Botulinum Toxin Type A Exerts Direct Trans-Synaptic Action at Bilateral Spinal Nociceptive Circuits

Botulinum toxin type A (BoNT-A) induces a bilateral analgesic effect following unilateral injection in rodent bilateral or mirror pain models. This occurs either by indirect plasticity-related actions, or by the toxin's direct central action in bilateral spinal circuits. Herein, we aimed to resolve this question by assessing the role of trans-synaptic toxin traffic in a bilateral inflammatory pain model. The analgesic effect of the toxin was examined in rats pre-treated with unilateral intraplantar BoNT-A (7 U/kg) and subsequently challenged with bilateral carrageenan-evoked hind-paw inflammation (2%, 50 µL/paw, 6 days post BoNT-A). Specific neutralizing antitoxin injected into the lumbar intrathecal space (2 IU, 24 h post BoNT-A), aimed at preventing the spinal trans-synaptic traffic of BoNT-A, abolished its bilateral analgesic effect. The toxin trans-synaptic effect was associated with reduced c-Fos neuronal activation and BoNT-A-mediated cleavage of synaptosomal-associated protein 25 (SNAP-25) in the bilateral dorsal horn. Here, we showed that, in bilaterally occurring pain, BoNT-A exerts a direct contralateral analgesic action extending beyond the level of the dorsal root ganglion sensory neuron that directly links the hindlimb injection site to the primary sensory region. This points to the crucial role of the toxin's central trans-synaptic traffic, and its direct action at propriospinal nociceptive circuits in its pain-relieving efficacy.