Effects of A2 type botulinum toxin on spontaneous miniature and evoked transmitter release from the rat spinal excitatory and inhibitory synapses.

We observed effects of newly developed A2 type botulinum toxin (A2NTX) on spontaneous miniature and evoked transmitter release from inhibitory (glycinergic or GABAergic), or excitatory (glutamatergic) nerve terminals in rat spinal cord, by use of 'synaptic bouton' preparations, under voltage-clamp condition. A2NTX (0.1-1 pM) initially augmented and then decreased amplitude and frequency of spontaneous miniature release of glycine or GABA (mIPSCs) concentration-dependently. At an increased concentration (1-10 pM), A2NTX suppressed the amplitude of glutamatergic mEPSCs. The rank order of the inhibitory effects was glycinergic > GABAergic >> glutamatergic synapses. Focal electrical stimulation of 'synaptic boutons' elicited eIPSC or eEPSC with larger amplitude and low failure rate (Rf). A2NTX (0.01-1 pM) initially enhanced the amplitude or decreased the failure rate of eIPSC or eEPSC, and then almost completely abolished the generation of eIPSC or eEPSC. The action of A2NTX on the evoked transmitter release was partially reversible. The rank order of the inhibitory effects on the amplitude or Rf were glycinergic eIPSC ≥ GABAergic eIPSC > glutamatergic eEPSCs. Excess extracellular K(+) or Ca(2+) (excess K(+) or Ca(2+)), and 4-AP restored spontaneous miniature glycinergic, GABAergic or glutamatergic postsynaptic currents suppressed by A2NTX. We conclude that A2NTX inhibits spontaneous miniature release at 0.1-10 pM and evoked release at 0.01-1 pM in rat spinal cord, and the inhibition was much efficient in the evoked rather than the spontaneous miniature release. Excess K(+), 4-AP and excess Ca(2+), which can raise the intracellular Ca(2+) concentration via the activation of voltage-dependent Ca(2+) channels, rescue the transmission suppressed by A2NTX poisoning, suggesting the transmitter release machinery became less sensitive to intracellular Ca(2+) in A2NTX poisoned 'synaptic boutons'.