Restoration of transmitter release in botulinum-poisoned skeletal muscle

Abstract

Botulinum toxin (BoTx) blocks acetylcholine (ACh) release from motor nerve terminals without affecting impulse conduction or ACh synthesis (see review by Simpson13). Since transmitter release is a calcium requiring process s it was of interest to examine the effect of calcium ions on the release process in BoTx-poisoned nerve terminals. In the present study we have examined the effects of the calcium ionophore A-23187 on spontaneous transmitter release and of tetraethylammonium (TEA) and guanidine on neurally evoked release. The results show that the aforementioned drugs, which are all believed to increase the intracellular concentration of calcium in nerve terminals, restore transmitter release to almost normal levels. The experiments were made in vitro on the extensor digitorum longus (EDL) muscle of adult male Sprague-Dawley rats with a body weight of about 180 g. BoTx type A was given as a single injection of 0.25 ml s.c. into the anterolateral region of the right hirtdleg. The toxin produced complete paralysis of the leg within 18 h which lasted for at least 3 weeks. At 2-9 days after BoTx the EDL muscle was removed from animals under ether anaesthesia and placed in a constant temperature bath (29 °C for mechanical recording and 37 °C for intracellular recording) and perfused with oxygenated medium as described by Liley 9. The nerve to the muscle was stimulated at 0.1 Hz by the use of a glass capillary suction electrode and supramaximal (8 V) current pulses of 0.05 msec duration. Isometric twitch tension was recorded with a Grass FT-03 transducer connected to an ink-writing oscillograph. The resting tension of the muscle was adjusted to give maximal twitch response. Conventional intracellular microelectrodes were used to record spontaneous transmitter release as miniature endplate potentials (mepps) and neurally evoked transmitter release as endplate potentials (epps). The calcium ionophore A-23187 (Eli Lilly Co.) was dissolved in ethanol and added to the bathing solution to give a final concentration of 10--:' M. TEA was used as the chloride and guanidine as the hydrochloride.