Ultrastructural changes in differentiating neuroblastoma x glioma hybrid cells

The ultrastructure of differentiating neuroblastoma x glioma hybrid cells NG108-15 was observed. Cells cultured in growth medium showed undifferentiated features, while cells treated with dBcAMP became round and large, and extended thick long neurites. After 1 week in culture, cells showed features similar to those of normal neurons. The dense cored vesicles with diameters ranging from 60 to 170 nm were observed in differentiated NG108-15 cells, but clear vesicles were usually rare. However, in the case of co-culture with striated myotubes, clusters of clear vesicles appeared in the neurites and terminals. The timecourse of the differentiation process was correlated with results obtained by the electrophysiology and freeze-fracture.     

Ultrastructural changes in differentiating neuroblastoma × glioma hybrid cells

The ultrastructure of differentiating neuroblastoma × glioma hybrid cells NG108-15 was observed. Cells cultured in growth medium showed undifferentiated features, while cells treated with dBcAMP became round and large, and extended thick long neurites. After 1 week in culture, cells showed features similar to those of normal neurons. The dense cored vesicles with diameters ranging from 60 to 170 nm were observed in differentiated NG108-15 cells, but clear vesicles were usually rare. However, in the case of co-culture with striated myotubes, clusters of clear vesicles appeared in the neurites and terminals. The timecourse of the differentiation process was correlated with results obtained by the electrophysiology and freeze-fracture.     

The endomembrane system of differentiating carposporangia in the red algaChondria tenuissima: Occurrence and participation in secretion of polysaccharidic and proteinaceous substances

Summary The endomembrane system during carposporogenesis inChondria tenuissima was studied using electron microscopy and histochemistry. Profiles of the nucleus are convoluted, resulting in a highly increased surface area. Stacked cisternae are found within the peripheral part of the nucleus. Vesicles, tubules and membrane bound fibrillar bodies occur within the nucleoplasm. The endoplasmic reticulum surrounds the nuclear envelope.The endoplasmic reticulum and the Golgi apparatus, together with small transition vesicles, represent a functional unit. They form two different secretory substances during carposporogenesis. In young stages, carbohydrates are produced by normal dictyosomes within large, normal exocytotic Golgi vesicles. They do not react positively with PAS or Thiéry method and are believed to represent cell wall material. In later stages, the central area of the Golgi cisternae becomes filled with electron dense material. The individual cisternae are transformed into cored vesicles at the trans-face of the dictyosomes. The dense core of the vesicles is proteinaceous and stains with coomassie brilliant blue R. The peripheral fibrillar material is polysaccharidic and reacts positively using the Thiéry method. The contents of the cored vesicles are believed to participate in carpospore attachment. The ER gives rise to cytolysosomes in which starch grains are sequestrated and digested. Mucilaginous sacs seem to be similarly formed.     

Cytochemical demonstration of amine-storing vacuoles and lysosomes in the chicken thrombocytes

Summary A combined electron microscopic and cytochemical study of the thrombocytes of the chicken has clearly identified the amine-storing organelles and lysosomes. A chormaffin positive-reaction product was observed on the inner surface and the granules of the large electronlucent vacuoles. No acid phosphatase activity was localized in these amine-storing vacuoles. However, the acid phosphatase activity was observed in the small vesicles, the primary lysosomes, and in the large electron dense inclusions with myelin which may be secondary lysosomes. The results of this study suggest that the large empty vacuoles, with one or two very dense osmiophilic peripherally-situated granules, in the chicken thrombocytes are comparable to the vesicles with electron dense materials called dense bodies in mammalian thrombocytes.To whom offprint requests should be sent     

Localization of phosphorylated TrkA in carrier vesicles involved in its nuclear translocation in U251 cell line

A number of transmembrane receptors are targeted to the nucleus and convincingly localized therein. However, what remains a conundrum is how these cell-surface receptors end up in the nucleus. In this study, we reported that the transmembrane receptor phosphorylated TrkA was located in a series of carrier vesicles, including ring-like vesicles near the plasma membrane, large core vesicles and small dense core vesicles around the nuclei, as well as in the nucleus in human glioma cell line U251 using immunocytochemistry and immunofluorescence staining. Meanwhile, we also showed that small dense core vesicles budded from large core vesicles, and interacted with the nuclear envelope. Accordingly, our results suggested that such a series of membrane compartments might be involved in the pathway of nuclear translocation of the transmembrane receptor TrkA.     

Localization of phosphorylated TrkA in carrier vesicles involved in its nuclear translocation in U251 cell line

A number of transmembrane receptors are targeted to the nucleus and convincingly localized therein.However, what remains a conundrum is how these cell-surface receptors end up in the nucleus. In this study, we reported that the transmembrane receptor phosphorylated TrkA was located in a series of carrier vesicles, including ring-like vesicles near the plasma membrane, large core vesicles and small dense core vesicles around the nuclei, as well as in the nucleus in human glioma cell line U251 using immunocytochemistry and immunofluorescence staining. Meanwhile, we also showed that small dense core vesicles budded from large core vesicles, and interacted with the nuclear envelope. Accordingly,our results suggested that such a series of membrane compartments might be involved in the pathway of nuclear translocation of the transmembrane receptor TrkA.     

Catecholamine-rich cells and varicosities in bovine splenic nerve,vesicle contents and evidence for exocytosis

The bovine splenic nerve trunk contins mast cells, ganglion cells, small intensely flurescent (SIF) cells, and varicosities which exhibit a brilliant fluorescence characteristic for noradrenaline (NA) and dopamine (DA) after formaldehyde exposure. All these catecholamine-rich structure could contribute particles to isolated nerve vesicle fractions. Mast cells are recognized ultrastructurally by their large (300–800nm) dense granules. SIF cells may be represented by cells and processes containing dense cored vesicles (120–140 nm) which are larger than the typical vesicles in axons and terminals. Terminal-like areas with typical large dense cored vesicles (LDV, 75 nm) and small dense cored vesicles (SDV, 45–55 nm) probably correspond to the fluorescent varicosities. The LDV constitute about 40% of all vesicle in terminal-like areas and terminals. Their staining properties indicate the presence of protein, phospholipids, and ATP. Tyramine depletes NA without loss of matrix density. The LDV can fuse with the terminal membrane, and released material outside omega profiles is interpreted to depict exocytosis. Large and small vesicles are easily distinguished from the very large mast cell granules and the moderately dense Schwann cell vesicles. Neither appear to contaminate the LDV fractions but the latter may contain a small population of SIF cell vesicles. Golgi vesicles from the Schwann cells mainly occur in the lighter zones of the gradient.     

Searching for Molecular Players Differentially Involved in Neurotransmitter and Neuropeptide Release

Neurotransmitters and neuropeptides are stored in small clear vesicles (SCVs) and large dense core vesicles (LDCVs), respectively. Many differences in the properties of SCVs and LDCVs suggest that these two classes of secretory organelles may employ different sets of molecules in exocytosis. Relatively little is known, however, about factors that differentially participate in SCVs and LDCVs release. This article briefly overviews some key molecules that are possibly involved in the differential regulation of the trafficking, docking, priming and fusion of SCVs and LDCVs. Special issue article in honor of Dr. Ji-Sheng Han.     

Investigations on the lectin-producing cells in the sponge Axinella polypoides (Schmidt)

Summary The localization of two carbohydrate binding proteins, so-called lectins, was studied in the sponge tissue of Axinella polypoides by light and immunofluorescence microscopy. They do not occur at the cellular surface of any cell type, but they are stored in vesicles of the spherulous cells. After short formaldehyde fixation spherulous cells can be isolated and they release the active lectins upon lysis in distilled water.Electron microscopical studies of spherulous cells show that they contain almost nothing else but a small nucleus and vesicles of different size and number. Small vesicles are full of an electron dense material, whereas the content of large vesicles has a fluffy and fibrillar structure. Spherulous cells are large and tightly packed in the outer layer of the ectosome and in the mesh work of the spongin fibres of the central axis. They are small and scattered in the inner layer of the ectosome, and they are found throughout the choanosome. The function of the lectins is not clearly defined, and different alternatives such as participation in glycoprotein synthesis, immunological defense, or carbohydrate transport are possible.This study was supported by a grant from the Deutsche ForschungsgemeinschaftWe are gratefully indebted to Dr. D. Keyser for his help in our electron microscopical studies     

Catecholamine-rich cells and varicosities in bovine splenic nerve, vesicle contents and evidence for exocytosis.

The bovine splenic nerve trunk contains mast cells, ganglion cells, small intensely fluorescent (SIF) cells, and varicosities which exhibit a brilliant fluorescence characteristic for noradrenaline (NA) and dopamine (DA) after formaldehyde exposure. All these catecholamine-rich structures could contribute particles to isolated nerve vesicle fractions. Mast cells are recognized ultrastructurally by their large (300-800 nm) dense granules. SIF cells may be represented by cells and processes containing dense cored vesicles (120-140 nm) which are larger than the typical vesicles in axons and terminals. Terminal-like areas with typical large dense cored vesicles (LDV, 75 nm) and small dense cored vesicles (SDV, 45-55 nm) probably correspond to the fluorescent varicosities. The LDV constitute about 40% of all vesicles in terminal-like areas and terminals. Their staining properties indicate the presence of protein, phospholipids, and ATP. Tyramine depletes NA without loss of matrix density. The LDV can fuse with the terminal membrane, and released material outside omega profiles is interpreted to depict exocytosis. Large and small vesicles are easily distinguished from the very large mast cell granules and the moderately dense Schwann cell vesicles. Neither appear to contaminate the LDV fractions but the latter may contain a small population of SIF cell vesicles. Golgi vesicles from the Schwann cells mainly occur in the lighter zones of the gradient.     

Chemical sympathectomy induced by 5.6-dihydroxytryptamine

Summary The effect of different doses of 5.6-dihydroxytryptamine—a serotonin analogue which produces a degeneration of serotonin containing nerve terminals in the rat brain—on the noradrenaline (NA) content and—storage sites of peripheral sympathetic nerves in the mouse and rat heart, spleen, rectum and vas deferens has been investigated by fluorescence—, electron microscopical and chemical methods. Moderate doses of 5.6-dihydroxytryptamine (5.6-DHT) (10–45 mg/kg ip.) cause a temporary, reversible displacement of noradrenaline from the adrenergic nerves concomitant with a significant increase in the number and opacity of small and especially large granular vesicles. The recovery of the neuronal NA concentration is, however, retarded after doses higher than 45 mg/kg (60 or 100 mg/kg ip.); a partial degeneration of varicose NA terminals is verified fluorescence- and electron microscopically. A combined treatment of animals with tyrosine hydroxylase inhibitors (-methyl-paratyrosine or -propyl-dopacetamide) and 5.6-DHT, in some instances also followed by reserpine, potentiates the destructive properties of 5.6-DHT; a similar potentiation is accomplished by reserpine posttreatment or by an additional pretreatment of animals with reserpine and nialamide.The results suggest that 5.6-DHT when given in moderate doses (up to 45 mg/kg) may be handled by sympathetic adrenergic nerves like a false neurotransmitter which displaces noradrenaline from the stores, but that it causes a chemical degeneration of noradrenaline containing nerve terminals when applied either in single high doses (60 or 100 mg/kg ip.), or when administered in moderate non-degenerative doses together with drugs that impair the neuronal inactivation mechanisms for 5.6-DHT (granular uptake and storage mechanism and/or monoamine oxidase activity) and thus provoke a temporary increase in the amount of free 5.6-DHT in the neuron's cytoplasm.The molar efficiency of 5.6-DHT in causing a chemical sympathectomy is clearly inferior to that caused by 6-hydroxydopamine. The differences are probably mainly due to differences in the affinity of both drugs to the amine uptake system located at the cell membrane and the membrane of the intraneuronal storage vesicles of the adrenergic nerve terminals.Supported by grants from the Deutsche Forschungsgemeinschaft.     

Preparation of noradrenaline-storing organelles from bovine sympathetic ganglia: Biochemical and morphological evaluation of partly purified large dense cored vesicles

Large dense cored vesicles from bovine sympathetic ganglia were isolated and partly purified. Biochemical and morphological evaluation of the present vesicle-preparation revealed that it represents a convenient fraction for the characterization of perikaryal noradrenergic vesicles.

Homogenates of bovine stellate ganglia were subjected to differential centrifugation and D₂O-sucrose density gradient centrifugation. Biochemical evaluation of gradient fractions was performed by measuring marker enzyme activities reflecting subcellular contamination, while morphological evaluation was performed by electron microscopic analysis of the isolated fractions. Both techniques revealed that the vesicle-preparation was, at first, still considerably contaminated by mitochondria and lysosomes.

An improved purification could be achieved by subjecting this fraction to an additional centrifugation under iso-osmotic conditions, also applied for the preparation of highly purified splenic nerve vesicles. The resulting vesicle-fraction was almost complete free of contaminating enzyme activities and consisted merely of large dense cored vesicles as revealed by electron microscopic observations (50–70% purity). Neuropeptide Y and chromogranin A were enriched more than 50 times as compared to the total homogenate.

Although the purity of these vesicles was still not satisfactory for direct chemical analysis, this vesicle-preparation seemed very well suited for immunological characterization of perikaryal large dense cored vesicles.     

Observations on the ultrastructure of nerve cells in the brain of the earthworm,Eisenia foetida,with special reference to neurosecretion

Summary The submicroscopic structure of nerve cells in the brain of the earthworm Eisenia was studied. Six types of neurons containing morphologically different inclusions are identified. Types 1, 2 and 3 contain vesicles filled with homogeneous materials of high electron density. These are essentially similar to elementary granules in neurosecretory systems of vertebrates and invertebrates. Type 4 shows dense-cored vesicles which resemble in size catechol-containing granules as described, for example, in chromaffin cells of the adrenal gland. Type 5 has clear vesicles with a mean diameter of 400 Å. Some of these vesicles have a dense osmium deposit. Type 6 contains electron lucent vesicles with diameters of 500–800 Å. Occasionally these have osmiophilic cores. Clear vesicles of types 5 and 6 are similar to classical synaptic vesicles, while granulated vesicles resemble in size and appearance those described in adrenergic nerve endings. All six vesicle types have the same mode of origin from Golgi membranes. All of these vesicles are considered to be discharged from the perikarya into the axons entering the neuropil.     

Synaptic and Extrasynaptic Secretion of Serotonin

Serotonin is a major modulator of behavior in vertebrates and invertebrates and deficiencies in the serotonergic system account for several behavioral disorders in humans.The small numbers of serotonergic central neurons of vertebrates and invertebrates produce their effects by use of two modes of secretion: from synaptic terminals, acting locally in hard wired circuits, and from extrasynaptic axonal and somatodendritic release sites in the absence of postsynaptic targets, producing paracrine effects.In this paper, we review the evidence of synaptic and extrasynaptic release of serotonin and the mechanisms underlying each secretion mode by combining evidence from vertebrates and invertebrates. Particular emphasis is given to somatic secretion of serotonin by central neurons.Most of the mechanisms of serotonin release have been elucidated in cultured synapses made by Retzius neurons from the central nervous system of the leech. Serotonin release from synaptic terminals occurs from clear and dense core vesicles at active zones upon depolarization. In general, synaptic serotonin release is similar to release of acetylcholine in the neuromuscular junction.The soma of Retzius neurons releases serotonin from clusters of dense core vesicles in the absence of active zones. This type of secretion is dependent of the stimulation frequency, on L-type calcium channel activation and on calcium-induced calcium release.The characteristics of somatic secretion of serotonin in Retzius neurons are similar to those of somatic secretion of dopamine and peptides by other neuron types. In general, somatic secretion by neurons is different from transmitter release from clear vesicles at synapses and similar to secretion by excitable endocrine cells.     

Ultrastructure of neuro‐spirocyte synapses in the sea anemone Aiptasia pallida (Cnidaria,Anthozoa, Zoantharia)

Using transmission electron microscopy of serially sectioned tentacles from the sea anemone Aiptasia pallida, we located and characterized two types of neuro‐spirocyte synapses. Clear vesicles were observed at 10 synapses and dense‐cored vesicles at five synapses. The diameters of vesicles at each neuro‐spirocyte synapse were averaged; clear vesicles ranged from 49–89 nm in diameter, whereas the dense‐cored vesicles ranged from 97–120 nm in diameter. One sequential pair of synapses included a neuro‐spirocyte synapse with clear vesicles (81 nm) and a neuro‐neuronal synapse with dense‐cored vesicles (168 nm). A second synapse on the same cell had dense‐cored vesicles (103 nm). An Antho‐RFamide‐labeled ganglion cell and three different neurites were observed adjacent to spirocytes, but no neuro‐spirocyte synapses were present. Many of the spirocytes also were immunoreactive to Antho‐RFamide. The presence of sequential neuro‐neuro‐spirocyte synapses suggests that synaptic modulation may be involved in the neural control of spirocyst discharge. The occurrence of either dense‐cored or clear vesicles at neuro‐spirocyte synapses suggests that at least two types of neurotransmitter substances control the discharge of spirocysts in sea anemones. J. Morphol. 241:165–173, 1999. © 1999 Wiley‐Liss, Inc.     

A monoamine in the gustatory cell of the frog's taste organ

Summary Fluorescence histochemistry reveals that in the frog's taste organ a yellow fluorescence is regularly observed at the most basal region of the sensory epithelium. The fluorescence has a strong intensity, but it fades rapidly upon the UV-irradiation. The peak of the emission spectrum is at 520 m. Following reserpine treatment the yellow fluorescence is markedly reduced, but not depleted completely. From these characteristics the monoamine fluorescence is regarded as representing 5-HT (serotonin).The ultrastructural study on sensory epithelia shows that the terminal portions of gustatory cell processes are localized at the basal region. These portions are filled with dense cored vesicles (700–1000 Å in diameter) and frequently opposed with nerve fibers penetrating into the epithelium. The gustatory cell processes are also interposed between the terminal portions or nerve fibers. The cytoplasm of the gustatory cell process is characterized by many mitochondria, fine filaments and glycogen particles, but contains few cored vesicles. The distribution of terminal portions of gustatory cell processes seems to correspond fairly well to that of the monoamine fluorescence observed discontinuously along the basal lamina. Accordingly it is concluded that the fluorigenic monoamine is localized in the cored vesicles of the gustatory cell.These results were reported in a preliminary form to the October, 1974 meeting of the Japan Society of Histochemistry and Cytochemistry.The authors gratefully acknowledge the support and helpful advice of Prof. Dr. T. Kanaseki.     

Vasoactive intestinal polypeptide (VIP)- and neuropeptide Y (NPY)-containing nerve fibres in the penile cavernous tissue of green monkeys (Cercopithecus aethiops)

Summary The cavernous body of green monkeys contains many unmyelinated and few myelinated axons. The unmyelinated axons form terminals in the adventitia of the arteries, between trabecular muscle cells, in the interstitium, and close to endothelium cells of the sinuses. All terminals displayed predominantly small clear vesicles and very few large granular vesicles; small granular vesicles were not seen. However, in rabbit penises, terminals with many large granular vesicles are prominent. Immunohistochemistry (PAP technique) showed a dense network of VIP- and NPY-reactive fibres around the arteries and around trabecular muscles. The density of nerve fibres was particularly high around the subendothelial cushions of the helicine arteries. Double staining for NPY and VIP revealed that both peptides were colocalized. Immunocytochemistry (preembedding PAP technique) showed VIP- and NPY-reactivity in terminals with small clear vesicles; the reaction product was bound to the cytoplasmic face of different membrane types. Although the intracellular localization of the reaction product is probably due to artefactual displacement during preparation, the uniformity of the terminals questions the view that large and small granular vesicles in all species characterize peptidergic and noradrenergic terminals, respectively. The essential findings can be summarized as (1) a high degree of uniformity of nerve terminals, (2) colocalization of VIP and NPY, (3) heavy innervation of the subendothelial cushions of the helicine arteries, and (4) possible innervation of endothelial cells.     

Different types of small granule-containing cells and neurons in the guinea-pig adrenal medulla

Summary An electron microscopic, histoand biochemical study was carried out on the adrenal medulla of newborn and adult guinea-pigs giving special emphasis to small granule-containing (SGC) cells. Adrenaline (A) was the predominating catecholamine (CA) both in newborn (70–90 % of total CA) and adult (85–90%) guinea-pig adrenals. In analogy to the biochemical findings electron microscopy revealed a high predominance of A cells, which contained large granular vesicles with an average diameter of 180 nm. Most noradrenaline (NA) storing cells showed granular vesicles of a considerably smaller average diameter (80 nm) and had a higher nuclear-cytoplasmic ratio. These cells were termed SGC-NA cells. NA cells with large granular vesicles (average diameter 170 nm) were extremely rare. Another type of SGC cells contained granular vesicles with cores of low to medium electron-density (SGC-NA-negative cells). Biochemical determinations made it unlikely that these cells contained predominantly dopamine (DA). SGC cells were scarcely innervated by cholinergic nerves. They formed processes, which were found both in the adrenal cortex and medulla contacting blood vessels including sinusoid capillaries, steroid producing cells of the reticularis and fasciculata zone and processes, which were interpreted to belong to medullary nerve cells.Two types of neurons were present in the guinea-pig adrenal medulla, one resembling the principal neurons in sympathetic ganglia, the other, which, according to its morphology, occupied an intermediate position between principal neurons and SGC cells.In adrenomedullary grafts under the kidney capsule, which were studied three weeks after transplantation, ordinary A cells resembled SGC-NA negative cells with respect to their ultramorphology. Processes of transplanted principal neurons showed uptake of 5-hydroxydopamine and, hence, were considered to be adrenergic. Despite the lack of extrinsic nerves to the transplants, few principal neurons received cholinergic synapses, the origin of which is uncertain to date.Supported by a grant from Deutsche Forschungsgemeinschaft (Un 34/4)Dedicated to Professor H. Leonhardt in honor of his 60th birthday.     

Subcellular Distribution of Mammalian Tachykinins in Rat Basal Ganglia

A combined differential and density gradient centrifugation procedure was used to study the subcellular localisation of the mammalian tachykinins in rat caudateputamen and substantia nigra. Substance P, neurokinin A, neuropeptide K, and neurokinin B were found to be concentrated in the synaptosomal fractions and in fractions containing heavy synaptic vesicles in both regions studied. In contrast, the catecholamines dopamine and noradrenaline had a more widespread distribution throughout the gradient. HPLC analysis of the immunoreactivity recovered showed that the tachykinin immunoreactivity coeluted with the relevant synthetic tachykinins, except in the soluble gradient fraction where neurokinin A immunoreactivity eluted in position consistent with neurokinin A3-10. These results suggest that, in the basal ganglia, the mammalian tachykinins are localised in fractions containing large dense cored synaptic vesicles. This vesicular localisation would be consistent with the proposed role of the tachykinins as neurotransmitters and neuromodulators.