We addressed the hypothesis that intraplantar Botulinum toxin B (rimabotulinumtoxin B: BoNT-B) has an early local effect upon peripheral afferent terminal releasing function and over time will be transported to the central terminals of the primary afferent. Once in the terminals it will cleave synaptic protein, block spinal afferent transmitter release and thereby prevent spinal nociceptive excitation and behavior. In mice, C57Bl/6 males, intraplantar BoNT-B (1U), given unilaterally into the hind paw had no effect upon survival or motor function but ipsilaterally decreased: i) intraplantar formalin evoked flinching; ii) intraplantar capsaicin evoked plasma extravasation in the hindpaw measured by Evans blue in the paw; iii) intraplantar formalin evoked dorsal horn SP release (NK1 receptor internalization); iv) intraplantar formalin evoked dorsal horn neuronal activation (cFos); v) ipsilateral DRG VAMP; vi) ipsilateral SP release otherwise evoked bilaterally by intrathecal capsaicin; vii) ipsilateral activation of cFos otherwise evoked bilaterally by intrathecal substance P. These results indicate that BoNT-B after unilateral intraplantar delivery is taken up by the peripheral terminal, is locally active (blocking plasma extravasation), is transported to the ipsilateral DRG to cleave VAMP and is acting presynaptically to block release from the spinal peptidergic terminal. The observations following intrathecal SP offer evidence for a possible transsynaptic effect of intraplantar BoNT. These results provide robust evidence that peripheral BoNT-B can alter peripheral and central terminal release from a nociceptor and attenuate downstream nociceptive processing via a presynaptic effect, with further evidence suggesting a possible postsynaptic effect.

Intrathecal (IT) studies have shown that several voltage sensitive calcium channels (VSCCs), such as the L-, N- and T-type may play roles in nociception and that of these only the N-type regulates primary afferent substance P (SP) release. However, the actions of other VSCCs at the spinal level are not well known. We investigated the roles of spinal P/Q- and R-type VSCCs, by IT administration of R-type (SNX-482) and P/Q-type (ω-agatoxin IVA) VSCC blockers on intraplantar formalin-evoked flinching, SP release from primary afferents and c-Fos expression in spinal dorsal horn. Intraplantar injection of formalin (2.5%, 50 µL) produced an intense, characteristic biphasic paw flinching response. In rats with IT catheters, IT SNX-482 (0.5 µg) reduced formalin-evoked paw flinching in both phase 1 and 2 compared with vehicle. Intraplantar formalin caused robust neurokinin 1 receptor (NK1r) internalization (indicating SP release) and c-Fos expression in the ipsilateral dorsal horn, which were blocked by IT SNX-482. IT ω-agatoxin IVA (0.03, 0.125 and 0.5 µg) did not reduce formalin-evoked paw flinching or c-Fos expression at any doses, with higher doses resulting in motor dysfunction. Thus, we demonstrated that blockade of spinal R-type, but not P/Q type VSCCs attenuated formalin-induced pain behavior, NK1r internalization and c-Fos expression in the superficial dorsal horn. This study supports a role for Cav2.3 in presynaptic neurotransmitter release from peptidergic nociceptive afferents and pain behaviors.

Delta-opioid receptors (DOR) are present in the superficial dorsal horn and are believed to regulate the release of small afferent transmitters as evidenced by the effects of spinally delivered delta-opioid preferring peptides. Here we examined the effects, a selective nonpeptidic DOR agonist, in three preclinical pain models, acute thermal escape, intraplantar carrageenan-tactile allodynia, and intraplantar formalin flinches, and on the evoked release of substance P (SP) from small primary afferents. Rats with chronic intrathecal catheters received intrathecal vehicle or SNC80 (100 or 200 mg). Intrathecal SNC80 did not change acute thermal latencies or carrageenan-induced thermal hyperalgesia.However, SNC80 attenuated carrageenan-induced tactile allodynia and significantly reduced both phase 1 and phase 2 formalininduced paw flinches, as assessed by an automatic flinch counting device. These effects were abolished by naltrindole (3 mg/kg i.p.), a selective DOR antagonist, but not CTOP (10 mg i.t.), a selective MOR antagonist. Furthermore, intrathecal SNC80 (200 mg) blocked formalin-induced substance P release otherwise evoked in the ispilateral superficial dorsal horn as measured by NK1 receptor internalization. In conclusion, intrathecal SNC80 alleviated pain hypersensitivity after peripheral inflammation in a fashion paralleling its ability to block peptide transmitter release from small peptidergic afferents, which by its pharmacology appears to represent an effect mediated by a spinal DOR.