyma of the neurohypophysis occupied by axons increased and typical elementary neurosecretory granules appeared in them. The data obtained are discussed with respect to the participation of HHNS in the regulation of water metabolism in the human foetuses.     

Glucagon-like immunoreactivity in hypothalamic neurons of the rat

Summary Antisera specific for three different regions of pancreatic proglucagon were used to examine the distribution of such immunoreactivity in rat hypothalamus. Neurons in the supraoptic and paraventricular nuclei were immunoreactive with an antiserum against glucagon, but not with antisera directed towards the aminoterminal region of proglucagon (glicentin) or the glucagon-like peptide I sequence in the carboxyl-terminal region of proglucagon. These findings confirm a previous report of glucagon-like immunoreactivity in the supraoptic and paraventricular nuclei, but indicate that, while this material is immunochemically related to glucagon, it is not derived from a proglucagon-like precursor.     

P granules in the germ cells of Caenorhabditis elegans adults are associated with clusters of nuclear pores and contain RNA

The germ cells, and germ cell precursors, in the nematode Caenorhabditis elegans contain distinctive granules called P granules. During early embryogenesis, P granules are segregated asymmetrically into those blastomeres that eventually produce the germ line. Because of the correlation between P granule distribution and the development of the germ line, P granules are widely thought to function in some aspect of germ line specification or differentiation. Most of the analysis of P granule structure and localization has focused on the early embryo, when P granules are located in the cytoplasm. However, during most of development P granules are associated with germ cell nuclei. We report here an ultrastructural analysis of the nuclear-associated P granules in the germ cells of the adult hermaphrodite gonad. We show that P granules are tightly associated with nuclear pores and that the positions of certain structures within the P granules correspond to the positions of pores on the nuclear envelope. We present immunocytochemical and ultrastructural data suggesting that P granules can associate, or remain associated, with pore-like structures even after they detach from the nuclear envelope during oogenesis. Finally, we show that nuclear-associated P granules in the gonad contain RNA, complementing previous studies showing that cytoplasmic P granules in embryos contain RNA.     

Immunohistochemical identification of neurons containing corticotropin-releasing factor in the rat hypothalamus

A specific rabbit anti-CRF serum and the immunoperoxidase technique were used to show that CRF-containing neurons are mainly distributed in the paraventricular and supraoptic nuclei of the rat hypothalamus. In addition, immunoreactive neurons are scattered in other hypothalamic regions. These neurons are 20--30 micrometers in diameter. From the present and previous investigations it may be concluded that the hypothalamic magnocellular nuclei, i.e., paraventricular and supraoptic, and other hypothalamic accessory nuclei, are the producing sites not only for vasopressin and oxytocin, but also for corticotropin-releasing factor.     

An immunocytochemical comparison of the angiotensin and vasopressin hypothalamo-neurohypophysial systems in normotensive rats.

In the present study we investigated the possibility that angiotensin II/III and vasopressin coexist in the hypothalamo-neurohypophysial pathway. For our experiments 8-week-old male rats not treated with colchicine were used. The anatomical orientation of the entire pathway for angiotensin and vasopressin was facilitated by examining a series of subsequent coronal, horizontal and sagittal sections. Arching fibre tracts are formed mainly by projections emanating from cell bodies in the paraventricular nucleus, the accessory magnocellular nuclei, the supraoptic nucleus and the retrochiasmatic part of the supraoptic nucleus. The majority extend as far as the median eminence and the neurohypophysis, where major terminal fields exist. However, there is a difference between the staining pattern within the suprachiasmatic nucleus and the hypophysis. The results clearly show the colocalization of angiotensin and vasopressin in neurones as well as in fibres of the hypothalamo-neurohypophysial system.     

Morphofunctional characteristics of neurosecretory cells of the supraoptic nucleus of rats with parathyroprival hypocalcemia]

Humori-positive neurosecretory cells of the supraoptic nucleus of the hypothalamus have been investigated during various time of parathyroprival hypocalcemia after extirpation of the parathyroid glands. Contents of total and ionized calcium, phosphorus in blood serum have been estimated. Volume of nuclei and nucleoli has been measured. In 5 days functional activity of the supraoptic nucleus increases (lightly stained cells predominate, volume of the nuclei and nucleoli increases). In subsequent 15-30 days its activity decreases (increase in amount of dark-stained cells, nucleolar volume decreases). In 60 days there is a tendency to restoration of neurosecretion.     

Granule populations in oxytocin and abnormal perikarya of the supraoptic nucleus of homozygous Brattleboro rats: Effects of colchicine administration

Summary Magnocellular neurones in the supraoptic nucleus of the homozygous Brattleboro rat, which are unable to produce vasopressin, were investigated by immunocytochemistry to identify both the oxytocin cells and the abnormal neurones, which in normal animals would produce vasopressin. The abnormal cell profiles were significantly more rounded than those of the oxytocin cells. Both cell types showed evidence of hyperactivity, but the Golgi apparatus was more extensive in the oxytocin cells, probably as a result of the failure of the abnormal cells to produce vasopressin and its neurophysin and the resultant reduction in hormone packaging. Neurosecretory granules (NSG) 160 nm in diameter were found in the oxytocin perikarya but were absent from the abnormal cell bodies. In addition, a population of small dense granules (SDG) 100 nm in diameter was observed in both types of neurone, in numbers equal to the NSG in oxytocin cells.Injection of a low, non-lethal dose of the axonal transport inhibitor colchicine resulted in a rapid and equal accumulation of both NSG and SDG in oxytocin perikarya and of SDG in the abnormal perikarya after one day. The effects of colchicine were reversed 2–3 days after administration. The SDG, which may contain a co-transmitter or co-hormone substance, are thus produced at a similar rate to NSG, and appear to be transported from the perikarya for subsequent release at the nerve endings.     

Ultrastructural studies on the hypothalamic neurosecretory neurons of the rat

Summary The general ultrastructural features of the hypothalamo-neurohypophysial system in rats with hereditary hypothalamic diabetes insipidus (DI-rats, Brattleboro strain) are described. There is no decisively distinguishing difference between the neurons of the supraoptic and paraventricular nuclei. The neurons of both nuclei show signs of active protein synthesis. The perikarya of the neurons are markedly hypertrophic, the nuclei are large and the nucleoli prominent. In the cytoplasm there are numerous ribosomes, abundant rough-surfaced endoplasmic reticulum and extensive Golgi complexes. However, very few neurosecretory granules are to be seen. The axons of the hypothalamo-neurohypophysial tract are likewise enlarged and the paucity of neurosecretory granules is a striking feature also in the area of the tract. The majority of nerve endings in the posterior pituitary of DI-rats are devoid of neurosecretory granules. Microvesicles are abundant in the nerve endings and there are findings which suggest that microvesicles are involved either in endoor exocytosis. The signs of active protein synthesis and the concomitant paucity of neurosecretory granules are interpreted to imply transportation of the secretory proteins in an extragranular phase. The possible mode of release of the secretory proteins from the nerve endings and the role of microvesicles therein are discussed.This study has been supported by grants from the Finnish Cultural Foundation and the Sigrid Jusélius Foundation. The collaboration of Professors Antti Arstila and Tapani Vanha-Perttula is gratefully acknowledged.The Brattleboro-rats were kindly provided by Dr. Heinz Valtin, to whom we express our thanks.     

Changes in metabolic activity of neurons in the anterior hypothalamus nuclei during hyperthermia, fever, and hypothermia

Differently directed changes in metabolic activity of anterior hypothalamic nuclei's neurons in rats during hyperthermia, fever, and hypothermia were revealed with histochemical methods. During hyperthermia, the activity of energy metabolism enzymes increased as well as RNA content in the neurons of supraoptic, paraventricular and median preoptic anterior hypothalamic nuclei. This is shown by an increase in the metabolic activity of neurons of these nuclei. Metabolic activity in neurons of median preoptic nuclei decreased and was not changed considerably in neurons of supraoptic and paraventricular nuclei during endotoxin-induced fever. The development of hypothermia was characterised by a decrease in metabolic activity of neurons of supraoptic, paraventricular and medium preoptic nuclei. It is supposed that differently directed metabolic activity changes in neurons of anterior hypothalamic nuclei during hyperthermia are connected with the mechanisms of body temperature regulation (median preoptic nuclei) and neurosecretory processes (supraoptic and paraventricular nuclei).     

Electron microscope immunohistochemical localization of neurophysin in the rat with hereditary diabetes insipidus

In order to study the subcellular distribution of neurophysin in the rat with hypothalamic hereditary diabetes insipidus (DI), an immunoelectron microscopic localization of neurophysin was performed in the hypothalamo-neurohypophysial system of both homozygous and heterozygous DI rats. Whereas in control rats neurophysin was localized in the granules present in the secretory neurons, in the homozygous DI rats neurophysin was found in the granules and outside the granules in the perikarya and axons of neurons of both supraoptic and paraventricular nuclei. In the heterozygous DI rats findings similar to those observed in homozygous DI rats were observed, although in the posterior pituitary, the exgranular material appeared to be less abundant than in homozygous DI rats. These results clearly demonstrated that in hyperstimulated neurons neurophysin was distributed in both granular and extragranular compartments.     

Cytological effects of colchicine on the grasshopper neuroblast in vitro with special reference to the origin of the spindle

The effectiveness of colchicine in destroying or preventing the development of the spindle is determined by the concentration of the colchicine and the degree of development of the spindle at the time of exposure; the greater the concentration of colchicine, the greater will be its effectiveness in destroying the spindle or interfering with its development; the more completely the spindle is developed at the time of exposure to colchicine, the greater is the concentration of colchicine required to destroy it or prevent its further development.The series of changes in chromosome orientation that take place during the course of colchicine action, namely, approximation of centromeres, formation of “stars,” the breaking up of one “star” into several, and complete chromosome disorientation, represent successive stages in the destruction of the spindle.Destruction of the completely formed spindle is typically accompanied by the accumulation of the spindle material outside the diminishing spindle in a hyaline globule. Similarly, interference of colchicine with spindle development leads to the accumulation of the presumptive spindle material, i.e., the karyolymph, in one or more hyaline globules. It is suggested that colchicine does not destroy the spindle material but merely alters its molecular orientation, so that it comes to comprise a spherical mass with no mitotic function.Strong concentrations of colchicine cause middle and late prophase nuclei to revert to early prophase. Somewhat lower concentrations applied to late prophase nuclei occasionally delay the breakdown of the nuclear membrane without altering the rate of chromosome contraction. In greatly retarded late prophase nuclei the chromosomes lose their intranuclear orientation, which lends support to the concept that the centromeres normally maintain a fixed position within the nucleus.     

The hypothalamic magnocellular system in the domestic fowl

Summary In the rostral hypothalamus of the domestic fowl, the magnocellular neurosecretory nuclei show a peculiar differentiation. Golgi studies of the supraoptic and paraventricular nuclei of the fowl reveal at least two major cell types: 1) large multipolar neurons, and 2) small interneurons. Golgi impregnations provide a detailed cytoarchitectural picture of the large-sized cells; the latter may well correspond to the neurosecretory cells demonstrated in the same regions by selective staining, and immunocytochemical and electron microscopical techniques.Electron microscopically, neuronal perikarya are observed to contain variable amounts of neurosecretory granules (100–200 nm in diameter; mean diameter of 160 nm) scattered throughout the cytoplasm. The diameters of these granules do not differ statistically in the two principal nuclear areas examined. The perikarya of these neurons display only a few axosomatic synapses containing electron-lucent and dense-cored vesicles (70–90 nm in diameter). Numerous nerve terminals of this type also end on the dendritic ramifications in the surrounding neuropil.     

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.     

Neurosecretory and microstructural changes in the hypothalamus of rats following administration of lithium chloride and 3H-thymidin

Adult male rats were intraperitoneally administered aqueous solution of lithium chloride (LiCl). Studies, including neurosecretory and microstructural changes within particular neurocytes in supraoptic (NSO) and paraventricular nuclei (NPV) were performed on hypothalamic sections. In the experimental rats the administered LiCl increased the level of GOMORI-positive neurosecretory material both in supraoptic and paraventricular nuclei. Great amounts of the neurosecretory material were markedly conspicuous in the above areas after 20 days of LiCl administration. Investigations carried out on cellular nuclei of particular neurocytes showed a significant enlargement of the nuclei, and statistical calculations revealed that, in comparison with the basic control, the difference was essentially significant (p less than 0.001). 3H-thymidin administration to the rats which had previously been on LiCl for 20 days demonstrated also that within supraoptic nuclei the incorporation of the isotope in cellular nuclei took a faster course than in control animals.