Relationship of the central ACTH-immunoreactive opiocortin system to the median eminence and the pituitary gland of the rat

Summary By immunocytochemical methods, the present study describes ACTH-immunoreactive fibers in the pituitary stalk and neural lobe. This opiocortin-hypothalamo-neural lobe projection arises in a bed nucleus of perikarya in the basal hypothalamus, follows supraoptico-hypophyseal fibers in the zona interna of the median eminence, and distributes throughout the neural lobe. No ACTH-immunoreactive fibers project to the zona externa; some are present in the subependymal layer and at the lateral margins of the median eminence. Further studies must identify the role of these fibers in posterior lobe function. It remains also to be determined whether this system terminates upon primary pituitary portal capillaries and delivers opiocortin neuropeptides to the adenohypophysis.Supported by NIH Grants HD-07962, NS-15345 and AM-22029The skillful technical assistance of Donna Wilson, Nancy Dembs and Jay Hocton is thankfully acknowledged     

Monoamine-containing neurons in the optic ganglia of crustaceans and insects

Summary With the fluorescence method of Falck and Hillarp, the presence and localization of monoaminergic neurons in the optic ganglia of several crustaceans and insects have been investigated. It was found that in both classes the monoaminergic terminals, when present, appeared (especially in the medullae externa and interna of the crustaceans and the medulla of the insects) in strata specific for each species. So far, the only monoamine (visualized by this technique) present in the crustacean optic ganglia is dopamine, whereas in the Insecta, the catecholamines dopamine and noradrenaline, and the indolamine, 5-hydroxytryptamine, are found in the optic lobe. But in the Insecta, different species show different content of these amines.This work was supported by grants 2760-3 and 2760-4 from the Swedish Natural Science Research Council (R.E.), by a fellowship from Deutsche Forschungsgemeinschaft, and a grant from the Swedish Medical Research Council B72-14X-712-D7B (N.K.). We are very grateful to the director of the Department of Histology, Faculty of Medicine, Lund, Professor Bengt Falck, who put all his facilities and knowledge at our disposal.     

Localization of monoaminergic neurons in the central nervous system of Astacus astacus Linné (Crustacea)

Summary The cellular localization of biogenic monoamines in crustaceans was studied by means of a highly specific and sensitive fluorescence method devised by Falck and Hillarp. It was found that neurons displaying specific fluorescence in the central nervous system were confined to the protocerebrum, the medulla externa and interna and the ventral nerve cord. The method allows a distinction between the fluorophores of 5-hydroxytryptamine (and 5-hydroxytryptophan), which emit the yellow light, and the fluorophores deriving from the catecholamines (and DOPA), which emit the green light. Green-fluorescent neurons occurred abundantly in the aforementioned parts of the central nervous system while yellow-fluorescent neurons were sparsely present in the same parts.The present work has been carried out at the departments of Histology and Zoology at the University of Lund. The authors take great pleasure in expressing their warmest thanks for laboratory facilities, provided by Professors Erik Dahl (Zoological Institute) and Bengt Falck (Histological Institute).The research reported in this document has been sponsored by the Air Force Office of Scientific Research under Grant AF EOAR 66-14 through the European Office of Aerospace Research (OAR), United States Air Force and by a grant from the Swedish Natural Science Research Council 99-32 (nr 5995).     

A method for the demonstration of adrenergic nerve fibres in peripheral nerves

Summary The adrenergic nerve fibres running from the ganglia to the innervated tissues usually have too low a content of noradrenaline to be clearly visualized with the histochemical fluorescence method of Falck and Hillarp. They can easily be demonstrated, however, as early as 24 hours after axotomy (crushing or constriction of the nerves) due to the rapid accumulation of what is probably noradrenaline taking place proximally to the lesion. The fibres can be visualized even more clearly if axotomy is combined with the administration of l-dopa and with monoamine oxidase inhibition. In this way the presence, distribution and direction of adrenergic fibres can be directly studied in peripheral nerves.For generous supplies of drugs we are indepted to Swedish Ciba, Stockholm (reserpine) and Swedish Pfizer, Stockholm (nialamide). The investigation has been supported by research grants from the United States Public Health Service (NB 02854-04), the Swedish Medical Research Council, and Knut and Alice Wallenbergs Foundation.     

Fluorescence microscopy of the 5-HTP turnover in the exocrine pancreas of mice and rats

Summary The turnover ofl-5-HTP,d-5-HTP and 5-HT in the exocrine pancreas have been studied by means of the fluorescence method ofFalck andHillarp. l- andd-5-HTP are easily taken up by the acinar cells, whereas 5-HT seems to pass into the cells only to a minor extent. After the administration ofl-5-HTP (and in some cases after 5-HT administration), specific fluorescence is seen in the form of apically located granules (probably identical with the zymogen granules) for a short period, which is prolonged, if the animals are pretreated with a MAO inhibitor. Decarboxylase inhibition prevents the appearance of these fluorescent granules. Administration ofd-5-HTP does not give rise to this granular fluorescence but to a diffuse fluorescence throughout the cells. Thus, there are reasons to assume that the granular fluorescence derives from 5-HT. The results obtained in this work correspond well with those from a similar study withl-DOPA and some of its analogues.abbreviations DOPA 3,4-dihydroxyphenylalanine - DA dopamine - NA noradrenaline - A adrenaline - 5-HTP 5-hydroxytryptophan - 5-HT 5-hydroxytryptamine - MAO monoamine oxidase This work was supported by grants from the Swedish Medical Research Council (B68-12X-712-03B and B68-14X-56-04B), the United States Public Health Service (06701-02) and the Faculty of Medicine, University of Lund, Lund, Sweden.     

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.     

Monoamine-containing fibres in the pituitary neuro-intermediate lobe of the pig and rat

Summary A rich system of monoamine-containing fibres is described in the neural lobe and pars intermedia of the pig and rat. a) A rich network of delicate varicose fibres is evenly distributed throughout the parenchyma of the neural lobe and surrounds the cells of the pars intermedia. b) Droplets or clusters of droplets are scattered throughout the neural lobe. Most of them probably constitute terminal swellings or end-apparatuses of smooth or varicose fibres. The number of droplets varies from animal to animal; they are found also in the pars intermedia. c) Coarse varicose fibres are mainly localized around larger vessels. At least some of these fibres are nerve fibres of sympathetic origin. A combination of fluorescence microscopy and aldehyde-fuchsin staining on the same sections demonstrated that the majority at least of these monoamine-containing structures were not identical with aldehyde-fuchsin positive neurosecretory fibres.This research was supported by a grant from the Swedish Medical Research Council (B68-12X-712-03B) and by the Faculty of Medicine, University of Lund.     

Ultrastructure of the developing gizzard epithelium in the chick embryo

Summary The fine structure of the gizzard epithelium is described at different stages in the development of the chick embryo. The elaborate apical processes, a characteristic feature, take part in secretion at thirteen days, but do not seem to have this function at nine and ten days. The formation of glands begins at thirteen days but the adult fine structure of the gland cells is not attained until hatching. The distinct surface layer present between thirteen and seventeen days may have a protective function. Acknowledgement. The author is grateful for research facilities provided by Professor G. M. Wyburn, Anatomy Department, The University of Glasgow.     

Kapillaren und perikapilläre Räume im Hypothalamus-Hypophysen-System und ihre Beziehungen zum Nervengewebe

Zusammenfassung Elektronenmikroskopische Beobachtungen an den Kapillarsystemen der Neurohypophyse des Meerschweinchens bestätigen, daß diese Gefäße eine Sonderstellung unter den Hirnkapillaren einnehmen. Ein Porenendothel zeichnet die Kapillaren des Portalplexus der Zona externa, des Hinterlappens und des Vorderlappens aus. Weite perikapilläre Räume umgeben die Kapillaren der Neurohypophyse im Gegensatz zu den übrigen Hirnkapillaren, die von einer Gliascheide dicht umschlossen sind. Die Baueigentümlichkeiten der neurohypophysären Kapillarsysteme weisen auf einen gesteigerten Austausch zwischen dem Blut und dem benachbarten inkretorisch tätigen Gewebe hin.Eine Besonderheit stellt das Vorkommen von Axonen und Axonendigungen in den perivaskulären Räumen um die Kapillaren der neurohämalen Kontaktzonen im Hinterlappen und der Zona externa dar. Die Nervenelemente unterschiedlichen Kalibers enthalten dort neben großen und kleinen Elementargranula auch synaptische Vesikel. Drei Axontypen lassen sich feststellen.Die Möglichkeit regeneratorischer Wachstumsvorgänge an neurosekretorisch tätigen Nerven wird diskutiert. Andererseits kommt der in der Neurohypophyse spezifisch differenzierten Glia (Tanyzyten, Pituizyten) auf Grund ihrer elektronenoptischen Struktur und ihrer engen Beziehung zu Nerven und Gefäßen eine besondere Bedeutung zu. Die lichtmikroskopisch bei Tieren beobachtete Zona granulosa (Hagen [1955]) zwischen Zona externa und Pars tuberalis der Adenohypophyse läßt sich in diesem Sinne deuten.

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Summary EM observations of the capillary system of the neurohypophysis of the guinea pig confirm that these vessels are unique among the capillaries of the brain. The capillaries of the portal plexus, the median eminence, the neural lobe and the pars anterior all possess a fenestrated endothelium. In contrast to the other brain capillaries, provided with a thick glial sheath, those of the neurohypophysis are surrounded by wide perivascular spaces. The structural peculiarities of the neurohypophysial capillary system point to an enhanced interchangeactivity between blood and surrounding endocrinous tissue.Especially noteworthy is the presence of axons and axon-endings in the perivascular space round the capillaries of the neurohaemal contact areas in the neural lobe and in the median eminence. These neural elements are of varying calibre and they contain synaptic vesicles in addition to large and small elementary granules. Three types of axon are observable.The possibility of regenerative growth-processes taking place on neurosecretory-active nerves is considered. On the other hand, specially differentiated glial cells (tanycytes, pituicytes) in the hypophysis are of particular significance on account of their EM structure and their close relation to nerves and blood vessels. The lightmicroscopically observed zona granulosa in animals (Hagen [1955]), observed between the median eminence and the pars tuberalis of the adenohypophysis, could be explainable on this basis.

     

Observations on the cellular localization of dopamine in the caudate nucleus of the rat

Summary The cellular localization of dopamine in the caudate nucleus of the rat hat been studied with the highly sensitive and specific fluorescence method of Falck and Hillarp, and by electron microscopy. The histochemical studies provided strong support for the view that the dopamine is concentrated within very fine nerve fibres which have abundant varicosities with an intense fluorescence. The electron microscopical studies revealed the presence of a tightly packed plexus built up i.a. of abundant synaptic nerve terminals, many of which had a diameter below 0.4 . The terminals made synaptic contact mainly with processes that seemed to belong to an extensive dendrite net.The investigation was supported by research grants from the United States Public Health Service (02854-04), The Swedish Medical Research Council and the Knut and Alice Wallenberg Foundation.     

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.     

Adrenergic neurons in teleost retina

Summary The adrenergic retinal neurons of perch and trout were studied with the fluorescence microscopical method of Falck and Hillarp. Pilot studies were also performed on pike, plaice, cod, eel, goldfish, cunner, black moor, cichlid and carp. Only minor differences were noted between the species.Adrenergic varicose terminals occur in three sublayers of the inner plexiform layer. The layer adjacent to the ganglion cells is the most elaborate. Adrenergic perikarya occur in the innermost cell rows of the inner nuclear layer, sending branches to all sublayers of the inner plexiform layer. Adrenergic perikarya also occur among the ganglion cells, sending their branches to the innermost sublayer of adrenergic fibres in the inner plexiform layer. Weakly fluorescent adrenergic fibres can be seen running through the entire depth of the inner nuclear layer. They originate from the adrenergic perikarya of the inner nuclear layer, and they end in an elaborate plexus of adrenergic terminals among the horizontal cells. Either of the horizontal cell types can be in contact with adrenergic terminals, but the middle horizontal cells have the greatest density about them, being surrounded by baskets of adrenergic terminals of presumably synaptic character. It cannot be excluded that some horizontal cells contain a catecholamine.Microspectrofluometry revealed dopamine in the perch and trout retinal neurons.The research reported in this document has been sponsored by USPHS Grant No. 06092 and by a Research Professorship from Research to Prevent Blindness, Inc. to A.M.L. and by the Swedish Medical Research Council (B69-14X-712-04C and B68-14X-2321-01).     

Fluorescence microscopy of the catecholamine-containing neurons of the hypothalamohypophyseal system

Summary The hypothalamohypophyseal system of the mouse, rat, guinea-pig, cat, dog and monkey (Macaca mulatta) was studied with the fluorescence method for catecholamine-containing neurons developed by Falck et al. (1962). The fluorescent fibers are prominent in the external layer and around the primary portal plexus of the infundibulum and in the peripheral region of the neural lobe of these animals, particulary on the external surface and surrounding the primary capillary loops. These fluorescent fibers are connected with fluorescent cells in the arcuate nuclei, and this connection coincides with the tuberohypophyseal system. The neurons of this system have a particular affinity for dopamine, possibly due to their own content of dopamine. In the supraoptic and paraventricular nuclei, no fluorescent cells were found. In the pars intermedia, we also found catecholamine-containing fibers.The presence of catecholamine-containing fibers in the adeno- and neurohypophysis are considered in relation to other data derived from fluorescence and electron microscopy.     

A catecholaminergic neuron connecting the first two optic neuropiles (Lamina ganglionaris and Medulla externa) of the crayfish Pacifastacus leniusculus

Summary The crustacean optic neuropiles, the lamina ganglionaris and especially the medulla externa, show a specific pattern of green fluorescence with the fluorescence histochemical method of Falck-Hillarp. Normally, only the terminals and the cell bodies fluoresce, but in reserpine-treated animals exogenous catecholamines are taken up by the whole adrenergic neuron and are thus visualized as a whole. Incubating crayfish optic neuropiles in dopamine or -methylnoradrenaline after reserpine treatment demonstrated a tangential neuron connecting the lamina and the medulla externa. The morphology of this tangential neuron differs from the two types of tangential neurons, Tan₁ and Tan₂, previously characterized with Golgi techniques. The catecholaminergic neuron thus constitutes a third tangential neuron type. Acknowledgement. The present study has been supported by the Swedish Natural Science Research Council, grant B 2760-009, the Magnus Bergvall foundation, and the Swedish Medical Research Council, grant 04X-712, the latter to Prof. Bengt Falck to whom we extend our gratitude. We are also indebted to Mrs. Rita Wallén and Miss Maria Walles for their skilled technical assistance. Reserpine (Serpasil^®) was generously given to us by Hässle-Ciba-Geigy AB     

A Catecholamine storage in the pancreatic A2-cells of owl monkey (Aotes trivirgatus)

Summary The fluorescence method ofFalck andHillarp was used to study the occurrence of biogenic monoamines in the islets of Langerhans of monkeys. A storage of a catecholamine was demonstrated in the A₂-cells of owl monkey, whereas no histochemically demonstrable amount of monoamines could be seen in the islet cells of marmoset,Rhesus monkey, squirrel monkey, andCebus monkey.Supported by grants from the Swedish Medical Research Council (No. B69-14x-712-04C) and by the National Institutes of Health (No. 06701-02).     

Electron microscopic localization of monoamine oxidase in the rat liver

Summary Two methods have been employed to localize monoamine oxidase activity in the cells of rat liver, using either 2-(2′-benzothiazolyl)-5-stryl-3-(4′-phtalhydrazidyl) tetrazolium chloride (BSPT) or ferricyanide as electron acceptor. With both methods monoamine oxidase activity was found both in the inner and the outer mitochondral membrane, although the outer membrane appeared the most probable location. In addition the BSPT method but not the ferricyanide method, revealed monoamine oxidase activity in the endoplasmatic reticulum. The results obtained by the two methods have been compared and are discussed in view of available biochemical data on monoamine oxidase. Supported by research grants from the National Research Council of Canada (A 3651), The Swedish Medical Research Council (4145) and M. Bergwall's Foundation, Stockholm.     

DNA synthesis in the embryonic chick central nervous system

Summary DNA synthesis has been studied in chick embryos age between 2 and 10 days, using labelling with tritiated thymidine and stripping film autoradiography. The observations made earlier in the literature on a premitotic migration of the nuclei in the neural epithelium have been verified. In young stages (before day 7) peripherally migrated cells do not synthesize DNA, but after day 7 such a synthesis occurs. In spite of this, few mitoses are seen. The interpretation of these facts is discussed.The costs of this investigation were defrayed by grants from the Swedish Medical Research Council, the Medical Faculty of Lund, and the Royal Physiographic Society.     

Control of the pars intermedia of the lizard,Anolis carolinensis

Summary The ultrastructure of the neural lobe of the lizard, Anolis carolinensis, was studied after fixation in a threefold aldehyde solution. The neural lobe appeared as narrow vertical diverticula separated from one another and from the pars intermedia by a continuous vascular septum. No nerves passed through this septum. The ependymal, fibrous and external layers were readily recognized. Peptidergic fibres were the main component of the fibrous layer. The peptidergic endings were in intimate contact with the ependymal cells, suggesting that the ependyma mediates the release of neural lobe peptides. The external layer contained ependymal end-feet and numerous aminergic terminals, ending directly on the perivascular basal lamina and/or on the ependymal end-feet. The functional aspects are discussed in terms of intermediate lobe control. The findings suggest that aminergic substances take part in the control of the intermedia, but do not exclude the involvement of peptide hormones.Supported by grants from the Swedish Natural Science Research Council and the Royal Physiographic Society of LundThe authors are indebted to Mrs. Lena Sandell for valuable technical assistance and to Miss Inger Norling and the late Mr. Lajos Erdös for photographic aid     

Monoaminergic mechanisms in the nervous system of Lumbricus terrestris (L.)

Summary The localization and intraneuronal distribution of the monoaminergic transmitters in the nervous system of the earthworm, Lumbricus terrestris, have been investigated in detail with the aid of the histochemical fluorescence method of Falck and Hillarp.In the ventral nerve cord, many yellow fluorescent, 5-hydroxytryptamine containing neurons are found, but only few green fluorescent noradrenaline containing cell bodies, which, however, are numerous in the peripheral nervous system. There is an abundance of both fibre types in the neuropile.The 5-hydroxytryptaminergic neurons probably have a motor (possibly inhibitor) function; the adrenergic neurons in the body segments are supposed to have a receptor (exteroceptive and possibly proprioceptive) function.In the cerebral ganglion, both 5-hydroxytryptamine and noradrenaline containing neurons are found in large numbers, and there are closely packed numerous fibres of both types in the neuropile. Their function is more obscure, though an associative function can be presumed for some adrenergic neurons; smaller 5-hydroxytryptaminergic neurons might have a motor (perhaps inhibitor) function.Adrenergic sensory cells are found in the body integument, most frequently in the clitellum segments, in the prostomium, and in the roof of the buccal cavity. These cells give off varicose fibres that form a basi-epithelial network which is in communication with the green fluorescent sensory fascicles in the ventral nerve cord via the epidermal nerves, the ring nerves, and the segmental nerves. No direct adrenergic sensory-effector innervation of either circular and/or longitudinal musculature or gland cells seems to exist. No adrenergic free nerve endings in the body integument have been observed. Instead, there must be a synaptic contact with the motoneurons, either directly in the neuropile or via an interjacent neuron.No synaptic contacts have been observed in the ventral nerve cord between adrenergic or 5-hydroxytryptaminergic fibres and either the giant fibres or fluorescent or nonfluorescent perikarya.An adrenergic innervation of the pharynx musculature has been found, and sensory cells of a different type are present in and below the epithelium; here, a direct senso-motoric innervation of the pharyngeal musculature cannot be excluded. It is established that the adrenergic neurons in the stomatogastric nervous system have an exciting function on the pharynx, whereas a direct monoaminergic influence of the muscular movements of the intestine probably does not exist.Abbreviations Used A adrenaline - CA catecholamine - DA dopamine - 5-HT 5-hydroxytryptamine - MA monoamine - NA noradrenaline The research reported in this document has been sponsored by the Air Force Office of Scientific Research under Grant AF EOAR 67-15 through the European Office of Aerospace Research (OAR), United States Air Force, by the Swedish Natural Science Research Council (99-34, 6627), and by the Swedish Medical Research Council (B67-12X-712-02A).     

Enzymhistochemische Untersuchungen an Infundibulum und Hypophysenhinterlappen der normalen und beidseitig adrenalektomierten Ratte

Zusammenfassung Das Verhalten verschiedener Enzyme (NADH-Cytochrom-c-Reduktase, Laktatdehydrogenase, Glukose-6-Phosphatdehydrogenase, Monoaminoxydase, -Naphthylacetatesterasen, 5-Bromindoxylacetatesterasen, Acetylcholinesterase, Lipasen, alkalische Phosphatasen und saure Phosphatasen) in Infundibulum und Hypophysenhinterlappen wurde an normalen und an beidseitig adrenalektomierten Ratten untersucht. Die Aminopeptidasen-Aktivität der Zona externa infundibuli nimmt als Folge des Eingriffs zu, während die Aktivität der -Naphthylacetatesterasen im Bereich der gesamten Neurohypophyse abnimmt. Die weitere Differenzierung der das -Naphthylacetat spaltenden Fermente ergab, daß es sich um Aliesterasen mit lipaseartigem Charakter handelt. Die möglichen Zusammenhänge zwischen diesen Befunden und dem Verhalten der Gomori-positiven Substanz der Zona externa infundibuli nach beidseitiger Adrenalektomie werden diskutiert.

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Enzyme-histochemical investigation of the infundibulum and neural lobe in the normal and bilaterally adrenalectomized rat

Summary The behavior of various enzymes in the infundibulum and neural lobe of the hypophysis of the normal and bilaterally adrenalectomized rat was investigated. As a result of the operation the activity of the aminopeptidases in the zona externa infundibuli increases, whereas the activity of the -naphthylacetate-esterases in the whole neurohypophysis decreases. Further analysis of the -naphthylacetate-splitting enzymes showed that these enzymes belong to the group of ali-esterases and have a lipase-like character. The possible connection between these findings and the changes in the Gomori-positive substance in the zona externa following bilateral adrenalectomy is discussed.

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Herrn Prof. Dr. Dr.E. Horstmann zum 60. Geburtstag gewidmet.