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  • 1
    ISSN: 1460-9568
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: A period of hypersensitivity to N-methyl-d-aspartate (NMDA) has been described during the early development of different types of neuron. Since activation of NMDA receptors can also induce rapid neuron death, the hypersensitivity to NMDA may be tightly controlled. In the present study we show that mouse cerebellar granule neurons become transiently hypersensitive to NMDA between days 10 and 14 after plating in a culture medium containing 30 mM K+. The NMDA sensitivity is higher when cells are cultured in the presence of an NMDA receptor antagonist [30 mM K+ plus 100 μM 3-((±)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid (CPP)], and no hypersensitivity is observed when cells are cultured in the continuous presence of NMDA (12.5 mM K+ plus 100 μM NMDA). The high NMDA sensitivity in control cells is associated with a higher density of NMDA receptors than that measured in NMDA-treated cells, suggesting that the sensitivity to NMDA may be partly controlled by activity-dependent NMDA receptor down-regulation. We also examined the level of NMDA-ζ1 mRNA and found no correlation between this parameter and the transient pattern of NMDA sensitivity. Such NMDA receptor plasticity may be of importance in the central nervous system, protecting developing cells from excitotoxicity at critical developmental stages.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Journal of neurocytology 13 (1984), S. 503-518 
    ISSN: 1573-7381
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Summary Cholinergic synapses form between ciliary neurons and cultured myotubes. We have identified these synaptic contacts using α-bungarotoxin conjugated to horseradish peroxidase (αBTX-HRP). The enzymatic reaction product was limited to a small portion of the sarcolemma in direct apposition to the nerve terminal. Multiple neuronal processes contact the region of the myotube containing acetylcholine receptors (AChRs). At the early stages of nerve-muscle contacts neuronal processes protrude into the coated pits of the myoplasm. Numerous coated pits and coated vesicles were found beneath these early contacts. These vesicles may be involved in the transport of protein molecules at the newly formed cholinergic structures.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Cell & tissue research 139 (1973), S. 29-45 
    ISSN: 1432-0878
    Keywords: Nerve cells ; Hydra ; Basal disk ; Epithelial cells ; Light and electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary In addition to glandulomuscular cells, three other cell types are found in the basal disk of Hydra. These are interstitial cells, cnidoblasts and nerve cells. Although only a few of the two former cell types are recognized in this region, the observations from this study refute previous statements to the contrary. Nerve cells are concentrated in the basal disk where they form a type of “network” system, due to the presence of bipolar, tripolar and multipolar cells. In some specimens, they assume a circular arrangement around the base of the polyp. Using morphological criteria for identifying the three types of epidermal nerve cells (neurosensory, neurosecretory and ganglionic) in other body regions, only neurosensory and neurosecretory cells are observed in the basal disk. These are indistinguishable ultrastructurally from their respective counterparts in other regions. It is possible that ganglionic cells are also present in the basal disk, but there may be few such cells. It is suggested that the three cell types originate from the budding region and these precursor cells are then forced proximally. Interstitial cells, escaping their differentiative function, do not develop apparently into other cell types of the basal disk. Cnidoblasts contain normal nematocysts but their functional ability is uncertain. Neurosensory and neurosecretory cells arise directly and independently from interstitial cells in the budding region, as evidenced by the appearance of immature nerve cells in the peduncle and their absence in the basal disk. Although viable cells may be discarded from the basal disk, it is believed that most cells die in situ and are then eliminated. The possible role of nerve cells is discussed briefly.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Cell & tissue research 150 (1974), S. 213-229 
    ISSN: 1432-0878
    Keywords: Nerve cells ; Regeneration ; Neurosecretion ; Hydra viridis, littoralis ; Light microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Using whole mount preparations, nerve cells at the cut surface (head region) and also the peduncular-basal disk region were studied during various stages of regeneration (zero hour — 96 h) in Hydra. Leucomethylene blue staining technique allows excellent stainability of nerve cells and thus a statistical count of them at the specified stages of regeneration was obtained. Within 1 h after transection a significant increase (P 〈 0.001) in release of neurosecretory droplets occurs. Between 4 and 15 h of regeneration the number of perikarya remains relatively constant, but the number of processes increases by 200%. This indicates that neurons in Hydra have the capacity to regenerate their processes. Their growth in length is dramatically illustrated at 18 hrs. of regeneration. This degree of anastomosing arrangement is not observed in any other stage of regeneration. After this time the majority of processes break down releasing the neurosecretory materials. A study of the number of perikarya, the number of neurites and the number of neurosecretory droplets in regenerating animals reveals a continuous increase in the number of nerve cells and neurosecretory droplets released for up to 24 h. With the accumulation of nerve cells at the cut surface (24 h of regeneration) there is a simultaneous appearance of tentacle outpushings. The tentacles increase in number and length during the subsequent periods of regeneration. As the regenerative process approaches completion (72–96 h) the number of neurosecretory droplets released decreases, approaching the pre-transection levels as seen in normal animals. It is suggested that the neurosecretory material may act as a “trophic” agent which stimulates differentiation of interstitial cells into nerve cells and thus influences the regenerative process.
    Type of Medium: Electronic Resource
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  • 5
    Electronic Resource
    Electronic Resource
    Springer
    Cell & tissue research 150 (1974), S. 231-247 
    ISSN: 1432-0878
    Keywords: Regeneration ; Nerve cells ; Neurosecretion ; Hydra ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Major ultrastructural changes in neurons were studied during sequential periods of hypostomal regeneration in Hydra. Some neurons remain unaffected except that at certain periods following amputation, they become more active in neurosecretory production. Other neurons in various stages of differentiation were also observed. Most emphasis was placed on degenerating neurons showing a loss of organelles and destruction of the perikarya. Certain large, membrane-bounded structures (up to 1.7 μ in diameter) suggested tentatively to be of a lysosomal-like nature, may be partly responsible for the degenerative process. The neurites of these cells first assume a beaded appearance and/or contain bulbous endings. The eventual isolated fragments of neurites contain typical membrane-bounded neurosecretory droplets (850/1700 Å in diameter) which disintegrate forming particulate materials (350 Å in diameter). Following complete disruption of the neurites, some of the granules accumulate in the extracellular spaces before they are disintegrated. From the data presented in this and the preceding paper, it is suggested that these particles, derived from neurosecretory droplets, may be responsible for the stimulation of: 1) interstitial cell differentiation into neurons, 2) the increased activity in neurosecretory production in normal cells, 3) the conspicuous increase in neurite length and consequently the exaggerated degree of anastomosis, and 4) the possible capacity of neurons to regenerate neurites.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Cell & tissue research 220 (1981), S. 573-588 
    ISSN: 1432-0878
    Keywords: Lysosomotropic agents ; ACh receptor-coated vesicles ; Cultured myotubes ; Endocytosis ; Exocytosis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Treatment of cultured muscle cells with the inhibitors of lysosomal function, leupeptin, and chloroquine, decrease the degradation of acetylcholine receptors (AChR) and causes accumulation of undegraded receptors intracellularly. Under these conditions the number of cytoplasmic coated vesicles, i.e. structures that appear to transport this receptor within the cultured muscle cell, increases in parallel. This study investigates the effects of leupeptin and chloroquine on the morphology of cultured myotubes in order to learn more about the turnover of acetylcholine (ACh) receptors and the origin of the coated vesicles. Chloroquine causes involution of the plasma membrane, disorganization in the arrangement of sarcomeres, vacuolization, and enlargement of dense lysosome-like bodies in myotubes. The diameter of dense bodies in untreated myotubes is 0.36 ± 0.01 μm (mean ± SEM) compared with 2 ± 0.12 μm after 48 h of incubation with chloroquine. Leupeptin does not disrupt the normal architecture of sarcomeres and does not cause vacuolization of the myotubes. However, leupeptin does enlarge the dense bodies, although to a lesser extent than chloroquine (average diameter after 48 h treatment, 1.0 ± 0.06 μm, p 〈 0.01). Untreated myotubes appear to contain equal numbers of large and small coated vesicles. After chloroquine treatment 95% of coated vesicles are large (80–120 nm in diameter), whereas after leupeptin treatment the majority of coated vesicles are small (40–70 nm in diameter). After incubation with horseradish peroxidase (HRP) 62% ± 9 of coated vesicles in chloroquinetreated cells contain the tracer, whereas in control cells only 11% ± 4 of coated vesicles contain HRP reaction product. These observations indicate that chloroquine causes accumulation of coated vesicles and interferes with degradation of AChR by preventing fusion of lysosomes with coated vesicles originating by endocytosis.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1432-0878
    Keywords: Acetylcholinesterase ; Calcium ionophore ; Myotubes ; Diisopropyl fluorophosphate ; Domestic fowl
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary Calcium (Ca2+) and calcium-transporting ionophores stimulate protein secretion in many cellular systems. We demonstrate here that increases in intracellular calcium concentration induce a time- and concentration-dependent deposition of extracellular matrix and an increase in acetylcholinesterase secretion. Scanning and transmission electron-microscopy revealed that treatment with the calcium ionophore A23187, or high extracellular Ca2+ levels (5 mM to 15 mM) produce significant deposits of extracellular matrix around the myotubes, as well as a marked increase in the acetylcholinesterase reaction-product. Blocking muscle contraction was not necessary for the induction of AChE secretory activity. Sucrose density-gradients of media conditioned by muscle cells revealed 3 separate acetylcholinesterase molecular forms. However, incubation with A23187 increased only the 4.5 S and the 7.2 S molecular forms, whereas the 12.0 S form showed no significant differences from controls. Polyacrylamide gel electrophoresis, and autoradiography using [3H]diisopropyl fluorophosphate revealed a broad band at 65000 daltons. This band was broader than for controls when medium was obtained from A23187-treated cells. Our results show that increasing intracellular Ca2+ concentration induces marked deposition of extracellular matrix and increased acetylcholinesterase secretion, with an apparent selectivity for the monomeric and dimeric acetylcholinesterase molecular forms.
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    Springer
    Cell & tissue research 248 (1987), S. 535-540 
    ISSN: 1432-0878
    Keywords: ACh receptor ; Coated vesicles ; Endocytosis ; αBTX ; Cultured myotubes
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The removal of the acetylcholine receptors (AChRs) from the surface of muscle cells serves as an important mechanism in the regulation of the AChR turnover rate. Our previous studies have shown that cultured myotubes contain coated pits and vesicles bearing α-bungarotoxin (αBTX)-binding sites (Bursztajn 1984; Bursztajn and Fischbach 1984). In this study we have used αBTX conjugated to horseradish peroxidase (HRP) and quantitative electron microscopy to determine the intracellular pathway(s) of acetylcholine receptors during the internalization process. To accomplish this, cultured rat myotubes were incubated with αBTX-HRP at 4° C after which cells were washed and incubated at 37° C for 0 min to 2 h. After warming the cells, coated pits, coated vesicles and smooth membraned vesicles containing the peroxidase reaction product were present. A threefold increase in coated vesicles containing the reaction product was observed 1 min after warming the cells. The number of smooth-membraned vesicles remained constant at this time point. However, 5 to 15 min after warming the cells, a fivefold increase in the number of smooth membraned vesicles was observed. After 1 h at 37° C the reaction product was present in the lysosomal like bodies, but was not observed in the Golgi complex or the small coated vesicles associated with the Golgi complex. Our observations indicate that there is a size segregation between those coated vesicles containing αBTX-HRP reaction product and those in which reaction product is absent. Our studies also suggest that within minutes of AChR internalization coated vesicles lose their coat and become smooth-membraned vesicles.
    Type of Medium: Electronic Resource
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  • 9
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 53 (1993), S. 251-264 
    ISSN: 0730-2312
    Keywords: coated vesicles ; monoclonal antibodies synapse formation ; nerve-muscle cultures ; immunofluorescent localization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Coated and noncoated vesicles participate in cellular protein transport. Both acetylcholine receptors (AChR) and acetylcholinesterase (AChE) are transported via coated vesicles, some of which accumulate beneath the neuromuscular synapse where AChRs cluster. To investigate the mechanisms by which these proteins are transported during postsynaptic remodeling, we purified coated vesicles from the bovine brain via column chromatography (Sephacryl S-1000) and raised monoclonal antibodies to epitopes of the vesicular membranes enriched in AChE. We assayed for AChE (coated vesicle enriched), hexosaminidase (lysosomal contaminants), NADH cytochrome C reductase (mitochondrial containing), and protein and demonstrated electron microscopically using negative staining that the vesicular fraction contained 95% pure coated vesicles. We then injected coated vesicle fractions and the fractions from which the coat was removed intraperitoneally into mice and obtained three monoclonal antibodies: C-33, C-172, and F-22. On immunoblots of purified vesicles and cultured skeletal muscle, mAb C-33 stained a 180 Kd band and mAb C-172 stained a 100 kd band. MAb F-22 stained 50 kd and 55 kd bands and was not characterized further. Immunofluorescent microscopy with C-33 and C-172 revealed punctate fluorescence whose distribution depends upon the stage of myotube development. Four days after plating, myotubes showed punctate fluorescence throughout the myotube, whereas those stained 8 days after plating showed a punctate perinuclear distribution. Myotubes innervated by ciliary neurons show punctate fluorescence limited to the nuclear periphery and most concentrated around nuclei which line up beneath neuronal processes. This differential vesicular distribution, observed during myotube differentiation and innervation, suggests that these vesicles participate in vesicular membrane traffic.
    Additional Material: 7 Ill.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 58 (1995), S. 22-38 
    ISSN: 0730-2312
    Keywords: in situ hybridization ; desmin ; nuclear diversity ; MyoD ; cell fusion ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Muscle progenitor cells differentiate to myoblasts, and subsequently myotubes, upon expression of muscle specific genes. We and others have previously shown that myotude nuclei, even in the absence of nerve, express AChR α subunit RNA at varying levels, with a small subset (about ten percent) of the nuclei expressing at high levels. These findings raised two important quwstions:(1) is the observed heterogeneneity a unique property of the α subunits, and (2) when does the heterogeneneity begin? In particular, is it induced only at or afer the time of fusion, or does it exist at the myoblast stage? We hasve, therfore, extended our observations to the γ and δ subunits and we also have examined the distributions of AChR α,γ, and δ subunit RNAs in both myoblasts and myotubes. We used intron and intron-exon probes to detect prespliced trascripts or mature mRNAs in the cells. Because inton-containing transcripts are not transported out of the nuclei, the distributions of these transcripts can indicate their expression patterns among nuclei in the same myotubes. Our results show that both myotubes and myotubes have distribution of the AChR α,γ, and δ subunit RNAs which differ sharply from that of the U1 RNA or Myo D. Thus, the heterogeneous expression of AChR genes is not only an intrinsic property of muscle cell nuclei (in the sense that it does not require the presence of nerves), but it also exists prior to fusion. Our results suggest that muscle nuclei attain individualized capacities for AChR subunit mRNA production early in their development. Conceptual models consistend with individuality imply an additional level of regulation beyond the known diffusible transcriptional factors. © Wiley-Liss, Inc.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
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