Effects of botulinum toxin on neuromuscular transmission in the rat.

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Abstract
Botulinum toxin [from Clostridium botulinum] (BoTx) type A partially blocked spontaneous transmitter release from nerve terminals in the rat. Miniature end-plate potentials (MEPP) were present at all end-plates, initially with a low frequency but increasing with time after poisoning. Their amplitude distribution was at first skew with a predominance of very small MEPP, but after a few days larger than normal MEPP appear. Tetanic nerve stimulation, black widow spider venom [from Latrodectus mactans], the Ca2+-ionophore A 23187 or mechanical damage to nerve terminals increased the frequency of MEPP and altered the amplitude distribution of MEPP towards a normal Gaussian one; the MEPP size approached that seen at normal end-plates. This was seen at any time after poisoning. Nerve stimulation gave rise to end-plate potentials (EPP) of low amplitude and high failure rate. Statistical analysis indicated that evoked release was quantal in nature and followed Poisson statistics, quantum size being initially very small, but after a few days approaching normal size. Short-term tetanic nerve stimulation reversibly increased the quantum content of EPP, and during early stages of paralysis long-term (2 h) stimulation caused an apparently permanent increase in quantum size. Raising the extracellular Ca2+ concentration from 2 to 16 mM increased the frequency of MEPP in normal muscle but not in BoTx poisoned ones. K+-free medium or ouabain, believed to raise the intracellular Ca2+ concentration in nerve terminals, similarly increased MEPP frequency in normal but not in poisoned muscles. When the Ca2+-ionophore A 23187 was used together with high extracellular Ca2+ (> 4 mM), massive release of transmitter occurred from poisoned terminals. The extracellular Ca2+ concentration which caused a certain level of transmitter release in response to nerve impulses was considerably higher at BoTx poisoned end-plates than at normal ones. The slope value for Ca2+ dependence of transmitter release was about 1.5 compared with about 3 at normal end-plates. Tetraethylammonium (TEA) greatly increased the amount of transmitter released by nerve impulses and restored neuromuscular transmission during all stages of poisoning, although it had no effect on spontaneous transmitter release. In the presence of TEA the power relation between Ca2+ concentration and quantum content at the BoTx poisoned end-plate was similar to that seen at normal end-plates. In BoTx poisoning the mechanism for transmitter release apparently has a reduced sensitivity to Ca2+, and the level for activation by intracellular Ca2+ may be elevated. Once the intracellular concentration of Ca2+ is raised to this level by tetanic nerve stimulation, mechanical injury to nerve terminals, the Ca2+-ionophore or the prolongation of the nerve action potential with TEA, augmented transmitter release occurs, similar to that which occurs in normal nerve terminals at a lower level of Ca2+.