Submicroscopic morphology of granular vesicles in sympathetic nerves of rat pineal body

Summary In the rat pineal body sympathetic nerve endings are characterized by numerous agranular and granular vesicles. The latter mainly include vesicles of about 450 Å diameter, each of which is bounded by a trilaminar membrane of about 75 Å total thickness. The cores of these granular vesicles appear structurally similar to the bounding membranes and are formed by a less dense, globular or vacuolar component around which a more dense component is distributed. The relative distributions of these two structural elements result in a variety of submicroscopic configurations which suggest a morphological continuum that may be related to the cyclical uptake, storage and release of biogenic amines. A tripartite classification of granular vesicles, such as proposed previously, is not supported by these data.This investigation was supported by United States Public Health Service Research Grants NB 05175-01 and AM-0699802. — The capable technical assistance of Mrs. Caroline Wilner is gratefully acknowledged.     

The epiphysis (pineal body) and the APUD system

Light and electron microscopic studies were conducted on 10 humans who died of the different cardiac diseases; and 20 guinea pigs pineal glands. Pinealocytes or secretory cells of the pineal gland have morphological likeness with the APUD system cells. They have a well-developed endoplasmic reticulum, Golgi complex, mitochondrial component and in cytoplasm dense-core vesicles are discovered. However the pinealocytes have a neuron-like structure and they are not separate cells as apudocytes, but they are a principal component of the pineal parenchyma in which pinealocytes are in tight interactions with glia, blood vessels and nerve terminations. Analysis of morphological and functional similarity and difference between pinealocytes and apudocytes allows to consider pineal gland as an APUD organ. A circadian rhythmicity of some secretory vesicles in pinealocytes of the guinea pig has been established.     

ISOLATION OF NERVE ENDINGS FROM THE POSTERIOR PITUITARY GLAND : Electron Microscopy of Fractions Obtained by Centrifugation

Bovine posterior pituitary glands were homogenized in 10 per cent sucrose and fractionated by differential centrifugation. The following centrifugation procedure resulted in the most satisfactory separation: 1000 g for 15 minutes—nuclei, connective tissue, basement membranes with associated endothelium, giant nerve endings, and whole pituicytes; 4200 g for 15 minutes—free nerve endings, including Herring bodies; 17,000 g for 15 minutes—mitochondria; 68,000 g for 15 minutes—neurosecretory granules. Electron microscopic examination was carried out on whole tissue and on the isolated fractions. Isolated nerve endings were examined also by negative staining techniques. Isolated nerve endings retain an apparently normal complement of mitochondria, neurosecretory granules, and microvesicles ("synaptic" vesicles). The free nerve endings closely resemble those observed in sections of intact posterior pituitary tissue. Free microvesicles were not observed in any of the fractions isolated and apparently sediment at centrifugal forces higher than those employed in this study.     

Sensory and secretory potencies and differentiations of the central nervous system

Secretory neurons capable of elaborating neuropeptides and biogenic amines are an integral component of nervous systems. This apparatus is more extended than assumed during an earlier period of investigations. It is involved in short- and long-range communication by means of paracrine, transmitter-like, modulatory and neurohormonal types of messages. This finely adjusted activity of secretory neurons serves the control of a variety of important biological functions. Secretory pinealocytes are derivatives of pineal photoreceptors, primary sensory cells of neuronal character. In contrast to these neuron-like or paraneuronal elements, the secretory cells of the subcommissural organ are of ependymal origin.     

THE DISTRIBUTION OF LABELED NOREPINEPHRINE WITHIN SYMPATHETIC NERVE TERMINALS STUDIED WITH ELECTRON MICROSCOPE RADIOAUTOGRAPHY

The distribution of radioactivity in association with sympathetic nerve terminals and intraneuronal organelles 30 min after the administration of tritiated norepinephrine (NE-³H) was studied by electron microscope radioautography with recently developed quantitative methods of analysis reported in the accompanying paper (Salpeter et al., 1969). Nerves from the pineal body and the adrenal capsule were examined. It was found that nerve terminals containing vesicles were heavily labeled. (These terminals were not necessarily in contact with some innervated structure.) There was no selective labeling of either the intraneuronal mitochondria or the relatively small population of large (~1000 A) dense core granules. Small vesicles (~500 A), some of which have a dense internal granule, could not be analysed separately because they are closely packed and occupy ~60% of the volume in terminals. Because of the extensive distribution of these small granular and agranular vesicles in the radioactive terminals, they remain the most likely site for norepinephrine binding. Yet although the vesicles were uniformly distributed within the nerve terminals, it appears that the radioactivity was not. There appeared to be a somewhat higher concentration of radioactivity at the periphery of the nerve terminals than in the center. The usefulness of the method of analysis used in this study for determining the location of bound H³NE pools in the nerve is discussed.     

Sympathetic innervation of the carotid bifurcation in the rabbit and cat: Blood vessels,carotid body and carotid sinus

Summary Two postganglionic branches of the superior cervical ganglion enter the area of the carotid bifurcation in the rabbit and the cat. The common and external carotid arteries receive a rich adrenergic nerve supply, which can be demonstrated by fluorophores of biogenic amines appearing after formaldehyde treatment. The internal carotid artery is only sparsely innervated; however, it shows a dense sympathetic supply at the site of pressor receptors. Following removal of the superior cervical ganglion, a total loss of fluorescent adrenergic nerves occurs and degeneration of nerve endings possessing dense core vesicles is conspicuous. These nerve terminals are situated mainly subendothelially in the carotid body sinusoids; they only rarely terminate on type I cells.     

Kinesin-related Smy1 enhances the Rab-dependent association of myosin-V with secretory cargo

The mechanisms by which molecular motors associate with specific cargo is a central problem in cell organization. The kinesin-like protein Smy1 of budding yeast was originally identified by the ability of elevated levels to suppress a conditional myosin-V mutation (myo2-66), but its function with Myo2 remained mysterious. Subsequently, Myo2 was found to provide an essential role in delivery of secretory vesicles for polarized growth and in the transport of mitochondria for segregation. By isolating and characterizing myo2 smy1 conditional mutants, we uncover the molecular function of Smy1 as a factor that enhances the association of Myo2 with its receptor, the Rab Sec4, on secretory vesicles. The tail of Smy1—which binds Myo2—its central dimerization domain, and its kinesin-like head domain are all necessary for this function. Consistent with this model, overexpression of full-length Smy1 enhances the number of Sec4 receptors and Myo2 motors per transporting secretory vesicle. Rab proteins Sec4 and Ypt11, receptors for essential transport of secretory vesicles and mitochondria, respectively, bind the same region on Myo2, yet Smy1 functions selectively in the transport of secretory vesicles. Thus a kinesin-related protein can function intimately with a myosin-V and its receptor in the transport of a specific cargo.     

Axon terminals with unusual vesicles in the brain of the polychaete,Ophryotrocha puerilis

Summary In the brain of Ophryotrocha puerilis swollen nerve endings filled with electron-lucent vesicles and aggregates of vesicles were observed. The vesicles do not resemble elementary neurosecretory granules. Tests for biogenic amines were negative; no dense-core vesicles were found. The vesicle type described here cannot be related to any of the types thus far found in nerve cells.Supported by Deutsche Forschungsgemeinschaft Pf116/3     

Ultrastructure of the pineal organ of the killifish,Fundulus heteroclitus,with special reference to the secretory function

Summary The pineal organ of the killifish, Fundulus heteroclitus, was investigated by electron microscopy under experimental conditions; its general and characteristic features are discussed with respect to the photosensory and secretory function. The strongly convoluted pineal epithelium is usually composed of photoreceptor, ganglion and supporting cells. In addition to the well-differentiated photosensory apparatus, the photoreceptor cell contains presumably immature dense-cored vesicles (140–220 nm in diameter) associated with a well-developed granular endoplasmic reticulum in the perinuclear region and the basal process. These dense-cored vesicles appear rather prominent in fish subjected to darkness. The ganglion cell shows the typical features of a nerve cell; granular endoplasmic reticulum, polysomes, mitochondria and Golgi apparatus are scattered in the electron-lucent cytoplasm around the spherical or oval nucleus. The dendrites of these cells divide into smaller branches and form many sensory synapses with the photoreceptor basal processes. Lipid droplets appear exclusively in the supporting cell, which also contains well-developed granular endoplasmic reticulum and Golgi apparatus. Cytoplasmic protrusions filled with compact dense-cored vesicles (90–220 nm in diameter) are found in dark-adapted fish. The origin of these cytoplasmic protrusions, however, remains unresolved. Thus, the pineal organ of the killifish contains two types of dense-cored vesicles which appear predominantly in darkness. The ultrastructural results suggest that the pineal organ of fish functions not only as a photoreceptor but also as a secretory organ.We thank Dr. Grace Pickford for the fishes.     

Occurrence of innervation in labral glands of Daphnia obtusa (crustacea,cladocera)

Electron microscopy discloses nerve endings in contact with gland cells situated in the labrum of Daphnia. Swellings of nerve fibers are in close contact with gland cell membranes, either on the cell surface or inserted into infoldings of plasma membrane. The axonal processes are single or double and lack glial wrappings. Inside the nerve fibers are vesicles of different sizes and electron density. These include granular vesicles, which often are dense-cored, and also clearer vesicles. Some presynaptic differentiations lie along the contact line of the axonal process with the gland cell membrane. The significance of the vesicles is discussed in terms of their possible content of biogenic amines, as described in other invertebrates.     

THE FINE STRUCTURE OF MEISSNER's TOUCH CORPUSCLES OF HUMAN FINGERS

Thin slices of the finger pads of six individuals were fixed in buffered 1 per cent osmic acid, embedded in deaerated, nitrogenated methacrylate, and cut into thin sections for electron microscopic study. Before embedding, the slices were trimmed so as to include several digital tactile corpuscles. Some thin sections were stained in 10 per cent aqueous phosphotungstic acid solution. The principal part of Meissner's corpuscle is made up of flattened laminar cells stretching across the corpuscle in irregular layers. The perinuclear cytoplasm of these cells contains numerous small mitochondria, a sparse granular endoplasmic reticulum, and a large number of small vesicles. Nerve fibers enter the side or base of the corpuscle, lose their myelin sheaths, and follow a meandering course between the laminar cell plates. The nerve endings enter into a close appositional relationship with the flattened portions of the laminar cells. In some areas the apposed axolemma and cell membranes are slightly thickened with small vesicles located along the cell membrane or on both surfaces. These regions are interpreted as synapses. The most prominent feature of the nerve endings is an extraordinary accumulation of small mitochondria which vary in size and internal density. The nerve endings also contain vacuoles, groups of dense concentric membranes, and small dense vesicles of irregular distribution. The laminar cells are separated from one another by a dense intercellular substance of uniform thickness which also envelops the entire corpuscle. This material contains randomly oriented collagen fibers and fine fibrils bound together by a dense material at nodal points recurring at regular intervals of approximately 120 mµ. These findings are discussed in relation to the problems of the function of Meissner's corpuscle, neural material loss and replacement, and the presence of synapses.     

Monoamine oxidase activity measurements using radioactive substrates

The use of Amberlite CG-50, Dowex 50 and solvent extraction for separation of the oxidation products of the biogenic amines are compared, and measurements of monoamine oxidase activity using ¹⁴C-labeled biogenic amines are described. K_m data for tyramine, dopamine, tryptamine, and serotonin for monoamine oxidase activity of rabbit brain mitochondria are reported. Rates of product formation from [¹⁴C]tyramine are compared with polarographic measurements of oxygen utilization using purified MAO and intact mitochondria from rabbit liver and brain. Difficulties in comparative measurements of monoamine oxidase activity and some reasons for wide variations in published data are discussed.     

ULTRASTRUCTURE OF THE SPOON TYPE SYNAPTIC ENDINGS IN THE NUCLEUS VESTIBULARIS TANGENTIALIS OF THE CHICK

The fine structure of the "spoon" type synaptic endings of the chick tangential nucleus was studied with the electron microscope. These endings often measure ~18 µ in length by ~3–4 µ in width. The axoplasm of the endings contains very few synaptic vesicles, a large number of neurofilaments oriented parallel to the long axis of the nerve fiber, and microtubules and numerous mitochondria. The synaptic membrane complex shows areas of localized occlusion of the synaptic cleft with the formation of an external compound membrane. It has not been decided whether these areas have a disc shape; their length measures between 0.04 and 0.47 µ. The five-layer pattern characteristic of an external compound membrane is shown in specimens fixed with formalin—OsO₄, glutaraldehyde—acrolein—OsO₄, and acrolein KMnO₄ but it does not appear in the glutaraldehyde-OsO₄-fixed specimens. The over-all thickness of the external compound membrane varies depending upon the fixative used. The synaptic clefts in the regions between the external compound membrane discs are widened and measure ~300 A. A condensation of dense material occurs in pre- and postsynaptic cytoplasms all along the synaptic membrane complex. The morphological relationships described in the spoon endings are suggestive of electrical transmission.     

Ultrastructure,innervation et fonction de l'épiphyse de l'orvet (Anguis fragilis L.)

Summary The pineal organ of Anguis fragilis contains two essential cell types: pinealocytes and interstitial cells. The scarce outer segments are of different appearance; they show cyclic degenerative changes. Only one intraepithelial ganglion cell has been identified in the material of this study. Although an intracommissural pineal nerve is present, the absence of synaptic junctions of pinealocytes with ganglion cells indicates a loss of photosensory function. Three kinds of vesicles (also dense-core vesicles) originating from Golgi complex are described in the pinealocytes. These secretory vesicles show a vascular polarity. The pinealocyte processes extend to the basement membrane. Secretory material is released into the peripineal space. Efferent sympathetic nerve fibers are described near the pineal epithelium. The nerve endings of these fibers contain three types of vesicles. It is suggested that the pineal organ of Anguis fragilis has a well established secretory function.

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Zusammenfassung Die Epiphysis cerebri von Anguis fragilis enthält zwei wesentliche Zelltypen: Pinealocyten und Zwischenzellen. Die Außenglieder, die nur selten vorkommen, sind verschiedenartig gestaltet und erleiden einen zyklischen Degenerationsprozeß. Im Pinealepithel ließ sich nur eine einzige Ganglienzelle mit Sicherheit nachweisen. Obwohl eine intracommissurale Nervenbahn vorhanden ist, spricht das Fehlen von Synapsen zwischen Pinealocyten und Ganglienzellen für einen Schwund der Lichtsinnesfunktion. In den Pinealocyten werden drei aus dem Golgiapparat stammende Bläschenarten, darunter auch solche mit einem elektronendichtem Inhalt (Granula), beschrieben. Diese Sekretbläschen sind polar auf die Blutgefäße ausgerichtet. Fortsätze der Pinealocyten stehen mit der Basallamina in Verbindung; allem Anschein nach werden hier Substanzen in den periepiphysären Raum abgegeben. Efferente sympathische Nervenfasern sind in der Nähe des Epiphysenepithels zu beobachten. Die Endigungen dieser Fasern enthalten drei verschiedene Bläschentypen. Die Epiphyse der Blindschleiche scheint eine sekretorische Funktion zu haben.

     

The effects of niamid and reserpine on the nerve endings of the pineal gland

Summary Kitten pineal glands were studied cytochemically under normal conditions, after reserpine injection, and after niamid administration. Adrenergic nerve elements were in perivascular spaces while cholinergic terminals were adjacent to pinealocytes, often times in synaptic contact. BA reactions are primarily in dotted vesicles of adrenergic terminals with some reaction in granular vesicles. Positive reaction occurs along neurotubules and membranous structures of adrenergig nerve fibers and terminals indicating membrane-bounded BA's. Niamid increased the number and density of dotted vesicles, and some granular vesicles are increased in density and size. Reserpine produced a loss reaction in dotted vesicles and a loss of vesicle matrix, producing elliptical vesicles. There is loss of reaction of the dotted vesicles, but occasionally, the positive granular reaction remains. Cholinergic terminals demonstrate no changes with either niamid or reserpine. These findings indicate BAs are stored in reserpine sensitive dotted vesicles and membraneous structures. The findings also show that the dotted vesicle matrix is reserpine sensitive and is necessary for storage of the BA's. Possibly biogenic amines cannot be stored or synthesized in terminals unless the matrix of the dotted vesicle is intact.Supported by: HEW Grant No. NS-10326. The University of Texas Medical School at Houston. — Special appreciation to Mrs. Charlotte Smith for her valuable technical assistance. Appreciation to Ciba-Geigy Corporation for supply of Serpasil (reserpine).     

THE FINE STRUCTURE OF THE GALL BLADDER EPITHELIUM OF THE MOUSE

Sections of mouse gall bladder epithelium fixed by perfusion with buffered osmium tetroxide have been studied in the electron microscope as an example of simple columnar epithelium. The free surface presents many microvilli, each presenting a dense tip, the capitulum, and displaying a radiating corona of delicate filaments, the antennulae microvillares. Very small pit-like depressions, representing caveolae intracellulares, are encountered along the cell membrane of the microvilli. The free cell surface between microvilli shows larger cave-like depressions, likewise representing caveolae intracellulares, containing a dense material. The lateral cell borders are extensively folded into pleats, which do not interdigitate extensively with corresponding folds of the adjacent cell membrane. The terminal bars are shown to consist of thickened densities of the cell membrane itself in the region of insertion of the lateral cell wall with the free cell surface. This thickening is associated with an accumulation of dense cytoplasmic material in the immediate vicinity. The terminal bar is thus largely a cytoplasmic and cell membrane structure, rather than being primarily intercellular in nature. The basal cell membrane is relatively straight except for a conical eminence near the center of the cell, projecting slightly into the underlying tunica propria. The basal cell membrane itself is overlain by a delicate limiting membrane, which does not follow the lateral contours of the cell. Unmyelinated intercellular nerve terminals with synaptic vesicles have been encountered between the lateral walls of epithelial cells. A division of the gall bladder epithelial cell into five zones according to Ferner has been found to be convenient for this study. The following cytoplasmic components have been noted, and their distribution and appearance described: dense absorption granules, mitochondria, Golgi or agranular membranes, endoplasmic reticulum or ergastoplasm, ring figures, and irregular dense bodies, perhaps lipoid in nature. The nucleus of these cells is also described.     

Evidence for basal secretion in the subcommissural organ of the adult rabbit

Summary Morphological evidence is presented supporting the possibility of basal secretion into hypendymal capillaries of the adult rabbit subcommissural organ (SCO). The synthetic apparatus of the SCO cell is described as well as the heterogeneous granules and vesicles which are concentrated in the basal processes bordering a widened perivascular space. The origin of the electron dense granules, of which two fairly distinct subgroups are found, is discussed.A binding of secretory sacs to the lateral plasma membrane is seen. The possibility of a lateral secretion is supported by the presence of a system of extracellular channels between SCO cells which are filled with a flocculent material resembling that of the secretory sacs.Nerve perikarya which are separated from the SCO by only a few glial fibers are demonstrated. Synapses are described in nerve fascicles bordering on the hypendymal capillaries. The possibility of an innervation of the hypendymal region is discussed as well as possible nervous connections with the pineal gland.This work was supported by grants from Statens almindelige Videnskabsfond, Copenhagen.     

Granulated vesicles in the pineal gland of the mouse

Summary Pineal glands of normal adult mice, 7 to 42 days after bilateral superior cervical gangliectomy and 5 and 16 hours after one dose of reserpine (10 mg/kg) were studied under the electron microscope. The architecture of the gland is basically similar to that of other mammalian pineal glands previously studied. Mouse pinealocytes are polymorphic cells with perivascular and intercellular processes. Its most prominent feature is the presence of abundant granulated vesicles with a mean diameter of 1100 Å and a dense core of about 800 Å intermingled with clear vesicles of similar size distributed throughout the cytoplasm and more concentrated in perivascular and intercellular processes. These processes were seen in continuity with the perykaryon and remained after bilateral superior cervical gangliectomy. Processes containing the plurivesicular component of adrenergic nerves situated in perivascular and intercellular spaces disappeared after bilateral superior cervical gangliectomy. Reserpine depleted small granulated vesicles of nerves but the larger ones of perikaryon and polar processes remained almost unchanged.The histochemical technique of Wood was positive for catechol- and indolamines in the nerves. The reaction was negative in the perikaryon and polar processes.The significance of these findings is discussed.This work has been supported by grants of the Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina and U.S. Air Force AF-AFOSR 67-0963 A.I am greatly indebted to Miss Haydee Agoff and Mr. Alberto Saenz for their skillful technical assistance.     

Synaptic ribbons during postnatal development of the pineal gland in the golden hamster (Mesocricetus auratus)

Summary Synaptic ribbons, functionally enigmatic structures of mammalian pinealocytes, were studied during the postnatal development of the pineal gland in the golden hamster (Mesocricetus auratus). On day 4 post partum, synaptic ribbons appear in cells that have already started to differentiate into pinealocytes. Between days 4 and 9, an increase in the number of synaptic ribbons occurs, concomitant with the continuing differentiation of the pineal tissue. Between days 9 and 16, when differentiation of this tissue is almost completed, the number of synaptic ribbons decreases and approaches that characteristic of the adult pineal gland. During development, the synaptic ribbons increase in length, and dense core vesicles are frequently found in the vicinity of these structures. It is assumed that a functional relationship exists between dense core vesicles and the synaptic ribbons, which are considered to be engaged in a certain form of secretory activity of the mammalian pineal gland.Supported by the Deutsche Forschungsgemeinschaft     

Cytochemical studies on cytoplasmic granular elements in the hamster pineal gland

Summary The pineal gland of adult golden hamsters (Mesocricetus auratus) was studied by various cytochemical methods at the electron microscopic level: (1) the modified chromaffin reaction specific for 5-hydroxytryptamine (5-HT), (2) argentaffin reaction, (3) zinc-iodide-osmium (ZIO) mixture reaction and (4) acid phosphatase reaction. In the pinealocytes, the dense-cored vesicles (80–160 nm in diameter) show both chromaffinity and argentaffinity, while the population of dense bodies (150–400 nm in diameter) is reactive to ammoniacal silver solution and ZIO mixture but not to the modified chromaffin reaction. After incubation for demonstration of acid phosphatase activity, reaction products are localized in some, but not all, of the dense bodies, in some of the small vesicles in the Golgi region and in one or two inner Golgi saccules. In nerve fibers in the pineal gland, small granulated vesicles are also reactive to the modified chromaffin reaction and ZIO mixture. Based upon these cytochemical results the following conclusions have been reached: (1) dense cored vesicles in the pinealocytes and small granulated vesicles in the nerve fibers of the hamster pineal gland contain 5-HT, and (2) the population of dense bodies in the pinealocytes is heterogenous, some are lysosomes and the others are possibly the granules responsible for the secretion of pineal peptides.Supported in part by a grant from the National Science Council, Republic of ChinaDedicated to Professor Doctor Huoyao Wei on the occasion of his 70th birthday