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.     

Morphology of the neural lobe of the hypophysis in rats treated with furosemide. II. Pituicytes

Following three days of furosemide administration, the characteristics of the pituicytes in the neural lobe of the rat hypophysis were studied. The pituicytes of the animals which were treated with furosemide and had access to water during the treatment showed scarce cytoplasm organelles, conferring them with a clear appearance (light pituicytes), and a frequent finding was neurosecretory axons enclosed in their own cytoplasm. In the animals deprived of water during treatment, the pituicytes were dense (dark pituicytes) elongated, with a well-developed Golgi apparatus, numerous profiles of smooth endoplasmic reticulum and frequent annular gap junctions. It is concluded that the dehydration induced by the diuretic agent is the cause of the prevalence of the dark pituicytes.     

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.     

Glial fibrillary acidic protein and differentiation of neonatal rat pituicytes in vitro

Summary The appearance and intracellular localisation of glial fibrillary acidic protein (GFAP) in pituicytes in neural lobe cultures of newborn rats aged 7 to 30 days were investigated by use of the indirect immunofluorescence method. GFAP-immunoreactive cells were observed mostly in the outgrowth zone. GFAP was localised in the perikaryal cytoplasm as well as in pituicyte processes. GFAP-positive pituicytes showed considerable morphological polymorphism. The presence of GFAP — astrocytic marker — in pituicytes in vitro and the evident morphological similarity to cultured astrocytes suggest the astroglial character of these cells.     

Ultrastructural changes of the neurohypophysis of the rat after castration

Summary The infundibular processes of the neurohypophysis of male and female rats were studied after different periods of castration. After seven days an increase in neurosecretory granules was observed. Two types of neurosecretory nerve endings were identified: dark ones, with dense neurosecretory elementary granules of 1600 A, and clear ones, with lighter neurosecretory granules of 1800 A. Protoplasmatic pituicytes showed a large increase in lipid granules together with a general hypertrophy. After one week of castration but with hormonal therapy the protoplasmatic pituicytes appeared normal or even showed less lipid granules than in the controls.With one month of castration the changes already mentioned in the nerve endings and pituicytes were more pronounced and after six months even more accentuated. Two types of neurosecretory nerve endings were clearly identified and the protoplasmatic pituicytes were loaded with lipid granules.The probable significance of the two different neurosecretory axons was discussed in relation to recent studies on the isolation of neurosecretory terminals from the neurohypophysis. The changes in the protoplasmatic pituicytes were considered in relation to the possible significance of the lipid granules.Supported by grants from the Consejo Nacional de Investigaciones Cientificas 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.Associated Investigator, Consejo Nacional de Investigaliones Científicas y Técnicas, Argentina.     

Morphological characteristics of pituicytes in different functional stages

Summary Most of the pituicytes of normal rats show relatively few indications of metabolic activity. This situation remains unchanged after two days of dehydration. From the third to the eighth day of dehydration, a considerable hypertrophy of the majority of pituicytes takes place. During this period proliferative and degenerative processes occur. After eight days of dehydration the pituicytes show an increase of degenerative processes and indications of decreased activity. Only one type of pituicytes in three different morphofunctional stages is considered to represent the basic cellular component of the neural lobe. A relationship between these stages and the release of neurosecretory material from the nerve endings is discussed.Partially supported by Unesco and Ibro post-doctoral fellowships.     

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.     

Proliferation of glial cells induced by lithium in the neural lobe of the rat pituitary is enhanced by dehydration

Rats dehydrated by 6 days of water deprivation had a low level of mitotic activity in the astrocytes ('pituicytes') of the neural lobe of the pituitary. Mitotic activity in the pituicytes was greatly increased when isotonic lithium was administered in the last 3 days of water deprivation. Rehydration on the last day of the experiment produced a further increase in mitoses. Isotonic solutions of sodium, potassium or rubidium chloride did not increase mitoses. This model of cell proliferation is of interest because the mitotic activity is related to a physiological attempt to maintain homeostasis rather than a response to injury or the development of neoplasia.     

Canada goose posterior lobe pituicytes: seasonal ultrastructural changes in relation to axonal release of neurosecretion

Changes in ultrastructure of the posterior lobe pituicytes during six phases (Spring premigratory, Spring postmigratory, breeding, moulting, Fall premigratory and Fall postmigratory) of the annual life cycle of the migratory Canada goose, were studied. Pituicyte nuclear volume in females was significantly greater than that in males during the Spring premigratory and moulting phases. In the Fall postmigratory phase, nuclear volume in males was greater than that in females. During Spring premigratory phase, the axonal endings in males contained fewer neurosecretory granules (NSG) than those in the Fall postmigratory phase. In females, however, the number of NSG was relatively more than that in males in the Spring premigratory phase but fewer in the moulting phase. Nuclear volume is correlated with pituicyte function in releasing NSG containing arginine vasotocin (AVT) from axonal endings. The significance of AVT release is discussed in relation to initiation of reproductive and migratory behaviour, and to osmotic and metabolic regulation.     

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.     

The hypothalamic magnocellular neurosecretory system of the guinea pig. III. Ultrastructure of the fetal neural lobe.

The ultrastructure of the fetal guinea pig neural lobe was studied from day 40 to day 60 of gestation. Pituitaries were taken every five days and at least four glands from each gestational period were examined. Bundles of nerve fibers had invaded the periphery of the gland on day 40. By day 50 axon profiles were distributed throughout the entire posterior pituitary though pituicyte processes continued to act as a barrier between axons and the perivascular space of capillaries. Neural processes established contact with the capillaries between days 55 and 60. Neurosecretory granules (NSG) were present within a few axons on day 40. The number of axons with NSG and the total quantity of granules increased gradually throughout fetal development. Electron-lucent granules (microvesicles) were observed infrequently until the day of birth. A population of dense-cored vesicles, 70-80 mmu in diameter, was present from day 50 onward; a second population with larger diameters was also present throughout the developmental sequence and these increased from 90-130 mmu in diameter to 170-220 mmu in diameter between days 40 to 60. The presence of neurosecretory granules is discussed in relation to the onset of synthesis and storage of neurohypophysial hormones.     

Reserpine inhibits release of vasopressin from the neural lobe of the pituitary in dehydrated rats

Summary The effects of reserpine on the osmotically induced release of pituitary vasopressin were studied (i) by measuring the urinary excretion and the vasopressin content of the neural lobe of the pituitary, and (ii) by examining the ultrastructural morphology of axons in the neural lobe of dehydrated rats. After water deprivation for two days, control rats displayed characteristic antidiuretic response including a 75% reduction of urinary excretion and a sixfold decrease in vasopressin content of the neural lobe associated with a dramatic depletion of neurosecretory granules in corresponding axons. In contrast, when they received two daily injections of reserpine, animals dehydrated for two days showed both urinary excretion and vasopressin contents in the neural lobe that remained at levels comparable to those measured in the normally hydrated rats. Additionally, neural-lobe axons of such dehydrated, reserpine-treated rats displayed a normal amount of neurosecretory granules. These data indicate that reserpine inhibits release of vasopressin from the neural lobe and favour the concept of a facilitatory role of the catecholaminergic innervation in the control of hypothalamo-neurohypophysial vasopressin-secreting neurons.     

Postnatal development of glial fibrillary acidic protein (GFAP) immunoreactivity in pituicytes and tanycytes of the Mongolian gerbil (Meriones unguiculatus)

Summary The postnatal development (day of birth up to the end of the third month) of neurohypophyseal pituicytes and tanycytes of the median eminence (ME) and the medial basal hypothalamus (MBH) was studied immunohistochemically in the Mongolian gerbil (meriones unguiculatus) with antibodies directed against glial fibrillary acidic protein (GFAP; the major protein subunit of glial filaments). Weak GFAP-immunoreactivity (IR) was scattered in the neural lobe (NL), the ME and the lining of the ventral 3rd ventricle at the first postnatal days. By the end of the second postnatal week, the intensity of the IR had reached a level comparable to that of adult animals. Generally, in the whole neurohypophysis a cytoarchitectonic pattern, which essentially corresponded to adult conditions, was reached around the beginning of the second month. During the first week postnatum, solely perinuclear stainings, mostly unipolar pituicytes with short processes and isolated fibers were discernible in the NL. In the course of the second and third postnatal week, a growing number of the densely arranged pituicytes appeared in form of bi- and multipolar cells. Thickness and length of pituicyte processes, as well as their degree of branching, increased progressively in the first month. The number of GFAP-positive tanycytes in the ventral 3rd ventricle and in the ME most markedly augmented in the first week postnatum. In the MBH, long tanycyte processes emerged from the ventricular lining to cross the arcuate nucleus in large bows, delimiting groups of neurons. Ependymal and subependymal tanycytes in the ME gave rise to radial processes extending to the external zone. Moreover, in this zone several tanycyte-like cells, whose number increased considerably in the second postnatal week, contributed to the palisadal arrangement of glial processes. In the third month, the arcuate nucleus was still penetrated by many immunopositive tanycyte processes. It was characteristic of the adult staining pattern that the highly branched processes of tanycytes and tanycyte-like cells in the ME were thicker and more variable in size than the less densely arranged tanycyte processes of the MBH. The postnatal increase of GFAP-IR intensity was paralleled by a similar development of vasopressin-IR in the ME and NL, indicating that the maturation of neurohypophyseal glia is closely linked to the functional differentiation of the neurosecretory axons.     

Mitotic Figures in the Median Eminence of the Hypothalamus

The median eminence of the hypothalamus is part of the avenue by which neurosecreted hormones from the hypothalamic nuclei reach the pars nervosa (neural lobe) of the pituitary and eventually the bloodstream. Lithium treatment and osmotic stress increases the transport of neurosecretory hormones to the pituitary in the adult rat. Specialized astrocytes termed pituicytes in the pars nervosa of the pituitary participate in the secretory process and also develop considerable mitotic activity. The present work reveals similar mitotic figures in cells within the median eminence following 3 days of lithium treatment. The location and appearance of these mitoses add to the evidence that pituicytes are present in the median eminence. Moreover, mitoses occur within the ependymal (tanycyte) layer of the median eminence. Thus, the present results suggest that the tanycyte layer may contain pituicytes, indicating that the hypothalamus possesses specialized cells for modulating neurosecretion in response to osmotic challenges.     

Involvement of accessory neurosecretory nuclei of hypothalamus in the formation of hypothalamohypophyseal system during prenatal and postnatal development in rats

We studied the development of direct axonal connections of the accessory neurosecretory hypothalamic nuclei with the posterior pituitary lobe on the fixed rat brain from day 15 of embryogenesis until day 10 of postnatal development using the retrograde diffusion method of the lipophilic fluorescent carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate along the neuronal membranes. The marker was applied onto the posterior pituitary lobe and, after incubation in a fixative, fluorescing bodies of nerve cells were visualized in the hypothalamus. Neuronal axons of the retrochiasmatic nucleus were the first of the accessory nuclei to ingrow in the posterior pituitary lobe (on days 16-17 of embryogenesis). Neurons of the circular and dorsolateral nuclei and the nuclei of the median bundle of the forebrain sent their axons to the posterior pituitary lobe starting from the first days of postnatal development. No direct connections of the anterior commissure and perifornical accessory nuclei with the posterior pituitary lobe were found in perinatal development. These facts are discussed in the light of concepts about the different functional role of accessory peptidergic hypothalamic nuclei in rats.     

Organization and ultrastructural identification of the catecholamine nerve terminals in the neural lobe and pars intermedia of the rat pituitary

Summary The central catecholamine innervation of the pituitary neural lobe and pars intermedia of the rat have been identified ultrastructurally and their organization has been investigated in a combined fluorescence histochemical and electron microscopical study. The dopamine analogues, 5-hydroxydopamine and 6-hydroxydopamine, were used to label the catecholamine terminals, and to enable the direct correlation between the fluorescence microscopical and the electron microscopical pictures.The fibre type that was identified as catecholamine-containing was ultrastructurally chiefly characterized by dense-cored vesicles, 500–1200 Å in diameter, intermingled with varying numbers of small empty vesicles. 5-hydroxydopamine was selectively accumulated in these fibres and caused an increased electron density of the granular vesicles as well as of some small normally agranular vesicles, and systemically administered 6-hydroxydopamine caused a selective degeneration of these fibres, most prominently within the neural lobe. The dopaminergic terminals of the neural lobe showed frequent close contacts (80–120 Å), without real membrane thickenings, to neurosecretory axons and to pituicyte processes. It is suggested that these close contacts might signify a direct dopaminergic influence on the neurosecretory axons and/or on the pituicyte processes. The identified central catecholamine fibres were also found to make common synapse-like contacts on the pars intermedia cells, whereas the innervation by neurosecretory fibres was very rare. This suggests that the direct central nervous control of the rat pars intermedia is exerted by the catecholamine neurons. A very special feature of the catecholamine fibres in the pituitary is the occurrence of peculiar, large dopamine-filled droplet-like swellings. Electron microscopically, such large axonal swellings (more than 2 in diameter) were found to contain, in addition to the characteristic vesicles and organelles, strongly osmiophilic lamellated membrane complexes resembling myelin bodies and multivesicular bodies encircling disintegrated vesicles, suggesting that these droplet fibres represent dilated stumps of spontaneously degenerating dopaminergic axons. It is suggested that the dopaminergic neural lobe fibres are undergoing continuous reorganization through degeneration—regeneration cycles, a phenomenon previously suggested for the neurosecretory axons of the neural lobe.Supported by the Deutsche Forschungsgemeinschaft.Supported by Svenska Livförsäkringsbolags Nämnd för Medicinsk Forskning, by The Medical Faculty, University of Lund and by the Ford Foundation.     

Cellular and Subcellular Aquaporin-4 Distribution in the Mouse Neurohypophysis and the Effects of Osmotic Stimulation

Water channel aquaporin-4 (AQP4) is the most abundant water channel in the rodent brain and is mainly expressed in cerebral areas involved in central osmoreception and osmoregulation. The neurohypophysis is the release site of hypothalamic neurohormones vasopressin and oxytocin, which are involved in the regulation of the water balance. The authors investigated the cellular and subcellular distribution of AQP4 in the mouse neurohypophysis before and after chronic osmotic stimulation, using immunofluorescence microscopy and immunoperoxidase electron microscopy. They showed that AQP4 was abundant in the mouse hypophysis, mainly in the neural lobe. AQP4 was discontinuously distributed along pituicytes plasma membranes, in the dense neurosecretory granules and microvesicles of nerve endings and fibers, and along the luminal and abluminal membranes of fenestrated capillary endothelial cells. After chronic osmotic stimulation, AQP4 immunolabeling was enhanced. Taken together, these results suggest that AQP4 could be involved in the pituicyte sensor effect during osmoregulation, the modification and/or maturation mechanism of neurosecretory granules during neurohormone release, and the blood perfusion of the hypophysis.     

Ultrastructural features of rat pituicytes in organotypic culture

Summary Neural lobes of adult rats have been explanted and organ-cultured for 5–10 days. While perivascular cells and neurosecretory fibers undergo a progressive degeneration, scattered pituicyte-like cells are observed mostly associated with each other in number of two or three cells for group. Cultured pituicytes are quite similar to in vivo pituicytes except for some particular features as the shape of the nucleus and the number of dense bodies. Furthermore they share with the in vivo pituicytes the phagocytic capacity which appears even increased as far as neurosecretory fibers are concerned. Finally, degenerating pituicytes and free lipid droplets are observed in the intercellular spaces: the significance of these facts is discussed and tentatively interpreted. The organotypic culture of the neural lobe seems to represent an experimental model useful to obtain a relatively pure population of pituicytes.With the technical assistance of Vincenzo Panetta.     

Involvement of Accessory Neurosecretory Nuclei of Hypothalamus in the Formation of Hypothalamohypohysial System during Prenatal and Postnatal Development in Rats

We studied the development of direct axonal connections of the accessory neurosecretory hypothalamic nuclei with the posterior pituitary lobe on the fixed rat brain from day 15 of embryogenesis until day 10 of postnatal development using the retrograde diffusion method of the lipophilic fluorescent carbocyanine dye 1,1"-dioctadecyl-3,3,3",3"-tetramethylindocarbocyanine perchlorate along the neuronal membranes. The marker was applied onto the posterior pituitary lobe and, after incubation in a fixative, fluorescing bodies of nerve cells were visualized in the hypothalamus. Neuronal axons of the retrochiasmatic nucleus were the first of the accessory nuclei to ingrow in the posterior pituitary lobe (on days 16–17 of embryogenesis). Neurons of the circular and dorsolateral nuclei and the nuclei of the median bundle of the forebrain sent their axons to the posterior pituitary lobe starting from the first days of postnatal development. No direct connections of the anterior commissure and perifornical accessory nuclei with the posterior pituitary lobe were found in perinatal development. These facts are discussed in the light of concepts about the different functional role of accessory peptidergic hypothalamic nuclei in rats.     

Golgi-like immunostaining of pituicytes and tanycytes positive for glial fibrillary acidic protein in the neurohypophysis of the Mongolian gerbil (Meriones unguiculatus)

Summary Glial cells that contain the glial fibrillary acidic protein (GFAP; the major protein constituent of glial filaments) were stained immunohistochemically in thick frozen sections of the neurohypophysis of the Mongolian gerbil (Meriones unguiculatus). The resulting Golgi-like images provided informations on cytological features and distributional patterns of tanycytes and pituicytes. In the proximal median eminence, numerous bundled processes of tanycytes were revealed. They emerged from the ependymal and subependymal layer and mostly reached the brain surface. Several tanycytic processes extended into the anatomical neural stalk. In the whole neural lobe, a dense network of GFAP-immunoreactive pituicyte processes was visualized. Stained pituicytes were highly asymmetric and exhibited a great morphological variability. Immunopositive fibers which were encountered in the intermediate lobe might be derived from pituicytes. Electron-microscopically further evidence was obtained that GFAP-positive pituicytes correspond to filament-rich fibrous pituicytes at the ultrastructural level.