Action of black widow spider venom on quantized release of acetylcholine at the frog neuromuscular junction: dependence upon external Mg2+.

4. In 2-10 mM-Mg2+, the lowest average quantal contents were markedly higher than the fourth power prediction. This discrepancy may occur either because Mg2+ somehow elevates intracellular [Ca2+], or because Mg2+ is itself a weak activator of transmitter release.5. Even at very low rates of evoked release, increasing the stimulus frequency to 5-50 Hz caused a progressive increase in both evoked release and the rate of 'background' quantal release during the interstimulus interval. The frequency-dependent enhancement of both evoked and background release was more pronounced in solutions containing 10 mM-Mg2+ than in solutions containing 2 mm-Mn2+.

Section: Possible Mechanisms For Deviations From Fourth Power In Mg2+mentioning

confidence: 97%

Section: Possible Mechanisms For Deviations From Fourth Power In Mg2+mentioning

confidence: 99%

Calcium dependence of evoked transmitter release at very low quantal contents at the frog neuromuscular junction.

Andreu¹,

Barrett²

1980

“…This solution was renewed once or twice during 10 min, then BWSV (0 5 glands from Italian spiders or 1 gland from American spiders) or a-LTX (0 5-1 ag) was added to the chamber, the bath gently stirred and the preparation incubated for 25-35 min. Quantal secretion is not stimulated under this condition (Misler & Hurlbut, 1979;Misler & Falke, 1987), and the toxin can be allowed to penetrate the muscle completely and bind to its receptor (Valtorta, Madeddu, Meldolesi & Ceccarelli, 1984). A wellsynchronized discharge of quanta from the various junctions in the muscle was started by applying Mg2+ and stopped by withdrawing it, and the rate of discharge was regulated by controlling the Mg2+ concentration (Misler & Hurlbut, 1979;Misler & Falke, 1987) or the temperature .…”

Section: Methodsmentioning

confidence: 99%

Correlation between quantal secretion and vesicle loss at the frog neuromuscular junction.

Hurlbut¹,

Iezzi²,

Fesce³

et al. 1990

Self Cite

SUMMARY1. We measured the rate of occurrence of miniature endplate potentials (MEPPs) at identified endplates in frog cutaneus pectoris muscles treated with crude black widow spider venom (BWSV) or purified ac-latrotoxin (a-LTX) in calcium-free solutions, and we examined the relationship between the length of the nerve terminal and the total number of quanta secreted, and the relationship between the number of quanta secreted and the number of vesicles remaining at different times.2. The venom, or toxin, was applied in a modified Ringer solution with tetrodotoxin, 1 mM-EGTA and no divalent cations, and quantal secretion was started by applying Ca2+-free solutions with Mg2+. This was done to synchronize the quantal discharge at the various junctions in a muscle. Ringer solution was applied after the MEPP rate had declined to low levels, and then the muscle fibre was injected with Lucifer Yellow, the endplate stained for acetylcholinesterase and the length of the nerve terminal and the length of a sarcomere were measured on the fluorescent fibre.3. The total number of quanta secreted by a terminal was measured under a wide variety of experimental conditions: the weights of the frogs ranged from 13 to 68 g, the temperature from 9 to 28 0C, and the concentration of Mg2+ from 2 to 10 mM. In one series of experiments the Mg2+ was withdrawn after 3-4 min and reapplied 35-40 min later in order to divide the total output of quanta into two approximately equal bouts of secretion that were well separated in time.4. The total number of MEPPs recorded at a junction was loosely correlated with the length of its nerve terminal, but it was not affected by the temperature, the concentration of Mg2+ or the division of secretion into well-separated bouts of quantal release. The average total secretion per unit length was about 3700 quanta/sarcomere or about 1200 quanta/,#m.5. The average time course of quantal secretion per micrometre of terminal was determined at single junctions in muscles held at 22-23 0C or at 9-10 TC. Other muscles were fixed at various times during the course of secretion at each temperature and the number of synaptic vesicles remaining in cross-sections of the terminals were counted on electron micrographs. The number of vesicles remaining MS 7979 W. P. HURLBUT AND OTHERS per micrometre of terminal was determined from the number per cross-section and the section thickness.6. The average number of vesicles remaining per micrometre of terminal was negatively correlated with the average number of quanta secreted per micrometre; the slope of the regression line was -14 to -12 vesicles/quantum and its correlation coefficient was -0-91. It is concluded that a quantum of transmitter is derived from a single vesicle.

“…Since black widow spider venom and a-LTx probably increase the permeability of nerve terminals to monovalent and divalent cations (Finkelstein, Rubin & Tzeng, 1976;Misler & Hurlbut, 1979;Nicholls, Rugalo, Scott & Meldolesi, 1982;Fritz & Mauro, 1982;Meldolesi, Scheer, Madeddu & Wanke, 1986;Misler & Falke, 1987), all these effects of extracellular Mg2+ may result from its entry into the axoplasm. The first effect might result from interactions between increased levels of Mg2" and the cytoskeleton, and the second from the ability of high concentrations of Mg2+ to stabilize clathrin coats (Woodward & Roth, 1979) and to uncouple the clathrin 'uncoating ATPase' (Braell, Schlossman, Schmid & Rothman, 1984 …”

Section: Electrophysiologymentioning

confidence: 99%

Effect of alpha‐latrotoxin on the frog neuromuscular junction at low temperature.

Ceccarelli¹,

Hurlbut²,

Iezzi³

1988

Self Cite

SUMMARY1. a-Latrotoxin (a-LTx) was applied to frog cutaneus pectoris muscles bathed at 1-3°C in either Ringer solution, Ca2"-free Ringer solution with 1 mM-EGTA and 4 mM-Mg2+ or Ringer solution plus 4 mM-Mg2+, and its effects on miniature end-plate potential (MEPP) frequency, nerve terminal ultrastructure and uptake of horseradish peroxidase (HRP) were studied.2. Large concentrations (2 ,ug/ml) of a-LTx increased MEPP rates to levels above 100/s at all junctions, but the time course of the increases depended upon the divalent cation content of the bathing solution. However, similar numbers of MEPPs (0-3-407 x 106) were recorded at all junctions during 2 h of secretion.3. Nerve terminals exposed to a-LTx for 2 h lost 60-75 % of their synaptic vesicles and were swollen; their presynaptic membranes were deeply infolded and they often contained many large vesicular structures. Terminals in Ringer solution retained the largest number of synaptic vesicles; terminals in Ringer solution plus Mg2+ swelled the least and contained the largest number of coated vesicles. The average number of synaptic vesicles lost was approximately equal to the average number of MEPPs recorded.4. Few vesicles became loaded with HRP when this extracellular tracer was present in the bathing solution and the muscles were fixed near the peak of secretion.5. When the terminals were warmed to 20°C, those in the Caa2+-free solution with Mg2+ secreted additional quanta and lost almost all their residual vesicles; those in Ringer solution without Mg2+ secreted few additional quanta and retained most of their residual vesicles.6. These results suggest that recycling was blocked at these terminals and that for each quantum secreted a vesicle became permanently incorporated into the axolemma.