Comparison of the Action of Types A and F Botulinum Toxin at the Rat Neuromuscular Junction

Abstract

A comparison of types A and F botulinum toxin (BoTx) was made using both in vivo and in vitro techniques. In vivo mouse and rat 50% lethal dose endpoint levels (LD505) suggest some differences in potency between the toxin types in terms of species susceptibility and route of administration. The mouse subcutaneous (s.c.) LD50 was similar for BoTx A and F. In contrast, the rat intraperitoneal (i.p.) LD50 was 20 times greater for type F than for type A BoTx, based on mouse i.p. LD505. Furthermore, the s.c. LD50, in rats, for type F BoTx was 150 - 200 times greater than that for type A BoTx. To study the effect of these toxins on transmitter release, blockade of neuromuscular transmission was produced in the lower hind limb of the rat by local s.c. injection of either type A or F BoTx. At 1, 3, 7, and 10 days after injection, the extensor digitorum longus (edl) nerve-muscle preparation was excised and analyzed for either alterations in muscle mechanical properties (in vivo) or for alterations in spontaneous and nerve stimulus-evoked quantal transmitter release (in vitro). Muscles from animals treated with type A toxin were completely paralyzed 1 and 3 days after s.c. injection and remained paralyzed 10 days after injection. To have produce complete paralysis with type F BoTx 1 and 3 days after injection, greater than 4 times more F than A mouse i.p. LD50s had to been injected. 7 Muscles from rats receiving these concentrations of F BoTx twitched in response to nerve stimulation by days after injection. Both BoTx types induced a marked decrease in the frequency of miniature end-plate potentials (MEPPs), but Botx type A induced a more dramatic reduction and had a duration of effect on the frequency much longer than did BoTx type F. Between 1 and 3 days after s.c. BoTx injection, nerve impulse-evoked transmitter release was also reduced, regardless of whether the BoTx type was A or F. Three interesting impulse-evoked transmitter release findings are reported and discussed: 1) one day after a type F s.c. injection of 200 or 2000 mouse i.p. LD505 (non-lethal levels in the rat), no end plate potentials (EPPs) could ever be found, while EPPs could always be found after non-lethal injection of type A BoTx, A 2) 3,4 - Diaminopyridine (3,4-DAP), a compound which increases nerve-evoked transmitter release by increasing calcium influx, was more effective in reversing the paralysis of type BoTx injection. 3,4-DAP induced the appearance of asynchronous EPPs in response to nerve stimulation in muscles from animals which were paralyzed by type F BoTx, while it induced twitching in muscle from animals which were paralyzed by type A BoTx, the blockade of nerve-impulse evoked transmitter release was 3) decreased by a reduction of temperature in muscles from animals which received s.c. type A BoTx injection, but was inversely effected by temperature in muscles from animals which received s.c. type F BoTx injection. Also of interest is the finding that amidination of the amino groups (presumably lysine) of the toxins, via ethylacetimidate treatment, increased the potency and paralytic efficacy of only type F BoTx. The results show that type F BoTx differs from type A mainly by its lower potency, by its shorter duration of action, by being less effectively antagonized by 3,4-DAP, by being inversely effected by temperature, and by being more effectively modified by ethylacetimidate treatment.