Zinc iodide-osmium tetroxide reaction: its effect on the synaptic vesicles in locust neuromuscular junctions and its chemical basis

1978 ◽  
Vol 36 (2) ◽  
pp. 614-615
Author(s):  
M. Reinecke ◽  
Ch. Walther
Keyword(s):  
Synaptic Vesicles ◽  
Osmium Tetroxide ◽  
Neuromuscular Junctions ◽  
Motor Nerve ◽  
Locusta Migratoria ◽  
Nerve Terminals ◽  
Neurobiological Research ◽  
Zinc Iodide ◽  
Electron Lucent

The zinc iodide-osmium tetroxide reaction (ZIO) was first used in neurobiological research by Maillet (Bull. Ass. Anat. 53, 233; 1968). Subsequently several authors have shown that, under appropriate conditions, ZIO stains mainly the interior of synaptic vesicles. The substrate of this reaction is under discussion, since ZIO can also react with other subcellular structures in a variety of tissues, e. g. mitochondria, endoplasmic reticulum, dictyosomes and lysosomes. Additionally, in vitro substances as different as some aminoacids, catecholamines, aldehydes and phospholipids (Pellegrino de Iraldi, Experientia 33, 1; 1977) can yield black precipitations with ZIO.Our studies were done with the motor nerve terminals at the femoral retractor unguis muscle of the locust (Locusta migratoria). These terminals are chiefly the endings of excitatory motoraxons and are characterized by the presence of electron lucent vesicles and by an accumulation of mitochondria.

scholarly journals Aspects of turnover and biogenesis of synaptic vesicles at locust neuromuscular junctions as revealed by zinc iodide-osmium tetroxide (ZIO) reacting with intravesicular SH-groups.

1978 ◽  
Vol 78 (3) ◽  
pp. 839-855 ◽  
Author(s):  
M Reinecke ◽  
C Walther
Keyword(s):  
Synaptic Vesicles ◽  
Osmium Tetroxide ◽  
Developmental Stages ◽  
Neuromuscular Junctions ◽  
Smooth Endoplasmic Reticulum ◽  
Motor Nerve ◽  
Average Volume ◽  
Experimental Conditions ◽  
Sh Groups ◽  
Zinc Iodide

Retractor unguis nerve muscle preparations from the locust were subjected to the zinc iodide-osmium tetroxide reaction (ZIO) after pre-fixation in glutaraldehyde. Applied for 18 h at 4 degrees C in the dark, ZIO reacts at pH 4.2--4.0 fairly selectively with the matrix of synaptic vesicles. Approximately 53% of the vesicles are completely and 4% partially stained. The percentage of ZIO-positive vesicles is increased to nearly 90% and reduced to 4% or less by pretreatment with SH-protecting (dithiothreitol) or SH-blocking (N-ethylmaleimide, p-chloromercuriphenyl sulfonic acid) and SH-oxidizing (azodicarboxylic acid-bis-dimethylamide) reagents, respectively. Stimulation of the motor nerve at 20 Hz for 7 min, partially fatiguing synaptic transmission, reduces the number of vesicles per square micrometer of terminal area by approximately 52%; 2 min of rest restores this number of its pre-stimulation level. These changes are chiefly accounted for by changes in the number of completely ZIO-positive vesicles. 2 min after the end of stimulation, partially ZIO-positive vesicles are three times more frequent than before. With all experimental conditions, the average volume of vesicles was as follows: ZIO-negative less than partially ZIO-positive less than completely ZIO-positive. The average volume of ZIO-positive vesicles is almost unaffected by stimulation; that of ZIO-negative vesicles is decreased by 25% immediately after stimulation, increasing with subsequent rest to the initial level after 1 h. It is suggested (a) that ZIO demonstrates intravesicular protein(s) containing SH-groups and (b) that the completely ZIO-positive vesicles represent the mature ones ready to be used for transmitter release. How the ZIO reaction differentiates between different developmental stages of vesicles which could arise from the smooth endoplasmic reticulum is discussed.

scholarly journals Depression of Synaptic Efficacy in High- and Low-OutputDrosophila Neuromuscular Junctions by the Molting Hormone (20-HE)

1999 ◽  
Vol 81 (2) ◽  
pp. 788-794 ◽  
Author(s):  
Marvin E. Ruffner ◽  
Stuart I. Cromarty ◽  
Robin L. Cooper
Keyword(s):  
Synaptic Vesicles ◽  
Neuromuscular Junctions ◽  
Motor Nerve ◽  
Nerve Terminals ◽  
Synaptic Efficacy ◽  
Molting Hormone ◽  
Synaptic Currents ◽  
Site Of Action ◽  
Excitatory Junction Potentials ◽  
High Output

Depression of synaptic efficacy in high- and low-output Drosophila neuromuscular junctions by the molting hormone (20-HE). The molt-related steroid hormone, 20-hydroxyecdysone (20-HE), was applied to muscles 6 and 7 of third instar larval of Drosophila melanogaster neuromuscular junction preparations to examine if rapid, nongenomic responses could be observed as was shown recently to occur in crustacean neuromuscular junctions. At a dose of 10 μM, the excitatory junction potentials were reduced in amplitude within minutes. To elucidate the site of action of the hormone, focal-macropatch recordings of synaptic currents were obtained over the neuromuscular junctions. The results showed that the high-output (Is) and the low-output (Ib) motor nerve terminals, which innervate muscles 6 and 7, released fewer synaptic vesicles for each stimulation while exposed to 20-HE. Because the size and shape of synaptic currents from spontaneous releases did not change, the effects of the 20-HE are presynaptic. The rapid effects of this hormone may account in part for the quiescent behavior associated with molts among insects and crustaceans.

scholarly journals Acetylcholinesterase from the motor nerve terminal accumulates on the synaptic basal lamina of the myofiber.

1991 ◽  
Vol 115 (3) ◽  
pp. 755-764 ◽  
Author(s):  
L Anglister
Keyword(s):  
Basal Lamina ◽  
Ache Activity ◽  
Neuromuscular Junctions ◽  
Motor Nerve ◽  
Synaptic Cleft ◽  
Motor Nerve Terminal ◽  
Nerve Terminals ◽  
Axon Terminals ◽  
Muscle Nerve ◽  
Almost All

Acetylcholinesterase (AChE) in skeletal muscle is concentrated at neuromuscular junctions, where it is found in the synaptic cleft between muscle and nerve, associated with the synaptic portion of the myofiber basal lamina. This raises the question of whether the synaptic enzyme is produced by muscle, nerve, or both. Studies on denervated and regenerating muscles have shown that myofibers can produce synaptic AChE, and that the motor nerve may play an indirect role, inducing myofibers to produce synaptic AChE. The aim of this study was to determine whether some of the AChE which is known to be made and transported by the motor nerve contributes directly to AChE in the synaptic cleft. Frog muscles were surgically damaged in a way that caused degeneration and permanent removal of all myofibers from their basal lamina sheaths. Concomitantly, AChE activity was irreversibly blocked. Motor axons remained intact, and their terminals persisted at almost all the synaptic sites on the basal lamina in the absence of myofibers. 1 mo after the operation, the innervated sheaths were stained for AChE activity. Despite the absence of myofibers, new AChE appeared in an arborized pattern, characteristic of neuromuscular junctions, and its reaction product was concentrated adjacent to the nerve terminals, obscuring synaptic basal lamina. AChE activity did not appear in the absence of nerve terminals. We concluded therefore, that the newly formed AChE at the synaptic sites had been produced by the persisting axon terminals, indicating that the motor nerve is capable of producing some of the synaptic AChE at neuromuscular junctions. The newly formed AChE remained adherent to basal lamina sheaths after degeneration of the terminals, and was solubilized by collagenase, indicating that the AChE provided by nerve had become incorporated into the basal lamina as at normal neuromuscular junctions.

scholarly journals An antiserum specific for cholinergic synaptic vesicles from electric organ.

1980 ◽  
Vol 87 (1) ◽  
pp. 98-103 ◽  
Author(s):  
S S Carlson ◽  
R B Kelly
Keyword(s):  
Synaptic Vesicle ◽  
Synaptic Vesicles ◽  
Membrane Fraction ◽  
Motor Nerve ◽  
Buoyant Density ◽  
Electric Organ ◽  
Rabbit Serum ◽  
Antigenic Determinants ◽  
Nerve Terminals ◽  
Isopycnic Centrifugation

Rabbit antisera to highly purified synaptic vesicles from the electric organ of Narcine brasiliensis, an electric ray, reveal a unique population of synaptic vesicle antigens in addition to a population shared with other electric organ membranes. Synaptic vesicle antigens were detected by binding successively rabbit antivesicle serum and radioactive goat anti-rabbit serum. To remove antibodies directed against antigens common to synaptic vesicles and other electric organ fractions, the antivesicle serum was extensively preadsorbed against an electric organ membrane fraction that was essentially free of synaptic vesicles. The adsorbed serum retained 40% of its ability to bind to synaptic vesicles, suggesting that about half of the antigenic determinants are unique. Vesicle antigens were quantified with a radioimmunoassay (RIA) that utilized precipitation of antibody-antigen complexes with Staphylococcus aureus cells. By this assay, the vesicles, detected by their acetylcholine (ACh) content and the antigens detected by the RIA, have the same buoyant density after isopycnic centrifugation of crude membrane fractions on sucrose and glycerol density gradients. The ratio of ACh to antigenicity was constant across the vesicle peaks and was close to that observed for vesicles purified to homogeneity. Even though the vesicles make up only approximately 0.5% of the material in the original homogenate, the ratio of acetylcholine to vesicle antigenicity could still be measured and also was indistinguishable from that of pure vesicles. We conclude that synaptic vesicles contain unique antigenic determinants not present to any measurable extent in other fractions of the electric organ. Consequently, it is possible to raise a synaptic vesicle-specific antiserum that allows vesicles to be detected and quantified. These findings are consistent with earlier immunohistochemical observations of specific antibody binding to motor nerve terminals.

Structural and functional changes of frog neuromuscular junctions in high calcium solutions

1971 ◽  
Vol 178 (1053) ◽  
pp. 407-415 ◽  
Keyword(s):  
Transmitter Release ◽  
Synaptic Vesicles ◽  
Neuromuscular Junctions ◽  
Motor Nerve ◽  
Secretory Response ◽  
Spontaneous Release ◽  
Functional Changes ◽  
High Calcium ◽  
Release Of Acetylcholine ◽  
Evoked Transmitter Release

When frog muscles are exposed for several hours to a solution of isotonic calcium chloride, the secretory response of the motor nerve terminals to imposed depolarization ultimately fails and the rate of spontaneous release of acetylcholine also declines towards zero. The failure of depolarization-evoked transmitter release is irreversible while spontaneous release reappears, though in highly abnormal fashion, when the muscle is returned to a normal ionic medium. Examination of motor end-plates, during various stages of calcium treatment, shows that there is gradual intra-axonal agglutination of synaptic vesicles which is only very incompletely reversible. This effect is presumably the consequence of gradual entry and intracellular accumulation of calcium ions. Analogous treatment with isotonic magnesium, while resulting in immediate loss of evoked transmitter release, does not lead to progressive agglutination of synaptic vesicles, nor to irreversible impairment of the secretory response of the nerve terminal. The possible relations between structural and functional changes during calcium and magnesium treatment are discussed.

Impairment of Synaptic Vesicle Exocytosis and Recycling During Neuromuscular Weakness Produced in Mice by 2,4-Dithiobiuret

2002 ◽  
Vol 88 (6) ◽  
pp. 3243-3258 ◽  
Author(s):  
You-Fen Xu ◽  
Dawn Autio ◽  
Mary B. Rheuben ◽  
William D. Atchison
Keyword(s):  
Synaptic Vesicle ◽  
Muscle Weakness ◽  
Nerve Stimulation ◽  
Synaptic Vesicles ◽  
Motor Nerve ◽  
Neuromuscular Disorders ◽  
Neuroaxonal Dystrophy ◽  
Nerve Terminals ◽  
Synaptic Vesicle Exocytosis ◽  
Vesicle Exocytosis

Chronic treatment of rodents with 2,4-dithiobiuret (DTB) induces a neuromuscular syndrome of flaccid muscle weakness that mimics signs seen in several human neuromuscular disorders such as congenital myasthenic syndromes, botulism, and neuroaxonal dystrophy. DTB-induced muscle weakness results from a reduction of acetylcholine (ACh) release by mechanisms that are not yet clear. The objective of this study was to determine if altered release of ACh during DTB-induced muscle weakness was due to impairments of synaptic vesicle exocytosis, endocytosis, or internal vesicular processing. We examined motor nerve terminals in the triangularis sterni muscles of DTB-treated mice at the onset of muscle weakness. Uptake of FM1-43, a fluorescent marker for endocytosis, was reduced to approximately 60% of normal after either high-frequency nerve stimulation or K+depolarization. Terminals ranged from those with nearly normal fluorescence (“bright terminals”) to terminals that were poorly labeled (“dim terminals”). Ultrastructurally, the number of synaptic vesicles that were labeled with horseradish peroxidase (HRP) was also reduced by DTB to approximately 60%; labeling among terminals was similarly variable. A subset of DTB-treated terminals having abnormal tubulovesicular profiles in their centers did not respond to stimulation with increased uptake of HRP and may correspond to dim terminals. Two findings suggest that posttetanic “slow endocytosis” remained qualitatively normal: the rate of this type of endocytosis as measured with FM1-43 did not differ from normal, and HRP was observed in organelles associated with this pathway- coated vesicles, cisternae, as well as synaptic vesicles but not in the tubulovesicular profiles. In DTB-treated bright terminals, end-plate potential (EPP) amplitudes were decreased, and synaptic depression in response to 15-Hz stimulation was increased compared with those of untreated mice; in dim terminals, EPPs were not observed during block withd-tubocurarine. Nerve-stimulation-induced unloading of FM1-43 was slower and less complete than normal in bright terminals, did not occur in dim terminals, and was not enhanced by α-latrotoxin. Collectively, these results indicate that the size of the recycling vesicle pool is reduced in nerve terminals during DTB-induced muscle weakness. The mechanisms by which this reduction occurs are not certain, but accumulated evidence suggests that they may include defects in either or both exocytosis and internal vesicular processing.

scholarly journals Interaction between Calcium Chelators and the Activity of P2X7 Receptors in Mouse Motor Synapses

2020 ◽  
Vol 21 (6) ◽  
pp. 2034 ◽  
Author(s):  
Anna Miteva ◽  
Alexander Gaydukov ◽  
Olga Balezina
Keyword(s):  
Neuromuscular Junctions ◽  
Motor Nerve ◽  
Control Level ◽  
Nerve Terminals ◽  
Quantal Content ◽  
Ach Release ◽  
P2x7 Receptors ◽  
Motor Nerve Terminals ◽  
Pannexin 1 ◽  
End Plate

The ability of P2X7 receptors to potentiate rhythmically evoked acetylcholine (ACh) release through Ca2+ entry via P2X7 receptors and via L-type voltage-dependent Ca2+ channels (VDCCs) was compared by loading Ca2+ chelators into motor nerve terminals. Neuromuscular preparations of the diaphragms of wild-type (WT) mice and pannexin-1 knockout (Panx1−/−) mice, in which ACh release is potentiated by the disinhibition of the L-type VDCCs upon the activation of P2X7 receptors, were used. Miniature end-plate potentials (MEPPs) and evoked end-plate potentials (EPPs) were recorded when the motor terminals were loaded with slow or fast Ca2+ chelators (EGTA-AM or BAPTA-AM, respectively, 50 μM). In WT and Panx1−/− mice, EGTA-AM did not change either spontaneous or evoked ACh release, while BAPTA-AM inhibited synaptic transmission by suppressing the quantal content of EPPs throughout the course of the short rhythmic train (50 Hz, 1 s). In the motor synapses of either WT or Panx1−/− mice in the presence of BAPTA-AM, the activation of P2X7 receptors by BzATP (30 μM) returned the EPP quantal content to the control level. In the neuromuscular junctions (NMJs) of Panx1−/− mice, EGTA-AM completely prevented the BzATP-induced increase in EPP quantal content. After Panx1−/− NMJs were treated with BAPTA-AM, BzATP lost its ability to enhance the EPP quantal content to above the control level. Nitrendipine (1 μM), an inhibitor of L-type VDCCs, was unable to prevent this BzATP-induced enhancement of EPP quantal content to the control level. We propose that the activation of P2X7 receptors may provide additional Ca2+ entry into motor nerve terminals, which, independent of the modulation of L-type VDCC activity, can partially reduce the buffering capacity of Ca2+ chelators, thereby providing sufficient Ca2+ signals for ACh secretion at the control level. However, the activity of both Ca2+ chelators was sufficient to eliminate Ca2+ entry via L-type VDCCs activated by P2X7 receptors and increase the EPP quantal content in the NMJs of Panx1−/− mice to above the control level.

Innervation of the ventral diaphragm of the locust (Locusta migratoria)

1977 ◽  
Vol 69 (1) ◽  
pp. 23-32
Author(s):  
M. Peters
Keyword(s):  
Electrical Stimulation ◽  
Electrical Properties ◽  
Electrical Activity ◽  
Neuromuscular Junctions ◽  
Motor Nerve ◽  
Locusta Migratoria ◽  
Posterior Segment ◽  
Muscle Fibres ◽  
Inspiratory Muscles ◽  
Motor Neurones

1. Innervation and some electrical properties of the locust ventral diaphragm were investigated with electrophysiological and histological methods. 2. Muscle fibres are coupled electrically. Electrical stimulation evokes a graded active membrane response. 3. Each segment is innervated by four motor neurones as follows. Two motor neurones are situated in each abdominal ganglion. Branches of their axons supply the ventral diaphragm in the respective and the next posterior segment. 4. This pattern of innervation was confirmed by axonal Co and Ni staining of the motor nerve endings. 5. Neuromuscular junctions are excitatory. EPSPs show summation but no facilitation. 6. Spontaneous electrical activity of the diaphragm is to a certain degree coupled to activity of the main inspiratory muscles.

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