Immuno-enzyme cytochemical demonstration of mesotocinergic nerve fibres in the pars intermedia of the amphibian hypophysis

Summary Immuno-enzyme cytochemical investigations showed that the whole amphibian pars intermedia of the hypophysis is innervated by an intercellular network of peptidergic varicose nerve fibres which contain mesotocin or (and) parts of the mesotocin molecule. The pars intermedia does not contain vasotocinergic fibres. The mesotocinergic fibres are branches of axons leaving the pituitary stalk and the neural lobe. In animals of which the hypothalamic magnocellular neurosecretory preoptic nuclei had been completely removed, the immuno-reactive mesotocinergic fibres of the pars intermedia had totally disappeared. From this result, it is concluded that the mesotocinergic fibres of the pars intermedia of the amphibian hypophysis are axons of neurosecretory perikarya located in the hypothalamic magnocellular neurosecretory preoptic nuclei.Dedicated to Professor Berta Scharrer on the accasion of her 70th birthdayThis investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

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.     

Immuno-cytochemical demonstration,in the external region of the amphibian median eminence,of separate vasotocinergic and mesotocinergic nerve fibres

Summary By means of single and double immuno-enzyme cytochemical staining techniques, it was shown that the external region of the amphibian median eminence contains separate vasotocinergic and mesotocinergic nerve fibres. Moreover, it was demonstrated that the vasotocinergic fibres also contain neurophysin. In animals in which the hypothalamic magnocellular neurosecretory preoptic nuclei had been completely removed, the immuno-reactive vasotocinergic and mesotocinergic fibres of the median eminence had disappeared. From this result, it is concluded that, at least the great majority of the vasotocinergic and mesotocinergic fibres of the external region of the amphibian median eminence are processes of neurosecretory perikarya located in the hypothalamic magnocellular preoptic nuclei. On the other hand, our results do not exclude the possibility that a minority of these neurosecretory fibres originate from small immuno-reactive perikarya which were found in the tuber cinereum. The observation that both kinds of processes accumulate around blood capillaries of the hypophysial portal system strongly suggests that they play a role in the control of the activity of the pars distalis of the hypophysis.This investigation was supported by a grant from the Belgian Nationaal Fonds voor Geneeskundig Wetenschappelijk Onderzoek     

The function of the hypophysis without preoptic neurosecretory control

Summary In a large number of adult female and male specimens of Rana temporaria, a total extirpation of the magnocellular neurosecretory preoptic nuclei was performed. Several months after operation, no regeneration of the neurosecretory nuclei had occurred. The results showed 1. that all A.F.-positive neurosecretory nerve fibres of the hypothalamo-hypophysial region originate from nerve cell bodies localized in the magnocellular preoptic nuclei; 2. that the magnocellular preoptic nuclei do not play an important part in the inhibitory control of the activity of the pars intermedia of Rana temporaria; 3. that the possible role of the magnocellular preoptic nuclei in the seasonal development of the gonads and of the secondary sexual characteristics of Rana, could be only of secondary importance; 4. that, in accordance with previous experiments, in Rana, ovulation is dependent on preoptic hypothalamic structures. Some results of the present and of other experiments can be interpreted as supporting the hypothesis of a possible dual higher control over the gonadotropic centre (Dierickx, 1965b, 1966, 1967) of the pars ventralis of the tuber cinereum.A part of these studies has been reported previously in abstract form (Dierickx, 1963a, 1963b, 1965a).     

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.     

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.     

Ultrastructure of the protocerebral neurosecretory cells of larval Galleria mellonella, in situ and after culture of the brain in vitro

Three major groups of neurosecretory cells are described in the larval brain of Galleria mellonella at two different times during the last larval instar and in larval brains after 72 hr of culture in vitro. The medial group in vivo consists of four distinct neurosecretory cell types, based on characteristic size and morphology, while the posterior and lateral groups each contain a single distinct type of neurosecretory cell. Morphological differences between the same neurosecretory cells at the different times during the last instar are most apparent in the lateral L-1 cells and in the medial M-2 cells, where pleiomorphism is particularly evident in the size, density and accumulations of neurosecretory granules. The only neurosecretory cells in which apparent synthesis of neurosecretory granules is still observed after culture of the brain in vitro are the medial M-2 cells. The other neurosecretory cell types show no accumulation of neurosecretory granules nor new synthesis of neurosecretory material, but are similar to neurosecretory cells in the brain in vivo in all other respects. The morphology of the neurosecretory cells in the larval brain in vivo and in vitro is discussed in relation to their appearance at the light microscopic level and to a known neurohormonal function of the brain which is maintained during 72 hr in vitro.     

Ultrastructure of neurosecretory cells in the pars intercerebralis of Rhodnius prolixus (Hemiptera).

Six neuron types are distinguished in the pars intercerebralis of the starved fifth instar of Rhodnius prolixus. All neuron types contain electron dense secretory granules derived from Golgi complexes which are of characteristic size and morphology in each type. The neuron types are not thought to represent stages in a secretory cycle. The variety of neuron types described is related to that revealed by staining sections of the same cells with paraldehyde fuchsin. Active synthesis of neurosecretory granules continues throughout starvation and the lysosomal system appears to be involved in the continual degradation of secretory granules. Some of the variations in granule morphology observed may be a consequence of granule fusion and the importance of cytoplasmic events in the development of neurosecretory granules is discussed.     

Hypothalamic accessory nuclei and their relation to the angiotensinergic and vasopressinergic systems.

The existence and colocalization of angiotensin II- and vasopressin-like immunoreactivity in individual magnocellular cell groups of the hypothalamus has been demonstrated by using immunocytochemical methods. These neurosecretory magnocellular groups consist of the paraventricular nucleus and the supraoptic nucleus, as well as different accessory cell groups. The fibers from the neurons of the accessory nuclei project directly to adjacent blood vessels and do not comigrate with the hypothalamo-neurohypophysial fiber pathway. On the basis of these findings it can be concluded that in the hypothalamus two different angiotensinergic and vasopressinergic neurosecretory systems exist: (1) an intrinsic hypothalamic and (2) a hypothalamo-neurohypophysial system. The distribution of the accessory cell groups in the hypothalamus is shown in a 3D reconstruction which includes the connection of these magnocellular nuclei with the vascular system in this area.     

A light and electron microscopic study of the preoptic nucleus of the grass frog (Rana pipiens), under normal conditions and following transection of the preoptico-neurohypophysial tract

Degenerative and regenerative processes occur in the preoptic neurons following transection of the preoptico-neurohypophysial tract. Three types of responses after transection were observed: affected, recovered, and degenerated neurons. However, transection of the tract did not stop the synthesis of neurosecretory granulated vesicles. The affected neurosecretory neurons showed nuclear changes, increased number of Golgi complexes, and dilated cisternae of rER, as well as, an increased number of dense bodies. The recovered neurosecretory neurons contained long non-dilated cisternae of rER which were organized in a concentric manner. Also seen were large nuclei with evenly distributed chromatin, less active Golgi complexes, and vesicles. The degenerated neurosecretory neurons exhibit pyknotic nuclei, a net of dilated cisternae of rER, dense bodies, and electron dense cytoplasm.     

Identification of the vasopressin producing and of the oxytocin producing neurons in the hypothalamic magnocellular neurosecretory system of the rat

Immuno-enzyme histochemical investigations showed that, in the magnocellular hypothalamo-hypophysial neurosecretory system of the rat, vasopressin and oxytocin are synthetized in separate neurons. Both the vasopressin neurons and the oxytocin neurons are present in both the supraoptic and the paraventricular nuclei in about the same number. Preferential location of the two kinds of rat neurosecretory neurons is not as obvious as in the bovine hypothalamus. Their perikarya do not show distinct morphological differences. The two kinds of neurosecretory perikarya are the origin of separate vasopressin-containing and oxytocin-containing axons respectively. In the neural lobe, the distribution of the two different types of axons is described.     

Ultrastructural visualization of galactose in the glycoprotein of gastric surface cells with a peanut lectin conjugate

Summary The ultrastructural localization of peanut lectin-binding sites in the gastric surface epithelial cell has been studied using a horseradish peroxidase-labelled peanut lectin (PL-HRP) conjugate and other cytochemical techniques. The PL-HRP procedure has visualized glycoprotein with presumed terminal galactose residues in the apical plasmalemma and secreted mucins and has localized such glycoprotein selectively in the intermediate Golgi cisternaé situated between the saccules of the maturing face and those of the forming face of the Golgi stacks. Other cytoplasmic organelles, including the forming and stored secretory granules, did not reveal glycoprotein with terminal galactosyl residues. These results demonstrate the applicability of the PL-HRP labelling technique at the electron microscopical level to localize not only extra but also intracellular peanut lectin-reactive sites. The observations afford information concerning the possible site in the Golgi apparatus where galactose residues are added to the growing oligosaccharide side-chain of mucous glycoprotein.     

Synaptology of neurosecretory cells in the nucleus paraventricularis of the domestic fowl

Summary Processes of magnocellular neurosecretory cells (MNCs) are easily identifiable on the basis of their content in neurosecretory granules in the neuropil of the rostral division of the paraventricular nucleus (PVN) of the domestic fowl. In specimens sacrificed during the winter the synaptic organization of the neuropil and the pattern of synapses ending on neurosecretory processes were studied at the ultrastructural level. Synapses in the rostral part of the PVN neuropil may be divided into three main categories on the basis of their morphology and their content of clear and dense-core synaptic vesicles. These different types of terminals can be attributed to aminergic, peptidergic or other types of synapses. The percent distribution of synapses within these categories differs when all synapses observed in the neuropil or only those ending on MNC processes are compared. Present ultrastructural data obtained in birds support two physiological hypotheses already suggested for mammals, i.e., the probable existence of a recurrent pathway to MNCs via an interneuron, and the importance of aminergic and peptidergic input in regulating the electrical activity of MNCs.This work was partly supported by a CNR grant (n. 81.00377.04)     

In vitro differentiation of hypothalamic magnocellular neurons of guinea pigs

Summary The development of neurophysin-oxytocin and neurophysinvasopressin containing neurons of the guinea pig was studied in vitro. Supraoptic (SO) and paraventricular (PV) nuclei were explanted from guinea pig foetuses at the 40th day of gestation and cultured in Maximov slides for nearly fifty days. The cultures were observed daily under a phase-contrast microscope. Explants were fixed every five days for observation with the electron microscope. At the time of explantation, magnocellular neurons were still immature. They acquired the morphologic characteristics of mature neurons, with axosomatic synapses, after about 10 days in vitro. After 15–20 days in vitro, they contained in addition neurosecretory granules (NSG), first in the Golgi region, then also dispersed in the cytoplasm. In the oldest culture (45–50 days), signs of granulolysis were regularly found. It appears that magnocellular neurosecretory neurons are able to differentiate in vitro from a primitive state in the absence of specific stimulation.The authors are indebted to M.J. Drian for help with tissue culture, and to D. Le Cren for photographic work     

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.     

Golgi apparatus, GERL, and secretory granule formation within neurons of the hypothalamo-neurohypophysial system of control and hyperosmotically stressed mice

The vasopressin-producing neurons of the hypothalamo-neurohypophysial system are a particularly good model with which to consider the relationship between the Golgi apparatus nd GERL and their roles in secretory granule production because these neurons increase their synthesis and secretion of vasopressin in response to hyperosmotic stress. Enzyme cytochemical techniques for acid phosphatase (AcPase) and thiamine pyrophosphatase (TPPase) activities were used to distinguish GERL from the Golgi apparatus in cell bodies of the supraoptic nucleus from normal mice, mice hyperosmotically stressed by drinking 2% salt water, and mice allowed to recover for 5-10 d from hyperosmotic stress. In nonincubated preparations of control supraoptic perikarya, immature secretory granules at the trans face of the Golgi apparatus were frequently attached to a narrow, smooth membrane cisterna identified as GERL. Secretory granules were occasionally seen attached to Golgi saccules. TPPase activity was present in one or two of the trans Golgi saccules; AcPase activity appeared in GERL and attached immature secretory granules, rarely in the trans Golgi saccules, and in secondary lysosomes. As a result of hyperosmotic stress, the Golgi apparatus hypertrophied, and secretory granules formed from all Golgi saccules and GERL. Little or no AcPase activity could be demonstrated in GERL, whereas all Golgi saccules and GERL-like cisternae were TPPase positive. During recovery, AcPase activity in GERL returned to normal; however, the elevated TPPase activity and secretory granule formation seen in GERL-like cisternae and all Golgi saccules during hyperosmotic stress persisted. These results suggest that under normal conditions GERL is the predominant site for the secretory granule formation, but during hyperosmotic stress, the Golgi saccules assume increased importance in this function. The observed cytochemical modulations in Golgi saccules and GERL suggest that GERL is structurally and functionally related to the Golgi saccules.     

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.     

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.     

The secretory cycle of supraoptic neurons in the rat

Summary After perfusion with formaldehyde and glutaraldehyde the supraoptic nucleus and infundibular process of the neurohypophysis of the rat were dissected and prepared for electronmicroscope observation. This study was carried out in specimens under normal water balance, in others fed on dry food and in rats submitted to forced hydration.Two extreme types of neurons with intermediary stages were recognized in the normal supraoptic nucleus. The main difference between them is in the content of ribosomes, development and dilatation of the vacuolar system and in the number of elementary neurosecretory granules. In both types lysosome-like particles are observed. The volume of the elementary granules increases 1.7 times along the hypothalamic-hypophyseal tract while the increase in the dense core of the granule is of the order of 4.3 times.After forty-eight hours on dry food there is a general depletion of secretory granules from the perikaryon and nearby axons, the ribosomes are numerous and the endoplasmic reticulum is dilated in all cells and contains a macromolecular filamentous material. With more prolonged dehydration the neurosecretory granules reappear in relation to the Golgi complex and the vacuolar system becomes progressively flattened. With forced hydration the number of granules in the perikaryon increases considerably.These observations are interpreted as indicative that the early stages of synthesis take place at the level of the ribosomes. The product, in a dilute macromolecular form, is transferred into the cisternae of the endoplasmic reticulum and then condensed into granules within the Golgi complex. The increase in size of the granules along the axon is discussed in relation to the progressive increase in hormone content.Supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas and by the Air Force Office of Scientific Research No 963-65.Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas.     

Ultrastructural changes in neurohaemal tissue of the stick insect,Carausius morosus,induced by the ionophores Br-X-537 A and A-23187

Summary The antibiotic ionophores Br-X-537A and A-23187 alter the ultrastructure of neurohaemal tissue on the transverse nerve of the stick insect, Carausius morosus. Br-X-537A induces dramatic changes in the ultrastructural appearance of all three types of neurosecretory fibres present in the neurohaemal tissue. The neurosecretory granules become more electron-lucent and the mitochondria become more electron-opaque. The bounding membrane of the granules is frequently ruptured. A-23187, on the other hand, has no effect on two of the three types of fibres, but does produce an increase in the number of exocytotic profiles in the third.The two ionophores therefore have different effects on the same tissue. The results are discussed in the light of previous work with the use of these ionophores.We wish to thank Mrs. J. Birch for assistance with the electron micrographs, and Roche Products Ltd. and Lilly Research Centre Ltd. for gifts of the ionophores Br-X-537A and A-23187. The work was supported by the Science Research Council