Use of a chemical model to study presynaptic neuromuscular diseases is proposed. Daily administration of small amounts (0.25-1.0 mg/kg) of the disulfide reducing agent 2,4-dithiobiuret (DTB) to rats produces a flaccid, delayed-onset, ascending skeletal muscle paralysis after 4-10 days of treatment. The paralytic state can be maintained for weeks by continued DTB treatment yet animals recover within 5-10 days once treatment is stopped. In animals paralyzed by chronic DTB treatment, neuromuscular transmission is impaired; single twitch and tetanic responses to electrical stimulation of the motor nerve in an in situ contracting hindlimb muscle preparation are decreased, and frequency-dependent fatigue is evident at stimulus frequencies greater than 50 Hz. Twitch and tetanic responses to direct electrical stimulation of the muscle are not affected by DTB. We propose therefore to study neuromuscular transmission in hindlimb muscles removed from DTB-poisoned rats during the onset, time of peak effect and recovery from paralysis using conventional intracellular microelectrode recording techniques. These techniques include recording of nerve-evoked and spontaneous transmitter release, focal extracellular recording of the nerve terminal action potential, iontophoretic application of acetylcholine (ACh) to the end-plate, and two microelectrode voltage-clamp to study decay kinetics of the end-plate currents. We wish to determine: (a) whether DTB depresses nerve-evoked transmitter release, spontaneous release, or both; (b) whether the presynaptic effect of DTB is related to effects on calcium ion concentration within the nerve terminal; (c) whether DTB affects propagation of the nerve action potential into the terminal; (d) whether DTB has a postsynaptic site of action in addition to its presynaptic site and, if so, whether this effect is mediated at the level of the acetylcholine receptor or the receptor-activated ionic channel; (e) whether DTB alters intracellular SH balance; and (g) whether ultrastructural effects are associated with paralysis. By studying neuromuscular transmission during the onset, peak and recovery from paralysis, we can determine which processes in neuromuscular transmission are most sensitive to DTB and, perhaps, whether compensatory changes occur to restore normal transmission. Results from the proposed experiments are anticipated to have three benefits: first, the DTB-treated rat should provide a unique animal model for study of a reversible presynaptic neuromuscular disease; second, DTB-poisoning may prove to have similar characteristics to specific human diseases such as myasthenic syndrome or familial infantile myasthenia and thus studies into the mechanism of action of DTB may increase our knowledge of these disorders; third, the results may shed new light on the role of sulfhydryl groups in ACh release at the neuromuscular junction.
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