Ultrastructural aspects of the inhibition of neurosecretion by puromycin

Summary The supraoptic nuclei and the infundibular process of the neurohypophysis were studied under the electron microscope in rats fed on dried food for 1 to 3 days and in similarly dehydrated animals but injected subarachnoidally with puromycin 4 to 24 hours prior to fixation. With 4 to 8 hours of puromycin the intracisternal filamentous material found in the control is greatly diminished. After 24 hours this inhibitory effect disappears in the perikaryon but the neurotubular material of the neuronal processes within the supraoptic nucleus and the neurosecretion in the infundibular process are greatly reduced. These results are discussed taking into consideration the inhibition of protein synthesis caused by puromycin and the neurosecretory cycle which characterizes the supraoptic neurons.Supported by grants from the Consejo Nacional de Investigaciones Cientificas y Técnicas and by the Air Force Office of Scientific Research (963–65).     

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.     

The caudal neurosecretory system of the teleostean Clupea melanostoma

Summary The caudal neurosecretory system of Clupea melanostoma is described. The urophyseal area in this species is merely a spinal cord enlargement divided into two distinct zones: a ventral and ventrolateral vascular zone where neurosecretory material is concentrated, and a dorsal cell-rich area where the perikarya of the neurosecretory cells are found.The hypothesis is advanced that the first-named vascular area has developed into the more differentiated urophysis of the less primitive teleosts while the dorsal cell-rich area has become part of the filum terminale. Two main types of neurosecretory cells are described.This work was supported by grant L 96 Z from the Consejo Nacional de Investigaciones Cientificas y Técnicas.     

The reticular substance of the medulla oblongata of the albino rat

Summary The region of the reticular giganto-cellular nucleus, perfused with formalin and postfixed in osmium tetroxide, was studied with histochemical and electron microscopic techniques. The perikarya of the neurons have two zones. The peripheral cytoplasm contains Nissl bodies, mitochondria, and free RNP particles. The juxtanuclear cytoplasm contains the Golgi complex, mitochondria, RNP particles and dense bodies. The nucleus is indented and has a prominent nucleolus and a paranucleolar body. Dense bodies are found along the axon and dendrites as well. Three different types of synapses are described and two types of synaptic vesicles (spherical and ellipsoidal) are shown.The capillary endothelium shows microvilli and marginal flaps. The endothelial cytoplasm contains vacuoles, micropinocytotic vesicles, and a few dense bodies. Processes of pericapillary cells, surrounded by a basement membrane, also contain dense bodies. The dense bodies found in the neurons and endothelial cells show acid phosphatase activity. On the basis of their morphology and their enzymatic activity these bodies are identified as lysosomes.Partially supported by a school grant No RF 62051 from the Rockefeller Foundation, New York, USA, and Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.Fellows of the Consejo National de Investigaciones Científicas y Técnicas, Argentina. The authors wish to thank Dr. Mario H. Burgos for his constant interest and criticism, and to Dr. Eduardo Rodriguez Echandia and Dr. Fabio L. Sacerdote for their valuable assistance.     

Differences between median eminence and neural lobe in amphibians

Summary The neurohypophysis of the toad Bufo arenarum Hensel can be subdivided into two well defined zones: the median eminence and the neural lobe. In its turn, the median eminence consists of two zones: a neural subependymal one, containing the axons of the hypothalamic-neurohypophysial tract; and a glandular one, made up of the capillaries of the primary plexus of the portal hypophysial system and of neurosecretory axons.Different staining techniques and certain experimental procedures (inanition, dehydration and adenohypophysectomy) showed that there are differences between the neurosecretory material of the neural lobe, which is a place of hormone liberation into the systematic circulation, and the neurosecretory material of the median eminence, which is the site of release of adenohypophysis-stimulating substances into the portal vessels.This work was introduced at the III Sesiones Científicas de Biología, Rosario, Argentina, March, 1965. The study was partially supported by the Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina) and The Rockefeller Foundation (School grant RF-58028).Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina. The author wishes to express his indebtedness to Dr. M. H. Burgos and Dr. F. Sacerdote for their help and criticism, to Miss B. Rodriguez. Miss M. Masot and Mr. L. Castro for their technical assistance.     

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.     

Treatment of dairy industry wastewater with an anaerobic filter

Summary Concentrations of up to 10.2 g COD/L were applied to an horizontal anaerobic filter at 40°C, obtaining efficiencies in COD removal of 85%. The contents of the reactor are kept mixed by recycling and at a pH value of 6.9.The addition of alkali to the influent increases the production of biogas reaching a maximum of 250 L of methane per kg of COD removed.Career Investigator of the Consejo Nacional de Investigaciones Cientificas y Técnicas (R. Argentina).Fellow of the Consejo Nacional de Investigaciones Cientificas y Técnicas (R. Argentina).     

Use of a packed-bed reactor for anaerobic treatment of stillage of sugar cane molasses

Summary The use of a packed-bed reactor for the treatment of stillage from sugar cane molasses is studied in a laboratory scale unit of 250 ml at 40°C. When stability is reached organic loads from 4 to 30 g COD/l/day are applied with COD reduction ranging from 85% to 55% depending on the load and the source of stillage. The production of biogas achieved is of 379 1 (72% CH₄) per Kg COD removed.Career investigator of the Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina).Fellow of the Consejo Nacional de Investigaciones Cientifícas y Técnicas (Argentina).     

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.     

The effects of adenohypophysectomy on the hypothalamic-hypophysial neurosecretory system and the adrenal gland of the toad Bufo arenarum Hensel

Summary The effects of adenohypophysectomy were studied on the hypothalamichypophysial neurosecretory system and the adrenal glands of Bufo arenarum Hensel. An increase in vascularization of the pars intermedia was found and the neurosecretory material (NSM) in the glandular region of the median eminence disappeared. Its reappearance later was accompanied by hypertrophy of the pars intermedia; differentiated chromophil cells appeared in the pars intermedia around the vessels and, at the same time, the adrenal reverted to normal. These findings are interpreted as hypertrophy and differentiation of the pars intermedia with replacement, at least partly, of the functions of the pars distalis. The probable mechanism of differentiation and the functions of chromophil cells of the pars intermedia are discussed.This paper was presented at the VI Reunión de la Asociación Latinoamericana de Ciencias Fisiológicas (A.L.A.C.F.), Viña del Mar, Chile, 1964. It was carried out under the auspices of the Consejo Nacional de Investigaciones Científicas y Técnicas (Argentina) and the Rockefeller Foundation (School grant RF-58028).Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.The authors wish to thank Prof. M. H. Burgos for his constant interest, Prof. B. A. Houssay, Prof. H. Heller and Dr. J. H. Tramezzani for their criticisms, Miss B. Rodriguez and Mr. L. Castro for their technical assistance.     

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).     

Seasonal variation of carrageenans in tetrasporic,cystocarpic and ‘sterile’ stages of Gigartina skottsbergii S. et G. (Rhodophyta,Gigartinales)

Cystocarpic and sterile plants of Gigartina skottsbergii produce -carrageenans, while tetrasporophytes produce -carrageenans. No seasonal variation in the carrageenan yields in the three stages was evident. Yields were clearly higher in cystocarpic (34.1–64.7%) and sterile samples (32.3–71.8%) than in tetrasporic plants (10.1–34.0%). Chemical characteristics of the polysaccharides for any stage are the same throughout the year.The authors are Research Members of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.The authors are Research Members of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Argentina.     

Histology and ultrastructure of the neurohypophysis of the south american lungfish,Lepidosiren paradoxa

Summary The neurohypophysis of the South American lungfish Lepidosiren paradoxa has been studied with light and electron microscopy, including the Falck-Hillarp technique for catecholamines. The pars nervosa hypophyseos is a well-marked, dorsally located subdivision of the pituitary gland composed of lobes or follicles, each one constituted of a central core of ependymal cells, a subependymal hilar region made up of nerve fibers and a peripheric palisade zone of nerve endings which contact capillary vessels. Four types of neurosecretory axons can be distinguished under the electron microscope. Type I, the most common, contains spherical elementary granules of high electron density, 1500–1800 Å in diameter. The scarce type II axons contain irregularly-shaped elementary granules. Type III contains only small clear vesicles, 400–600 Å in diameter. Type IV, mostly present in regions of the gland contacting the pars intermedia, contain large granulated vesicles, 900–1000 Å in diameter. The Falck-Hillarp technique revealed axons with a positive reaction for catecholamines at sites corresponding approximately to the location of type IV of the electron microscope.Ependymal cells are of large size, linking the cerebrospinal fluid, the nerve endings and the blood vessels. A conspicuous membrane-bound, spherical dense material, 1400–2000 Å in diameter, is observed in both the apical and vascular processes of these cells. The ependymal processes which traverse the hilar and palisade regions contain structures resembling degenerated neurosecretory axons. These results are discussed in relation with the currently available information on the comparative anatomy of the pars nervosa. The possible functional significance of ependymal cells and of each type of axon are also discussed.This study was aided by the following grants: NIH NS 06953 to Prof. De Robertis, Consejo Nacional de Investigaciones Científicas y Técnicas to Prof. Zambrano, Comisión de Investigaciones Científicas de la Provincia de Buenos Aires and Comisión de Investigaciones Cientificas de la Universidad Nacional de la Plata: to Prof. Iturriza.The authors are indebted to Prof. De Robertis for his generosity in granting us his laboratory facilities, and to Dr. F. J. J. Risso and Mr. A. Fernández (Resistencia, Chaco) who provided the specimens used in this study. The able microtechnical assistance of Miss L. Riboldazzi and Mrs. R. Raña and the photographic work of Mr. A. Saenz are much appreciated.Members of the Scientific Career, Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.     

On the presence of conspicuous electron dense bodies in the pinealocytes of the pig

Summary The pinealocytes of the pig contain a large number of aldehydefuchsin positive granules. In order to determine their nature an ultrastructural study was carried out. Numerous bodies having a maximal diameter of about 1,600 nm were found. These elements showed a great variety of internal structure, ranging from a homogeneous content and amorphous dense aggregates to lamellate bodies. Although only a few of them displayed a positive reaction for acid phosphatase, their morphological appearance strongly suggests that they belong to the lysosomal system.Aided in part by a grant from the Consejo Nacional de Investigaciones Cientificas y Técnicas de la República ArgentinaAssociate Professor of Embryology and Histology and Member of the Investigator Career of the CONICETProfessor and Chairman of Histology and Member of the Investigator Career of the CONICETCenter sponsored by the Universidad Nacional de La Plata and the Comisión de Investigaciones Cientificas de la Provincia de Buenos Aires     

ZIO staining in synaptic vesicles of the rat pineal nerves after inhibition of serotonin and noradrenaline synthesizing enzymes

Summary Two compartments have been defined in monoaminergic synaptic vesicles: the core or central compartment, storage site for monoamines, and the matrix or outer compartment, of unknown function. The outer compartment reacts with the mixture of zinc iodide-osmium tetroxide (ZIO). This reaction is temperature and time dependent and may be abolished by -SH reagents.The effect of drugs inhibiting the synthesis of serotonin and noradrenaline (stored in the core) on the ZIO reaction in the matrix was studied in synaptic vesicles of rat pineal nerves. The inhibitors of monoamine synthesis abolish or decrease the ZIO reaction directly or in combination with the administration of tyramine. This effect is temperature dependent suggesting that the drugs act on different components of the matrix that react with ZIO at different temperatures. A comparison of the present results with those obtained with -SH reagents seems to indicate that the drugs assayed act, at least in part, by changing the accessibility of-SH groups in vesicle proteins. (An abstract of this paper was presented at the 7th International Congress of Pharmacology, Paris, 1978.)Supported by grants from the Consejo Nacional de Investigaciones Cientificas y Técnicas, CONICET, and the Secretaría de Ciencia y Técnica, SECYT, ArgentinaWe are grateful to Margarita López de Cáceres for technical assistance, to Adriana Contreras for the electron micrographs and to María Aued de Rau for the illustrations. Rita Cardoni is a fellow of the Consejo Nacional de Investigaciones Cientificas y Técnicas, Argentina     

Light and electron microscopy of granules in the toad choroid plexus

Summary Two types of granules can be distinguished in the toad choroid plexus under the light microscope: pigment granules, mainly localized in the cells that line the free ends of the choroidal villi, and Gomori-positive granules, present in most epithelial cells.The ultrastructural analysis of the choroid plexus reveals three types of granules: multivesicular bodies (MVB), multigranulous bodies (MGB) and dense bodies (DB), and intermediate stages between the last two bodies. The pigment granules seen under the light microscope probably correspond to the DB of the electron micrographs, and the Gomori-positive granules to the MGB. The probable role of these bodies is discussed and so is the significance of the glycogen present in the choroidal cells, their processes and endothelium.This study was partially supported by the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina and the Rockefeller Foundation (School grant RF — 58028).Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas de la República Argentina. The author wishes to thank Prof. M. H. Burgos for his constant interest. His thanks are also due to Prof. H. Heller for providing certain facilities in his department and for his criticism.     

Appearance of granules in the Purkinje cells of dehydrated toads (Bufo arenarum Hensel)

Summary Seven days of dehydration produce alterations in the Purkinje cells of the toad, Bufo arenarum Hensel. Aldehyde-fuchsin-positive granules appear in the cytoplasm of the cells and at the same time modifications take place in cytoplasmatic and nuclear ribonucleoproteins. These modifications consist of an increase in nucleolar volume and appearance of a nuclear cap, polarized towards the granules.This study was aided by a grant of the Consejo Nacional de Investigaciones Cientificas y Técnicas of Argentina.     

Effect of exogenous application of gibberellic acid on color change and phenylalanine ammonia-lyase, chlorophyllase, and peroxidase activities during ripening of strawberry fruit (Fragaria x ananassa Duch.)

The effect of exogenously applied gibberellic acid (GA₃) on the postharvest color change of strawberry fruit was evaluated through their external color and surface color parameters. A significant delay on color evolution was observed in fruits treated with GA₃. The evolution of activities of phenylalanine ammonia-lyase (PAL), chlorophyllase, and peroxidase was also analyzed. PAL activity increased during strawberry ripening, but in fruits treated with GA₃ the increase in such activity was slower, and, probably as consequence, the development of red color was delayed. Moreover, the activity of chlorophyllase and peroxidase, enzymes possibly involved in chlorophyll metabolism, decreased during strawberry ripening. However, a delay was observed in the decrease of such activities in GA₃-treated fruits.Abbreviations PAL phenylalanine ammonia-lyase - GA₃ gibberellic acid₃ - PVPP polyvinylpolyprrolidone - CEAU chlorophyllase enzymic activity unit - PEAU peroxidase enzymic activity unit - LSD least significant difference. Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) de la República Argentina. Author for correspondence.Members of the Research Career of the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) de la República Argentina.     

Cytochemistry of 5-hydroxytryptamine at the electron microscope level

Summary The nerves of rat pineal gland are known to contain norepinephrine and 5-hydroxytryptamine. With the glutaraldehyde-dichromate reaction for the cytochemical localization of unsubstituted catechol- and indoleamines, dense reactive granules could be demonstrated in such endings. A similar reaction was observed in the adrenergic nerves supplying the vas deferens and storing exclusively norepinephrine. Formaldehyde fixation, prior to the glutaraldehyde-dichromate treatment, interferes with the reaction given by catecholamines not affecting the indolic reactive sites. After this combined procedure pineal nerves still exhibited the dense reactive granules, while these were not found in the nerves of the vas deferens. Following bilateral cervical sympathectomy reactive granules disappeared from the perivascular processes of the pineal gland. No reaction could be observed in the cytoplasm of parenchymal cells neither in their perivascular processes.These cytochemical results suggest that both catecholamines and 5-hydroxytryptamine are contained within the granulated vesicles of pineal nerves.This work has been supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina and U.S.Air Force (AF-AFOSR 963-66).Fellow of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina. We want to express our gratitude to Prof. E. De Robertis for his constant help and encouragement; and to Miss Nélida Fernández Oranges, Mr. Raúl Castelli and Mr. Alberto Sáenz for their skillful technical assistance.     

Diabetes-induced alteration in subcellular distribution of poly(A)-rich RNA from skeletal muscle

Summary Skeletal muscle ribosome preparations from diabetic rats have lower polysome content than those from normal animals. The ratio of poly(A) containing RNA between polysomes and postribosomal supernatant has been measured both in normal and diabetic rats. The results suggest that in diabetic animals there is a greater proportion of free poly(A) containing RNA, in postribosomal supernatant.This work was supported by Grants from the Consejo Nacional de Investigaciones Cientificas y Técnicas (República Argentina), Universidad Nacional de Buenos Aires, and partly under contract of the Ministére de la Politique Scientifique within the framework of the Association Euratom—University of Brussels—University of Pisa.