High-frequency stimuli preferentially release large dense-core vesicles located in the proximity of nonspecialized zones of the presynaptic membrane in sympathetic ganglia

We characterized the effect of a brief high-frequency stimulus on the number, distribution, and optical density of large dense-core vesicles (LDCVs) in the nerve terminals of the rat superior cervical ganglia. From 4.21+/-0.37 LDCVs/bouton detected in control nerve terminals, a stimulus of 40 Hz for 1 min released 41% of LDCVs, decreasing their number to 2.48+/-0.14 LDCVs/bouton (p=0.0009). In control ganglia, most dense vesicles were located close to the plasma membrane (at 相似文献   

The variety of synaptic vesicles in the interpeduncular nucleus (ITP) of the frog.

The electron microscope has revealed a large variety of synaptic vesicles in the interpeduncular nucleus (ITP) of the frog "Rana esculenta". They vary in shape, size and electron density. There are two types of synapses which show only translucent spherical vesicles: in one type the vesicles are 40 nm, in the other type they are 70 nm in diameter. In other types of synapses the translucent vesicles may be mixed with those with dense core. Large granules, 160 nm in diameter, already reported in the ITP (KEMALI 1977a), are also shown as well as tiny flat mixed with large flat dense core vesicles of dumb-bell shape. Two types of axo-axonic synapses are illustrated while no crest synapses have been demonstrated. The results suggest that the afferents to the ITP might be more numerous than those reported in the literature or that--as in the case of the habenular afferents which consist of cholinergic and peptergic fibres--each projecting nucleus to the ITP has different types of fibres with more than one type of transmitter. Furthermore, due to the vesicles sizes, we may consider the ITP as a site in the vertebrate central nervous system where conventional neurotransmitter structures coexist with probable neurohumoral elements.     

On different types of nerve endings in the frog median eminence after fixation with permanganate and glutaraldehyde-osmium

Summary In the frog median eminence, fixed with glutaraldehyde and osmium tetroxide, four types of nerve endings can be generally distinguished. These endings are in contact with the pericapillary spaces of primary portal vessels and can be identified by the internal structure and the size of their granules and vesicles. Type 1 contains large granules (1500–2400 Å in diameter) and small clear vesicles (300–500 Å in diameter), type 2 intermediate granules (about 1100–1700 Å in diameter) and small clear vesicles, type 3 small granules (about 600–1000 Å in diameter) and small clear vesicles, type 4 only numerous small clear vesicles. The mixed types containing the large, intermediate and small dense granules in the same ending are infrequently found.After KMnO₄ or LiMnO₄ fixation the granules and vesicles mentioned above are observed as follows. The large granules in the type 1 nerve ending appear mostly pale or less-dense. The intermediate granules in the type 2 also appear mostly pale or less-dense, but some frequently show granules of high density. The small granules in the type 3 consistently contain the dense substance and these endings can be subdivided into two different types according to the populations of different sizes of dense granules [type 3a (900–1000 Å) and type 3b (500–800 Å)]. Dense-cored and cleared-synaptic vesicles are frequently present with together in the type 3 endings. The small vesicles (300–400 Å), in the type 4, appear generally pale (type 4a), but some nerve endings contain small dense cored-vesicles (type 4b).The author wishes to thank Prof. H. Fujita for his advice and criticism.     

EGFP-Tagged Vasopressin Precursor Protein Sorting Into Large Dense Core Vesicles and Secretion From PC12 Cells

1. Hypothalamic magnocellular neurons synthesize, store, and secrete large quantities of the neuropeptides, vasopressin (VP) and oxytocin (OT), which are synthesized as protein precursors also containing proteins called neurophysins. These protein precursors are sorted through the regulated secretory pathway (RSP), packaged into large dense core vesicles LDCVs, and their peptide products are secreted from nerve terminals in the posterior pituitary.2. It has been hypothesized that this efficient packaging is dependent on the interaction of the peptide with neurophysin in a complex that forms the granule core. To test this, PC12 cells were transfected with vasopressin precursor DNA constructs that either contained or deleted the neurophysin moiety and tagged with enhanced green fluorescent protein (EGFP) as reporters. The intracellular routing and secretion of the EGFP-tagged VP precursor proteins were studied by in differentiated PC12 cells by fluorescence microscopy, electron microscopic immunocytochemistry, and fluorescent imaging techniques.3. The data showed that only when the neurophysin was present in the VP precursor construct did the fluorescent fusion protein become routed to the RSP and get efficiently packaged into LDCVs and secreted. These data are consistent with the view that routing of the precursor to LDCVs requires the amino acids that encode the intravesicular chaperone, neurophysin.     

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.     

An Ultrastructural Account of Otoplanid Turbellaria Neuroanatomy I. The cerebral ganglion and the peripheral nerve net

The otoplanid nervous system investigated in Otoplana truncaspina Lanfranchi, 1969 and Parotoplanella heterorhabditica Lanfranchi, 1969 consits of: (a) an ellipsoidal cerebral ganglion located between the gut and the cephalic intestine and invested by a fibrillar collagen-like capsule 0.3 μm thick; (b) anterior extracapsular ganglion cell clusters; (c) a peripheral nerve plexus locally thickened at the level of the epithelial sensory and glandular areas, with extensive synaptic connections. At least two neuron types can be identified within the ganglion: (a) an inner layer close to the central neuropile of the 1st type of neurons, showing a vesicular cytoplasm rich in RER and Golgi complexes processing both round, clear, 25–45 nm in diameter, and dense cored vesicles, 50–80 nm in diameter; (b) an outer layer of the 2nd type of neurons, adjoining the capsule and filled with uniformly dense vesicles, 60–90 nm in diameter. Synaptic endings in the neuropile are provided with clear vesicles and dense cored vesicles or uniformly dense vesicles. The presynaptic side has paramembranous projections channelling the vesicles to the active zone; omega-like profiles are also observed. Thin banded muscle fibres run within the brain. A comparison is drawn with the other turbellarian neuron types described in the literature, to suggest their possible function. The functional implications of the synaptic ultrastructure are discussed.     

以消散场成像和单微粒跟踪技术揭示GLUT4囊泡和分泌囊泡的不同动态过程

葡萄糖转运子蛋白4(glucose transporter 4,GLUT4)在维持体内葡萄糖动态平衡的过程中起着至关重要的作用。GLUT4贮存囊泡(GLUT4 storage vesicle,GSV)和神经内分泌细胞中的分泌囊泡含有许多相同的蛋白。研究证明这些蛋白调节了分泌囊泡的胞内转运过程,但是GLUT4囊泡和分泌囊泡是否具有相同的胞内动态过程还未阐明。文章以3T3-L1纤维原细胞中的GSV和神经内分泌细胞PC12细胞中的分泌囊泡:致密核心大囊泡(large dense core vesicle,LDCV)为研究对象,使用消散场显微成像技术和单微粒跟踪技术直观观察了活体细胞内单个GSV和LDCV的三维运动轨迹。通过以适当方程拟合单个囊泡的均方位移曲线,发现两种囊泡都具有三种运动模式。定量分析显示作自由扩散运动和方向性扩散运动的GSV数量明显多于LDCV。对比GSV和LDCV的三维扩散系数,发现GSV的扩散系数中值为7.2×10-4μm2/s,而LDCV的扩散系数中值仅为1.94×10-4μm2/s。这一结果说明GSV的活动性远大于LDCV,提示GSV的胞内转运过程涉及不同的分子机制。     

An acidic motif retains vesicular monoamine transporter 2 on large dense core vesicles

The release of biogenic amines from large dense core vesicles (LDCVs) depends on localization of the vesicular monoamine transporter VMAT2 to LDCVs. We now find that a cluster of acidic residues including two serines phosphorylated by casein kinase 2 is required for the localization of VMAT2 to LDCVs. Deletion of the acidic cluster promotes the removal of VMAT2 from LDCVs during their maturation. The motif thus acts as a signal for retention on LDCVs. In addition, replacement of the serines by glutamate to mimic phosphorylation promotes the removal of VMAT2 from LDCVs, whereas replacement by alanine to prevent phosphorylation decreases removal. Phosphorylation of the acidic cluster thus appears to reduce the localization of VMAT2 to LDCVs by inactivating a retention mechanism.     

Direct observation of membrane retrieval in chromaffin cells by capacitance measurements

This study was focussed on the identification of the endocytic organelles in chromaffin cells which retrieve large, dense core vesicle (LDCV)-membrane components from the plasma membrane. For this purpose, 'on-cell' capacitance measurements and electron microscopy were employed. We found capacitance steps and capacitance flickers, corresponding to single exo- and endocytic events. The analysis revealed that the total membrane surface of completely fused LDCVs is recycled by large endocytic vesicles and smaller, most likely clathrin-coated vesicles, at approximately the same ratio. These results were confirmed by rapid-freeze immuno-electron microscopy, where an extracellular marker was rapidly internalized into endocytic vesicles that morphologically resembled LDCVs.     

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.     

The activation of exocytotic sites by the formation of phosphatidylinositol 4,5-bisphosphate microdomains at syntaxin clusters

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a minor component of the lipid bilayer but plays an important role in various cellular functions, including exocytosis and endocytosis. Recently, PI(4,5)P2 was shown to form microdomains in the plasma membrane. In this study, we investigated the relationship between the spatial organization of PI(4,5)P2 microdomains and exocytotic machineries in clonal rat pheochromocytoma PC12 cells. Both PI(4,5)P2 and syntaxin, a soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein essential for exocytosis, exhibited punctate clusters in isolated plasma membranes. The number of PI(4,5)P2 microdomains colocalizing with syntaxin clusters and large dense core vesicles (LDCVs) was decreased after catecholamine release. Alternatively, the expression of type I phosphatidylinositol-4-phosphate 5-kinase (PIP5KI) increased the number of PI(4,5)P2 microdomains at syntaxin clusters with docked LDCVs and enhanced exocytotic activity, possibly by increasing the number of release sites. About half of the PI(4,5)P2 microdomains were not colocalized with Thy-1, a specific marker of lipid rafts, and the colocalization of transfected PIP5KI with syntaxin clusters was observed. These results suggest that the formation of PI(4,5)P2 microdomains at syntaxin clusters with docked LDCVs is essential for Ca2+-dependent exocytosis.     

Preferential localization of a vesicular monoamine transporter to dense core vesicles in PC12 cells

Neurons and endocrine cells have two types of secretory vesicle that undergo regulated exocytosis. Large dense core vesicles (LDCVs) store neural peptides whereas small clear synaptic vesicles store classical neurotransmitters such as acetylcholine, gamma-aminobutyric acid (GABA), glycine, and glutamate. However, monoamines differ from other classical transmitters and have been reported to appear in both LDCVs and smaller vesicles. To localize the transporter that packages monoamines into secretory vesicles, we have raised antibodies to a COOH- terminal sequence from the vesicular amine transporter expressed in the adrenal gland (VMAT1). Like synaptic vesicle proteins, the transporter occurs in endosomes of transfected CHO cells, accounting for the observed vesicular transport activity. In rat pheochromocytoma PC12 cells, the transporter occurs principally in LDCVs by both immunofluorescence and density gradient centrifugation. Synaptic-like microvesicles in PC12 cells contain relatively little VMAT1. The results appear to account for the storage of monoamines by LDCVs in the adrenal medulla and indicate that VMAT1 provides a novel membrane protein marker unique to LDCVs.     

The nervous system of Microstomum lineare (Turbellaria,Macrostomida)

Summary The nervous system (NS) of Microstomum lineare (Turbellaria, Macrostomida) was studied by electron and light microscopy, combined with fluorescence histochemistry (Falck-Hillarp method for biogenic monoamines). The NS is primitively organized, with a bilobed brain, two lateral nerve cords lacking commissures, and peripheral nerve cells scattered along the nerve cords. The stomatogastric NS, with a pharyngeal nerve ring, is joined to the central NS by a pair of connective ganglia. A green fluorescence in all parts of the NS indicates catecholaminergic neurons as the dominant neuron type.Ultrastructurally, two types of neurons were identified on the basis of their vesicle content: 1. Aminergic (catecholaminergic) neurons containing densecore vesicles of varying electron-density and size, i.e., small dense-core vesicles (diameter 50–100 nm), vesicles with a highly electron-dense core (60–140 nm), and vesicles with an eccentric dense-core. 2. Presumed peptidergic neuro-secretory neurons containing large granular vesicles (diameter about 200 nm) in the stomatogastric NS and peripheral parts of the central NS. In light microscopy, paraldehyde-thionin stained neurons were observed in the same areas.     

Localization and identification of catechol compounds in the ctenophore mnemiopsis leidyi

1. The ctenophore Mnemiopsis leidyi, was examined for monoaminergic nerves by glyoxylic acid-induced fluorescence.2. The meridional canals contained strands of fluorescent cells.3. High pressure liquid chromatography (HPLC), showed the presence of 2,3-dihydroxyphenylalanine (DOPA), 0.25–0.35 pmol/specimen, and 5-S-cysteinylDOPA, 0.3–0.85 pmol/specimen.4. Electronmicroscoplcal examination of tissues showing histofluorescence, revealed large cells (> 20 μm) containing irregular osmium-reducing vesicles, usually 100–500 nm in size. A second cell type, situated by the outside of the canal, contained dense core vesicles, 175–200 nm in diameter.5. It is suggested that the histofluorescence is caused by DOPA and 5-S-cysteinylDOPA. The presence of catecholamines is discussed.     

Ultracytochemical identification of catecholamine-containing vesicles in the ligated sciatic nerve of the rat. Comparison with sympathetic nerve terminals

Summary Using the fixation procedure of Tranzer, three kinds of granular vesicles were identified in certain unmyelinated fibres of rat sciatic nerves proximal to a ligature: (1) small vesicles (SGV: 30–60 nm in diameter), (2) large vesicles (LGV: 60–100nm in diameter), and (3) large elongated vesicles (LEV: 60–100nm in diameter). A comparative study concerning the distribution of these granular vesicles was carried out using a cytopharmacological method (reserpine) and employing different fixatives (aldehydes + OsO₄, or OsO₄ alone) in periarterial nerve plexus of the femoral artery, vas deferens and the pineal organ.Use of Tranzer's method allows preservation in almost all granular vesicles of a strongly electron-dense core, while with the other fixatives mainly small, eccentric dense cores occur in the vesicles. Two main features were observed in ligated sciatic nerves: (i) a clear increase in the number of LGV, and (ii) the presence of LEV, considered as a variety of LGV rather than a new population of granular vesicles. Reserpine caused the cores of SGV to disappear almost completely, while LGV and LEV remained only partly depleted. The original method combining Tranzer's fixation procedure with radioautography revealed radioautographic labelling only in the unmyelinated fibres of ligated sciatic nerves and mainly superimposed over SGV, LGV and LEV. It is suggested that (i) SGV, LGV and also LEV represent possible storage sites of catecholamines, and (ii) a local morphogenesis of SGV from the large vesicles occurs in ligated sympathetic nerve fibres.     

The last few milliseconds in the life of a secretory granule

We have monitored single vesicles (granules) in bovine adrenal chromaffin cells using an optical sectioning technique, total internal reflection fluorescence microscopy (TIRFM). With TIR, fluorescence excitation is limited to an optical slice near a glass/water interface. In cells located at the interface, granules loaded with fluorescent dye can be visualized near to or docked at the plasma membrane. Here we give evidence that (1) TIRFM resolves single vesicles and (2) the fluorescence signal originates from vesicles of roughly 350 nm diameter, presumably large dense core vesicles (LDCVs). (3) Diffusional spread of released vesicle contents can be resolved and serves as a convenient criterion for a fusion event. (4) We give details on vesicle properties in resting cells, such as lateral mobility of chromaffin granules, number density, and frequency of spontaneous fusion or withdrawal into the cytoplasm. (5) Upon stimulation with high extracellular potassium, TIRFM reports depletion of the `visible pool' of vesicles closest to the plasma membrane within hundreds of milliseconds, consistent with previous concepts of a release-ready pool. We conclude that TIRFM constitutes an independent assay for pool depletion. TIRFM will allow us to study aspects of secretion that have previously been inaccessible in living cells, in particular the spatial relations and dynamics of vesicles prior to and during exocytosis and re-supply of the near-membrane pool of vesicles. Received: 26 June 1997 / Accepted: 26 September 1997     

Electron microscopic autoradiography on the localization of serotonin in the frog median eminence

Summary For the identification of the nerve fiber containing serotonin (5-hydroxytryptamine) in the frog median eminence, an electron microscopic autoradiography was performed with 5-hydroxytryptophane-³H which is the precursor of serotonin. At 1 and 24 hours after the intraperitoneal injection, most silver grains were located over the nerve fibers and endings, and a few were also found over the glia cell and the perivascular space. A large number of silver grains were located over the type 3 nerve endings (Nakai, 1971) containing small dense granules about 600–1000 Å in diameter 1 and 24 hours after the injection. Some silver grains were localized over the nerve endings containing intermediate-size dense granules 1100–1700 Å in diameter. Silver grains were also frequently observed over the nerve fibers in the inner layer of frog median eminence. There is no significant difference in the pattern of distribution of silver grains between tissues of 1 hour and 24 hours after the injection.The authors wish to thank Prof. H. Fujita for his advice and criticism.     

Chromogranins A and B are key proteins in amine accumulation, but the catecholamine secretory pathway is conserved without them

Chromogranins are the main soluble proteins in the large dense core secretory vesicles (LDCVs) found in aminergic neurons and chromaffin cells. We recently demonstrated that chromogranins A and B each regulate the concentration of adrenaline in chromaffin granules and its exocytosis. Here we have further studied the role played by these proteins by generating mice lacking both chromogranins. Surprisingly, these animals are both viable and fertile. Although chromogranins are thought to be essential for their biogenesis, LDCVs were evident in these mice. These vesicles do have a somewhat atypical appearance and larger size. Despite their increased size, single-cell amperometry recordings from chromaffin cells showed that the amine content in these vesicles is reduced by half. These data demonstrate that although chromogranins regulate the amine concentration in LDCVs, they are not completely essential, and other proteins unrelated to neurosecretion, such as fibrinogen, might compensate for their loss to ensure that vesicles are generated and the secretory pathway conserved.     

Dominant-Negative Myosin Va Impairs Retrograde but Not Anterograde Axonal Transport of Large Dense Core Vesicles

Axonal transport of peptide and hormone-containing large dense core vesicles (LDCVs) is known to be a microtubule-dependent process. Here, we suggest a role for the actin-based motor protein myosin Va specifically in retrograde axonal transport of LDCVs. Using live-cell imaging of transfected hippocampal neurons grown in culture, we measured the speed, transport direction, and the number of LDCVs that were labeled with ectopically expressed neuropeptide Y fused to EGFP. Upon expression of a dominant-negative tail construct of myosin Va, a general reduction of movement in both dendrites and axons was observed. In axons, it was particularly interesting that the retrograde speed of LDCVs was significantly impaired, although anterograde transport remained unchanged. Moreover, particles labeled with the dominant-negative construct often moved in the retrograde direction but rarely in the anterograde direction. We suggest a model where myosin Va acts as an actin-dependent vesicle motor that facilitates retrograde axonal transport.     

A phosphorylation site regulates sorting of the vesicular acetylcholine transporter to dense core vesicles

Vesicular transport proteins package classical neurotransmitters for regulated exocytotic release, and localize to at least two distinct types of secretory vesicles. In PC12 cells, the vesicular acetylcholine transporter (VAChT) localizes preferentially to synaptic-like microvesicles (SLMVs), whereas the closely related vesicular monoamine transporters (VMATs) localize preferentially to large dense core vesicles (LDCVs). VAChT and the VMATs contain COOH-terminal, cytoplasmic dileucine motifs required for internalization from the plasma membrane. We now show that VAChT undergoes regulated phosphorylation by protein kinase C on a serine (Ser-480) five residues upstream of the dileucine motif. Replacement of Ser-480 by glutamate, to mimic the phosphorylation event, increases the localization of VAChT to LDCVs. Conversely, the VMATs contain two glutamates upstream of their dileucine-like motif, and replacement of these residues by alanine conversely reduces sorting to LDCVs. The results provide some of the first information about sequences involved in sorting to LDCVs. Since the location of the transporters determines which vesicles store classical neurotransmitters, a change in VAChT trafficking due to phosphorylation may also influence the mode of transmitter release.