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.

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.

Comparison of slow larval and fast adult muscle innervated by the same motor neurone

1980 ◽  
Vol 84 (1) ◽  
pp. 103-118
Author(s):  
M. B. Rheuben ◽  
A. E. Kammer
Keyword(s):  
Developmental Stages ◽  
Neuromuscular Junctions ◽  
Motor Nerve ◽  
Thick Filament ◽  
Motor Neurone ◽  
Muscle Fibre Types ◽  
Thin Filaments ◽  
Adult Muscle ◽  
Motor Neurones ◽  
Two Stages

1. Muscles innervated by an identified set of motor neurones were compared between larval and adult stages. 2. The structure of the larval muscle is typically tonic: long sarcomeres, irregular Z-bands, and 10-12 thin filaments around each thick filament. The structure of the adult muscle is phasic: 3-4 micrometers sarcomeres, regular Z-bands, 6-8 thin filaments around each thick filament, and large mitochondrial volume. 3. The tensions produced by these muscles were correspondingly different. The larval twitch was about 7 times slower and the tetanus/twitch ratio 10 times greater than those of the adult. 4. No structural or physiological differences were observed in the neuromuscular junctions of the two stages. 5. The relatively unchanging functional relationship of a single motor neurone with two different muscle fibre types during two developmental stages is compared with the converse situation in which it has been reported that implantation of a different type of motor nerve into a muscle modifies contractile properties.

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.

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.

Application of the zinc iodide-osmium tetroxide impregnation of synaptic vesicles in cephalopod nerves

1969 ◽  
Vol 15 (1) ◽  
pp. 1-16 ◽  
Author(s):  
R. Martin ◽  
J. Barlow ◽  
A. Miralto
Keyword(s):  
Synaptic Vesicles ◽  
Osmium Tetroxide ◽  
Zinc Iodide

Transmitter packaging at frog neuromuscular junctions exposed to anticholinesterases; the role of second-stage acetylcholine loading

1996 ◽  
Vol 76 (4) ◽  
pp. 2614-2625 ◽  
Author(s):  
L. A. Naves ◽  
W. Van der Kloot
Keyword(s):  
Neuromuscular Junction ◽  
Synaptic Vesicles ◽  
Time Course ◽  
Neuromuscular Junctions ◽  
Motor Nerve ◽  
Ache Inhibitor ◽  
Motor Nerve Terminal ◽  
Unexpected Finding ◽  
Extracellular Solution ◽  
Second Stage

1. This investigation was undertaken to explore an unexpected effect of vesamicol, an agent that inhibits active acetylcholine (ACh) uptake into isolated synaptic vesicles. Previous studies at the neuromuscular junction showed that vesamicol makes miniature end-plate currents (MEPCs) smaller only after tens of thousands of quanta have been released. Inhibiting acetylcholinesterase (AChE) makes the MEPCs larger than normal. Our unexpected finding was that with the AChE inhibitor present, adding 2 microM (-)-vesamicol decreases the size of the MEPCs by approximately 30%. The decrease was apparent within 15-30 min, during which only a few thousand quanta had been released. 2. Experimental tests showed that the (-)-vesamicol treatment is unlikely to be acting postsynaptically. For example, it did not slow the rise of MEPCs, which would occur if the endplate receptors were blocked. 3. When AChE was inhibited, three treatments expected to block active choline (Ch) uptake into the presynaptic terminals decreased MEPC size: 1) elevating extracellular K+ to diminish the Na+ electrochemical gradient required for Ch uptake; 2) replacing extracellular Na+ with methylamine+; and 3) adding hemicholinium-3 (HC-3), an inhibitor of the Ch transporter. These treatments did not act by reactivating AChE, blocking the endplate ACh receptor, or by enhancing the desensitization of the ACh receptor. 4. Previous evidence suggests that synaptic vesicles are formed and partially filled with ACh in the cytoplasm and then receive additional ACh when they attach to the active zones, a process that is called second-stage loading. We conclude that the MEPCs are becoming smaller when second-stage loading is blocked by (-)-vesamicol or when the supply of ACh in the cytoplasm of the motor nerve terminal is depleted. 5. To follow the time course of second-stage loading, we used the false transmitter precursor monoethylcholine (MECh). It enters the terminal and is transformed into acetylmonoethylcholine (AMECh). When 200 microM MECh was placed in the extracellular solution and the AChE was inhibited, MEPC size was significantly smaller after 10 min. MEPC size increased once again over a period of time when MECh was removed from the extracellular solution and replaced with Ch. 6. We conclude that at the neuromuscular junction second-stage loading is responsible for loading a significant fraction of the ACh into the quanta.

Interrelationships between Golgi, GERL and synaptic vesicles in the nerve cells of insect and gastropod ganglia

1976 ◽  
Vol 22 (2) ◽  
pp. 435-453
Author(s):  
N.J. Lane ◽  
L.S. Swales
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Complex ◽  
Synaptic Vesicles ◽  
Osmium Tetroxide ◽  
Positive Response ◽  
Schistocerca Gregaria ◽  
Multivesicular Bodies ◽  
Golgi Region ◽  
Limnaea Stagnalis ◽  
Zinc Iodide

In addition to demonstrating synaptic vesicles, staining with the zinc-iodide-osmium tetroxide (ZIO) method reveals the presence of positively reacting GERL membranes in association with the Golgi complex and lysosomes in the nerve cell bodies within ganglia from the locust Schistocerca gregaria and the gastropod molluscs, Limnaea stagnalis and Helix aspersa. A positive response to ZIO occurs in certain Golgi vesicles and saccules, in GERL (Golgi-endoplasmic-reticulum-lysosomes), in multivesicular bodies as well as residual bodies and in small vesicles and cisternae of axonal smooth endoplasmic reticllum (ER). The interrelationships between these organelles are considered in view of the similarity of the ZIO localization to phosphatase-rich sites in the neuronal perikarya and with respect to the possibility that components of the synaptic vesicles are formed in the Golgi region of the cell and migrate via the axonal smooth ER to the synaptic regions.

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