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.

Abstract

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~ in the dark, ZIO reacts at pH 4.2-5.0 fairly selectively with the matrix of synaptic vesicles. -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 -52%; 2 min of rest restores this number to 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 < partially ZIO-positive < completely ZIO-positive. The average volume of ZIOpositive 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 ZIOpositive 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. KEY WORDS syrlaptic vesicles 9 insectThe zinc iodide-osmium tetroxide method (ZIO), neuromuscular junction turnover 9 ZIO reaction first introduced by Maillet (33) into the field of intravesicular proteins neurohistochemistry, has been demonstrated by J. CaLL BmLOGY 9 The Rockefeller University Press 9

The effect ofin vivo stimulation on the cytology of neuromuscular junctions of locust flight muscles

“…There appears to be a good correlation between the events reported here and those reported to occur in other systems for stimulating nerve-motor synapses in vivo (Koenig et al, 1983;Palacios-Pru et al, 1983) and in vitro (Reinecke & Walther 1978Botham et al, 1979a, b;Maxwell, 1981;Titmus, 1981). The present system is thus a useful model for the study not only of synaptic transmission but also of the effects of various drugs and neurotoxins on this process.…”

“…Our studies demonstrate that the normal unflown flight neuromuscular junctions of Schistocerca gregaria are similar in ultrastructure to the resting neuromuscular junctions of other insect muscle (Reinecke & Walther, 1978Botham et al, 1979a, b;Titmus, 1981). In particular they resemble the excitatory motor axon terminals of the locust jumping leg retractor unguis muscle (Titmus, 1981) and the locust extensor tibiae muscle (Botham et al, 1979a) rather than the inhibitory terminals of locust muscle (Titmus, 1981).…”

The effect ofin vivo stimulation on the cytology of neuromuscular junctions of locust flight muscles

“…There appears to be a good correlation between the events reported here and those reported to occur in other systems for stimulating nerve-motor synapses in vivo (Koenig et al, 1983;Palacios-Pru et al, 1983) and in vitro (Reinecke & Walther 1978Botham et al, 1979a, b;Maxwell, 1981;Titmus, 1981). The present system is thus a useful model for the study not only of synaptic transmission but also of the effects of various drugs and neurotoxins on this process.…”

“…Our studies demonstrate that the normal unflown flight neuromuscular junctions of Schistocerca gregaria are similar in ultrastructure to the resting neuromuscular junctions of other insect muscle (Reinecke & Walther, 1978Botham et al, 1979a, b;Titmus, 1981). In particular they resemble the excitatory motor axon terminals of the locust jumping leg retractor unguis muscle (Titmus, 1981) and the locust extensor tibiae muscle (Botham et al, 1979a) rather than the inhibitory terminals of locust muscle (Titmus, 1981).…”

Ultrastructure of identified fast excitatory, slow excitatory and inhibitory neuromuscular junctions in the locust

“…Other studies on phasic NMJs in locusts have shown that higher frequencies of stimulation (20, 50 and 100 Hz) cause a greater reduction of vesicle numbers than that reported here without a greater reduction in EJP size (80-90%) (McKinlay & Usherwood, 1973;Botham et al, 1978a, b;Reinecke & Walther, 1978). These studies also report that vesicles with reduced volume appear in the endings immediately after stimulation.…”

“…From this study and others (McKinlay & Usherwood, 1973;Botham et al, 1978a, b;Reinecke & Walther, 1978), it appears that vesicle membrane movements during neuromuscular transmission in insects resemble those described at vertebrate central and peripheral synapses and in glandular cells (Heuser & Reese, 1973;review by Holtzman, 1977). Exocytosis is indicated by synaptic vesicle depletion in terminals during intense stimulation of the axons.…”

Structural plasticity of optic synapses in the rat suprachiasmatic nucleus: adaptation to long-term influence of light and darkness

“…Similarly, Lynch (1982) found larger mitochondria in neuromuscular synapses after long-term, low frequency stimulation. Conversely, shortterm, drastic stimulation causes an increase in number but not in area of mitochondrial profiles as reported, e.g., by Jones and Kwanbunbumpen (1970), Heuser and Reese (1973) and Reinecke and Walters (1978). However, continuous, high frequency activation of synapses in the hippocampal subfield CA1 for 10 min results in an increase in area of presynaptic mitochondria (Chang and Greenough 1984).…”

The effect ofin vivo stimulation on the cytology of neuromuscular junctions of locust flight muscles

“…There appears to be a good correlation between the events reported here and those reported to occur in other systems for stimulating nerve-motor synapses in vivo (Koenig et al, 1983;Palacios-Pru et al, 1983) and in vitro (Reinecke & Walther 1978Botham et al, 1979a, b;Maxwell, 1981;Titmus, 1981). The present system is thus a useful model for the study not only of synaptic transmission but also of the effects of various drugs and neurotoxins on this process.…”

“…Our studies demonstrate that the normal unflown flight neuromuscular junctions of Schistocerca gregaria are similar in ultrastructure to the resting neuromuscular junctions of other insect muscle (Reinecke & Walther, 1978Botham et al, 1979a, b;Titmus, 1981). In particular they resemble the excitatory motor axon terminals of the locust jumping leg retractor unguis muscle (Titmus, 1981) and the locust extensor tibiae muscle (Botham et al, 1979a) rather than the inhibitory terminals of locust muscle (Titmus, 1981).…”

Ultrastructure of identified fast excitatory, slow excitatory and inhibitory neuromuscular junctions in the locust

“…Other studies on phasic NMJs in locusts have shown that higher frequencies of stimulation (20, 50 and 100 Hz) cause a greater reduction of vesicle numbers than that reported here without a greater reduction in EJP size (80-90%) (McKinlay & Usherwood, 1973;Botham et al, 1978a, b;Reinecke & Walther, 1978). These studies also report that vesicles with reduced volume appear in the endings immediately after stimulation.…”

“…From this study and others (McKinlay & Usherwood, 1973;Botham et al, 1978a, b;Reinecke & Walther, 1978), it appears that vesicle membrane movements during neuromuscular transmission in insects resemble those described at vertebrate central and peripheral synapses and in glandular cells (Heuser & Reese, 1973;review by Holtzman, 1977). Exocytosis is indicated by synaptic vesicle depletion in terminals during intense stimulation of the axons.…”

Structural plasticity of optic synapses in the rat suprachiasmatic nucleus: adaptation to long-term influence of light and darkness

“…Similarly, Lynch (1982) found larger mitochondria in neuromuscular synapses after long-term, low frequency stimulation. Conversely, shortterm, drastic stimulation causes an increase in number but not in area of mitochondrial profiles as reported, e.g., by Jones and Kwanbunbumpen (1970), Heuser and Reese (1973) and Reinecke and Walters (1978). However, continuous, high frequency activation of synapses in the hippocampal subfield CA1 for 10 min results in an increase in area of presynaptic mitochondria (Chang and Greenough 1984).…”

The effect ofin vivo stimulation on the cytology of neuromuscular junctions of locust flight muscles

“…There appears to be a good correlation between the events reported here and those reported to occur in other systems for stimulating nerve-motor synapses in vivo (Koenig et al, 1983;Palacios-Pru et al, 1983) and in vitro (Reinecke & Walther 1978Botham et al, 1979a, b;Maxwell, 1981;Titmus, 1981). The present system is thus a useful model for the study not only of synaptic transmission but also of the effects of various drugs and neurotoxins on this process.…”

“…Our studies demonstrate that the normal unflown flight neuromuscular junctions of Schistocerca gregaria are similar in ultrastructure to the resting neuromuscular junctions of other insect muscle (Reinecke & Walther, 1978Botham et al, 1979a, b;Titmus, 1981). In particular they resemble the excitatory motor axon terminals of the locust jumping leg retractor unguis muscle (Titmus, 1981) and the locust extensor tibiae muscle (Botham et al, 1979a) rather than the inhibitory terminals of locust muscle (Titmus, 1981).…”

Ultrastructure of identified fast excitatory, slow excitatory and inhibitory neuromuscular junctions in the locust

“…Other studies on phasic NMJs in locusts have shown that higher frequencies of stimulation (20, 50 and 100 Hz) cause a greater reduction of vesicle numbers than that reported here without a greater reduction in EJP size (80-90%) (McKinlay & Usherwood, 1973;Botham et al, 1978a, b;Reinecke & Walther, 1978). These studies also report that vesicles with reduced volume appear in the endings immediately after stimulation.…”

“…From this study and others (McKinlay & Usherwood, 1973;Botham et al, 1978a, b;Reinecke & Walther, 1978), it appears that vesicle membrane movements during neuromuscular transmission in insects resemble those described at vertebrate central and peripheral synapses and in glandular cells (Heuser & Reese, 1973;review by Holtzman, 1977). Exocytosis is indicated by synaptic vesicle depletion in terminals during intense stimulation of the axons.…”

Structural plasticity of optic synapses in the rat suprachiasmatic nucleus: adaptation to long-term influence of light and darkness

“…Similarly, Lynch (1982) found larger mitochondria in neuromuscular synapses after long-term, low frequency stimulation. Conversely, shortterm, drastic stimulation causes an increase in number but not in area of mitochondrial profiles as reported, e.g., by Jones and Kwanbunbumpen (1970), Heuser and Reese (1973) and Reinecke and Walters (1978). However, continuous, high frequency activation of synapses in the hippocampal subfield CA1 for 10 min results in an increase in area of presynaptic mitochondria (Chang and Greenough 1984).…”