Abstract
Botulinum neurotoxin B (BoNT-B) mediates proteolytic cleavage of VAMP I/II (synaptobrevins I/II), which prevents vesicle-membrane fusion and blocks neurotransmitter release. In the present study, we investigated the effects of BoNT-B on neurotransmitter release in vivo from spinal primary afferent sensory fibers and the effects of this blockade on nociception. With intrathecally (IT) delivered BoNT-B in C57B/l6 mice, we characterized the effects of such block on the release of substance P (SP) from spinal afferent nociceptors (as measured by neurokinin-1 receptor, NK1-R, internalization), spinal neuronal activation (as indicated by spinal C-Fos expression) and nociceptive behavior after intraplantar (IPLT) formalin. In addition, we investigated the effect of IT BoNT-B on spinal nerve ligation-induced tactile allodynia. A single percutaneous IT injection of BoNT-B 0.5 U at 2 or 5 days prior to IPLT formalin reduced NK1-R internalization and C-Fos expression. These effects correlated with BoNT-B cleavage of VAMPI/II protein in tissue lysate. IT BoNT-B also produced a corresponding reduction in phase 2 of formalin-evoked flinching behavior for over 30 days after IT injection. In mice with spinal nerve ligation (SNL), tactile allodynia was observed, which was attenuated by IT BoNT-B 0.5 U over the next 15 days, as compared to vehicle animals. These effects were observed without effects upon motor function. The specificity of the IT BoNT-B effect is indicated by: i) IT co-injection of BoNT-B and anti-BoNTB antibody prevented effects on SP release, and ii) IT BoNT-B 50 U in the Sprague Dawley rats showed no effect on formalin-evoked flinching or SNL-induced tactile allodynia, which is consistent with rat resistance to BoNT-B. IT BoNT-B blocks transmitter release from spinal primary afferents, and attenuates inflammatory nociceptive response and spinal nerve injury-induced neuropathic pain, in the absence of motor impairment. These observations provide an initial assessment of the ability of IT BoNT-B to regulate spinal nociceptive processing.
Highlights
Botulinum neurotoxins (BoNTs) are metalloproteases produced by Clostridium botulinum
In order to demonstrate the functional significance of Botulinum neurotoxin B (BoNT-B) effects, we examined its effects upon IPLT formalin-induced flinching behavior and spinal nerve ligation-induced tactile allodynia
IT BoNT-B 0.5 U was Characterization of NK1-R expression and internalization Substance P binds to neurokinin-1 receptors
Summary
Botulinum neurotoxins (BoNTs) are metalloproteases produced by Clostridium botulinum. They mediate proteolytic cleavage of protein subunits in the family of soluble N-methylaleimidesensitive attachment protein receptor (SNARE) proteins in the synaptic terminal. Botulinum neurotoxin serotypes B, F, and G cleave VAMP/synaptobrevin protein [1]. Such enzymatic cleavage results in inhibition of vesicle-membrane fusion and prevents the release of neurotransmitters. Despite the widespread role of SNARE proteins in CNS and PNS, there has been little work on the effects of BoNTs on neurotransmitter release in systems other than neuromuscular junction. The majority of this work has focused on ex vivo systems and the effects on behavior have been described in a limited fashion
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