Acute high intensity stimuli lead to a pain state referred to the site of stimulation. This pain state reflects the intensity-dependent activation of populations of small primary afferents (A∂ and C fibers) by high intensity thermal or mechanical stimuli, which release excitatory transmitters (amino acids and peptides) from their terminals in the spinal dorsal horn. These transmitters activate second order spinal dorsal horn neurons that project supraspinally [97]. Absent the stimulus, the afferent traffic resolves and the pain sensation abates.

Intraplantar delivery of both BoNTs, A1 and B1, at doses that do not obstruct motor function, have no effect upon the behavioral escape response otherwise evoked by acute high intensity thermal or mechanical stimuli (pin prick), or chemical stimulation such as evoked paw withdrawal or local chemical irritants (e.g., phase 1 of the biphasic flinching response otherwise evoked by intraplantar or orofacial formalin) [66,67,68,69,70,71,72,73]. It is important to emphasize that nociceptive threshold testing must occur in the absence of disabling motor function. Such end points would include absence of changes driven by non-noxious stimulation, including corneal touch-evoked blink, hind paw placing and stepping reflexes, weight bearing or symmetrical ambulation. Adhering to such robust end points would indicate that any observed changes in escape latency or pain thresholds were not the result of a failure in the motor-dependent ability to produce the analgesic end point (e.g., paw withdrawal/flinching) [66,67,71,98]. It should be noted that in general the doses employed in these studies are typically in the range of 0.5–1 U, where 1 U is the dose that leads to mortality in half of the mice upon intraperitoneal injection. This reflects upon the fact that the intraplantar or subcutaneous injections result in a restricted redistribution as compared to that achieved with intraperitoneal delivery [67,84,99].

In human studies, subcutaneous BoNT/A1, as in the preclinical studies, had no effect upon normal thermal or mechanical pain thresholds in quantitative sensory testing paradigms [57,61,62,63]. As will be reviewed further below, given the effects of BoNT/A1 and BoNT/B1 on afferent transmitter release, these observations, showing no change in acute thresholds, are unexpected.

In the face of tissue injury and inflammation, the associated pain state is characterized by: i) an ongoing sensation that persists in the absence of the injuring stimulus; and, ii) the presence of a hyperalgesia where an innocuous or mildly noxious stimulus is perceived to be aversive or noxious (e.g., hyperalgesia). Typically, once the underlying pathology is resolved (e.g., wound healing, resolution of inflammation), the pain and hyperalgesia may also resolve. The ongoing pain state and enhanced responsiveness reflects two elements. First, nociceptive afferents innervating the damaged/inflamed tissue are acted upon by active factors released by vascular cells (neutrophils, lymphocytes, platelets) and resident cells of the innate immune response (macrophages and mast cells) [100]. These factors act upon eponymous receptors expressed by many small afferents to depolarize the terminals leading to continuous afferent input and an enhanced response of the terminal to subsequent afferent stimulation [101]. This terminal sensitization is secondary to the activation of terminal kinases which phosphorylate terminal receptors (such as for the bradykinin receptor) [102] and ion channels (e.g., TRPV1, voltage gated sodium and calcium channels) [103,104,105,106,107]. Secondly, spinal systems in the face of ongoing high frequency small afferent activation can display an enhanced input-output function, referred to as wind up and/or central sensitization [101,108]. The pharmacology of this central facilitation is equally complex and reflects: i) a progressive depolarization of the second order neuron; ii) activation (phosphorylation) of spinal kinases that enhance the reactivity of the second order membrane by phosphorylating membrane channels and receptors [109,110,111]; iii) changes in the transport of various excitatory receptor and channel subunits to the membrane [112,113,114], and iv) activation of dorsal horn non-neuronal (astrocyte and microglia) cells leading to release of a variety of pro excitatory products [115,116]. These cascades are ubiquitous and relevant to all inflammatory states, including those arising from the skin, muscle, bone, and visceral tissues such as the gut and the meninges. These pain states have several important characteristics. First, pain states arising from viscera (e.g., irritable bowel) and bone (e.g., cancer), for example, display a referred pain component, in which the visceral/bone afferent input is referred to the superficial (cutaneous) dermatome which projects by a somatic afferent to the same spinal second order neurons. This referral model is particularly relevant to phenomena such as migraine, wherein meningeal afferents activated by local meningeal inflammatory processes activate neurons in the nucleus caudalis that also receive input from cutaneous cranial afferents [117]. Second, it has become appreciated that, in the face of persistent inflammation, the inflammatory pain phenotype may progress to one that involves nerve injury. In the case of joint inflammation, pain may continue after resolution of the inflammation. The mechanism of this transition to a chronic pain state is not well understood, but several changes are relevant. Persistent peripheral inflammation may lead to: i) increased expression of ATF-3 in the dorsal root ganglion (indicative of an injury response); ii) spinal cord glial activation [118,119]; and, iii) peripheral terminals may begin to express nerve injury epitopes suggestive of sprouting [120]. Thus, mechanisms of pain associated with tissue and nerve injury may occur in conditions such as osteoarthritis where inflammatory markers (joint volume, neutrophils) may be minimally present [121,122,123].

Unilateral intraplantar delivery of BoNT/A1 and /B1 at doses of 0.5 U or less exhibit a local, typically homolateral anti-hyperalgesia as measured by thermal and mechanical thresholds in rodent models of inflammation (e.g., intraplantar carrageenan, or formalin) [66,67,76,78]. These effects, where reported, have an onset of 2 to 24 hours and a duration of action in excess of 14 to 21 days [67]. In horses, intra-articular BoNT/A1 can attenuate lameness in a model of acute synovitis [124]. Similarly, in dogs with chronic osteoarthritis, intra-articular BoNT/A1 was found to reduce indices of pain [81,82,83]. The typical ipsilateral effect emphasizes that the BoNTs did not exert a bilateral effect and thus were not likely due to a simple systemic redistribution at the employed doses. We note that, while ipsilaterally delimited effects have been observed, particularly at lower doses, bilateral anti-hyperalgesia [78,88,89] and bilateral muscle relaxation [125] after unilateral delivery into the paw have been reported. The likelihood of multi-segmental effects on motor and sensory function in humans has been expertly reviewed and discussed in detail elsewhere [126].

In humans, myofascial pain syndromes, characterized by activation of multiple bilateral local pain state trigger points, have been shown to be attenuated by local delivery of BoNT/A1 [127,128]. However, systematic trials appear to be lacking [129,130]. Intra-articular delivery of BoNT/A1 has been shown to have attenuating effects upon inflammatory and chronic joint pain in the shoulder [37,38,39,40,131]. Paraspinal muscular application of BoNT/A1 reduced refractory low back pain in the majority of patients [53].

To further characterize the effects of peripheral BoNTs on human pain behavior, validated experimental models have been examined. Subcutaneous capsaicin, resulting in persistent small afferent activation, evokes a local pain sensation referred to the site of injection, a primary and secondary hyperalgesia and an increase in local cutaneous blood flow (flare). Pretreatment with subcutaneous BoNT/A1 was reported to diminish pain (hyperalgesia) and blood flow (flare) [57]. Others reported either an effect only upon capsaicin induced flare [132] or no effect on any end point [61,62,63]. Studies examining the effects of pretreatment with subcutaneous BoNT/A1 for the pain and local increase in skin blood flow evoked by subcutaneous glutamate observed a significant inhibition with peak effects by seven days and a return to baseline by 60 days [64]. Following focal thermal injury, the associated primary and secondary hyperalgesia was not altered by BoNT/A1 in a double-blinded crossover study [65]. Taken together, these data reveal mixed indications of efficacy of BoNTA1 in reducing inflammatory pain states, and further studies are needed.

Injury to the peripheral nerve trunk or terminals (e.g., yielding mono and poly neuropathies, respectively) leads to a paradoxical pain state in which the individual reports an ongoing pain condition (dysesthesia) and an exaggerated response to light touch ipsilateral to the nerve injury or bilateral. The tactile hypersensitivity is mediated by the activation of large (low threshold) Aβ axons. In animal models, mononeuropathies arise from ligation of nerve roots (Chung model neuropathy), partial nerve ligations (Shir model) or nerve compressions (Bennett model) [133,134], and poly-neuropathies may arise from toxic effects from chemotherapy (vinca alkaloid, platin drugs) [135] or from diabetes [136]. Mechanistically, these changes in neuraxial function result from the development of ectopic activity in the sprouting terminals of the injured axons and in their DRG [137]. While not necessary for afferent conduction, DRG activation can be a robust source of afferent traffic into the dorsal horn [138]. This increased activity results from a number of neuro-inflammatory responses which contribute to development and maintenance of the neurogenic pain state: i) increased DRG transcription factor activity [139] associated with altered channel (e.g., increased sodium/decreased potassium) receptor (e.g., increased purine, glutamate) protein expression [140,141,142]; ii) migration of inflammatory cells into the DRG that release lipid mediators, cytokines and growth factors which can activate the neighboring sensory neurons [143,144,145]; iii) sprouting of sympathetic terminals into the neuroma and DRG, which can activate afferent input [146]; iv) activation of DRG satellite cells (glia) [144,147], and development of an enhanced excitatory coupling between these satellite cells and dorsal root ganglion neurons [148], reflecting increased trafficking of GAP junction and excitatory purine receptor expression [149]. Following nerve injury, the increased ongoing afferent input can lead to a central facilitatory response, as observed after tissue injury [150]. In addition, nerve injury may lead to changes in spinal processes resulting in a loss of inhibitory control over large afferent-evoked excitation that likely lead to the phenomena of enhanced sensitivity to light touch (tactile allodynia) [151,152]. Unlike the states associated with tissue injury, pain secondary to injury of the peripheral nerve rarely resolves, resulting in chronic pain states.

In rodent models, intraplantar delivery of BoNT/A1 and BoNT/B1 in doses of 0.5–1 U has been reported to reduce the allodynia observed in mononeuropathy models, including those of peripehral nerve ligation [84,85], ventral root transection [86,87], and infraorbital nerve constriction [74], and in polyneuropathy models, including those produced by chemotherpeutics [78,84] and diabetes [88]. As with the models of inflammation, these effects begin after ~1–2 days and last in excess of 21 days. In human case reports and clinical studies, the hyperesthesia observed in post-herpetic neuralgia [49,50,51], diabetic neuropathy [52], and peripheral nerve injury [41,42,43] is relieved by peripheral BoNT/A1 treatment. The use of local intramuscular, subcutaneous delivery of BoNT/A1 has also been reported to have efficacy in trigeminal neuralgia [44,45,46,47,48], which is a facial mononeuropathy. These data suggest that BoNT/A1 and /B1 can reduce neuropathic pain states resulting from peripheral nerve injury.

BoNTs can be taken up in the peripheral terminal of motor axons to cleave terminal SNAREs and block release of acetylcholine. It is now evident that BoNT/A1 and /B1 can also be locally taken up at peripheral terminals of sensory axons. In addition to in vitro work showing that BoNTs (primarily BoNT/A1/B1) can enter and cleave SNAREs in all neurons and not just motor neurons (reviewed in [166]), principal support for this hypothesis of a terminal action in sensory neurons is derived from the following observations:

i) Activation of the peripheral terminals of TRPV1 (+) C-fibers with capsaicin leads to the local vesicular release of neuropeptides such as SP and CGRP. This release results in vasodilation and plasma extravasation (e.g. neurogenic inflammation) [167]. In rodents, unilateral subcutaneous BoNT/A1 and /B1 injections into the paw at intervals as short as 15 min to 1 h will block the ipsilateral, but not contralateral, flare and plasma extravasation otherwise evoked by bilateral intraplantar injections of capsaicin. This indicates a local block of the release of these small afferent peptides after local BoNT/B1 treatment [67,75] (Figure 3, #1).

ii) A block of dural extravasation (from trigeminal afferents innervating the meninges) has been similarly demonstrated [74].

iii) Consistent with these findings, human studies also showed similar effects of BoNT/A1 in preventing local cutaneous flare and/or extravasation [57,58,91], though negative results have been reported [61,62,63]. The positive results are consistent with the ability of toxins to prevent afferent transmitter release at the peripheral terminal.

Locally delivered BoNTs may also affect mechanisms in inflammation other than the afferent terminal (Figure 3, #3). Neutrophils, macrophages and mast cells, when activated, will release local inflammatory products that activate small afferent terminals [168,169], and all employ SNAREs in their transport and exocytosis [170,171]. Macrophages employ a constitutive secretory pathway for the secretion of cytokines (e.g., TNF; IL-6 and IL-10), which mediates trafficking to the cell surface by SNARE-dependent membrane fusion [172]. Neutrophils store pro-inflammatory cytokines preformed in granular packaging and release a variety of highly cytotoxic and stimulatory products including myeloperoxidase and a variety of matrix metalloproteinases through a variety of SNARE- mediated processes [171,173,174]. Mast cells play a major role in the local inflammatory response and their degranulation leads to the local release of a variety of products, including histamine, high molecular weight heparins and cytokines through differential release of granule proteins and distinct secretory pathways, and other dilatory products that are similarly mediated by a variety of SNAREs [175] (Figure 3, #2). These SNARE dependent secretory processes [176,177] are potentially sensitive to effects by BoNTs, provided that the BoNT can enter the relevant cells and cleave a SNARE involved in such secretory processes. Entry of BoNTs into mast cells has not been demonstrated so far and the main SNAP isoform expressed in human mast cells is the BoNT/A1 insensitive SNAP-23. In addition, it appears that VAMP8, rather than the neuronal and BoNT sensitive VAMP1/2, is involved in the secretory process of mast cells [177]. However, local BoNT/A1 has been reported to reduce mast cell degranulation in rodents [178] and reduce burn-induced itching in humans [179]. Although involvement of SNARE proteins in the release of mediators from neutrophils and macrophages has been demonstrated, the direct effect of BoNT/A1 on this release thus far has not been adequately explored. Further research efforts are warranted to determine whether and how BoNTs may have an effect on neutrophils, macrophages and mast cells, and the role that they play in altering the local injury milieu.

In studies with dural C fiber afferents, chemical sensitization results in an enhanced afferent response [180]. This facilitated response is prevented by local BoNT/A1 [181]. Further, BoNT/A1 prevented the development of mechanical hypersensitivity of these afferents. Whether this effect reflects a direct effect upon mechanisms underlying the initiation of an excitable state in the afferent terminal (e.g., mobilization of protein kinases leading to phosphorylation of terminal channels/receptors) [168,169], or a potential block of release of active factors from local inflammatory cells (e.g., mast cells, macrophages and lymphocytes) remains to be determined (Figure 3, #3). In short, these findings emphasize that, overall, while BoNT/A1 may not directly alter the acute activation of afferent terminals (e.g., absent effect upon normal sensory thresholds), the toxin can be taken up in an active form in the homologous (ipsilateral) sensory afferent terminal and potentially in local pro-inflammatory cells to reduce the release of local pro-excitatory products otherwise mobilized by injury and inflammation.

SNAREs are localized in the lipid rafts [182] which play a role in the organization of membrane protein insertion [183]. Cleavage of SNARES in DRG/TG neuronal cell culture has been reported [92,93,94,95,184] and therefore, it would not be surprising if BoNTs had robust effects upon the trafficking of a variety of membrane proteins. One example would be the TRPV1 channels [185]. It has been shown that during hyperpathic states, TRPV1 receptor function is enhanced by increased trafficking of that protein to the plasma membrane at the peripheral and, likely, central terminals and the DRG. Evidence suggests that BoNT/A1 hinders recruitment of TRPV1 by affecting regulated exocytosis evoked by capsaicin in the trigeminal system [90]. This paradigm suggests a mechanism by which BoNT/A1 would be effective in attenuating hyperalgesia in capsaicin-induced trigeminal sensitization in humans [58] (Figure 3, #2). Other membrane proteins, the trafficking of which is relevant to facilitated pain states, warrant further study, including those for the glutamate ionophores [186] and the TRP channel [187,188].

While it is clear that BoNT/A1 and /B1 can exert an effect upon local terminal function, as reviewed above, the majority of work to date has not supported a direct effect of these BoNTs on peripheral terminal excitability in the normal state, as measured by baseline thermal and mechanical thresholds. As noted above, however, it was reported that BoNT/A1 inhibited responses to mechanical stimulation of the dura but only to supra-threshold forces [181]. These electrophysiological studies suggested that BoNT/A1 is selective for C- but not Aδ-trigeminal meningeal nociceptors. That report is consistent with the observation that BoNTs can block release secondary to C-fiber activation, e.g., intraplantar BoNT/A1 or /B1 block capsaicin-induced flare/plasma extravasation in animal [67,74] and human models [58,59,60,132], reflecting the local stimulatory effects mediated by the TRPV1 receptor on the peripheral terminals of the somatic peptidergic C-fiber. While these BoNTs have relatively little effect upon normal pain thresholds, they are noteworthy for their action on facilitated states as they occur after local inflammation and nerve injury. As reviewed above, local BoNT/A1 and /B1 can block the facilitated pain response generated by a variety of stimuli such as intraplantar irritants (e.g., capsaicin, formalin, histamine), but it is not apparent whether this peripheral action accounts for the “analgesic” actions of local BoNT/A1. Several points should be noted. While BoNT/B1 can have an acute onset at the terminal as measured by plasma extravasation after local injection, the effect, for example, on formalin-evoked flinching parallels the reduction in DRG SNAREs, which appears to require a longer interval (e.g., 24 h) [67]. Further, in the case of the formalin flinching model, local BoNT/A1 or /B1 have little or no effect upon the first phase flinching but produce a long-term reduction in the second phase, which is believed to reflect upon a centrally-mediated facilitation of spinal function generated by the conditioning discharge observed during the first phase [67,68,69,70,71,72,73,189]. These results suggest that local BoNT/A1 and /B1 may alter, with short latencies, the processes leading to changes in transduction and the discriminable process of terminal release, thereby preventing pain transmission to the second order neuron. There may be additional mechanisms that are at play and are reflected in part by the delayed onset of effects that are not local but mediated by transport from the site of delivery.

Several lines of evidence now indicate that, apart from a local action, BoNT serotypes A1 and B1 are taken up and transported in the neuron to distal terminals. Fast axonal transport for BoNT/A1 has been demonstrated in the brain [190,191,192]. However, in contrast, a slow movement of BoNT/A1 was reported as cleaved SNAP-25 appeared along neurites and accumulated in the soma over several weeks [193]. In the primary afferent, such transport after subcutaneous/intramuscular BoNT/A1/B1 delivery has been suggested by data derived from several experimental strategies.

i) Movement of radiolabeled (iodinated) BoNT/A1 and /B1 has been reported in spinal pathways [194,195,196];

ii) Cleavage of SNARE proteins (SNAP-25-BoNT/A1 / VAMP-BoNT/B1) has been detected in the ipsilateral afferent cell body (DRG/or trigeminal ganglia (TG)) after intraplantar delivery [67,71,197,198]. BoNT/A-cleaved SNAP-25 appeared bilaterally in the ventral and dorsal horns four days after injection of BoNT/A1 / BoNT/A2, suggesting that catalytically active BoNT/A1 and BoNT/A2 were transported via peripheral motor and sensory nerves and transcytosis [199].

iii) A block of dorsal horn substance P release from primary afferents in the nucleus caudalis evoked by supraorbital capsaicin [75] or in the spinal dorsal horn evoked by intraplantar formalin has been observed after BoNT/B1 treatment [67]. Further, the same authors showed that peripherally delivered BoNT/B1 blocked the evoked release of substance P in the ipsilateral but not contralateral dorsal horn (otherwise evoked bilaterally by spinally delivered capsaicin). This demonstrated that the BoNT/B1 delivered peripherally to one paw had to reach the ipsilateral central afferent terminals where the intrathecal capsaicin was acting.

iv) BoNT/B1 treatment resulted in a block of evoked dorsal horn activity as measured by a decreased incidence of evoked dorsal horn c-Fos (+) neurons [67,75]. These observations indicate that peripherally delivered BoNT/B1 is taken up at the peripheral afferent terminals and transported in an active form to central afferent terminals in both spinal and trigeminal systems, where they cleave SNAREs and prevent transmitter release (Figure 3, #5, #8).

v) Retrograde and anterograde transport of BoNT/A1 in non-acidic organelles has been shown by several groups, and estimates indicate speed profiles matching fast microtubule-dependent transport that overlaps with TeNT (tetanus neurotoxin) positive carriers in motor neurons and in vivo models [74,89,190,191,192,197,199,200,201,202]. However, it is currently unclear what fraction of the locally administered BoNT/A1 undergoes such intraneuronal transport, and whether the transport is concentration-dependent or differs mechanistically for different routes of administration.

The likelihood that BoNT/A1 and /B1 transported centrally in the afferent can block transmitter release is an important component of understanding their antinociceptive action. However, a common thread in the actions of the BoNTs on pain behavior is the general lack of effect upon acute nociceptive thresholds. Given the role of small afferents in pain transmission, it is puzzling that blocking central terminal release fails to alter acute transmission (if not producing an all-out anesthesia). This conundrum is not limited to the action of BoNTs. Studies with intrathecally delivered calcium channel blockers (e.g., for the N-type channel) showing effects on afferent transmitter release typically fail to block large afferent input (light touch) and have little effect upon acute pain thresholds, yet BoNT/A1 and/B1 delivery prominently reduces facilitated states in animal models and in humans [203,204,205,206,207,208,209]. The reason for this apparent discrepancy remains to be characterized. In short, however, the evidence for the time-dependent central transport of BoNT/A1 in an active form in the primary afferent and cleavage of SNAREs in the cell body (DRG/trigeminal ganglion) emphasizes the potential for a centrally mediated change in transmitter release from the central afferent terminal and, perhaps less appreciated, from its cell body (DRG/trigeminal ganglion) [126].

As noted above, transmitter release of glutamate, among other transmitters, occurs in isolated DRG/trigeminal ganglion cells and from associated glia (satellite cells) in cultured cells [96,210,211]. Such release likely also occurs in vivo [212]. Electrophysiological recording studies have indicated cross-excitation between large (low threshold tactile sensitive) afferents and small (high threshold, nociceptive) afferents. This interaction, potentially leading to tactile evoked activation of nociceptive input, is enhanced after peripheral injury [213,214,215]. Current work strongly suggests that such release could indeed activate other DRG cells through, for example, glutamatergic receptors [216], some of which are on DRG glia (satellite cells) [217] (Figure 3, #6, #7). Given the contribution of such DRG release to hyperpathic states initiated by nerve and tissue injury, along with the ability of peripherally injected BoNT/A1 and /B1 to cleave DRG and trigeminal SNAREs, it is an intriguing hypothesis that such an action by BoNTs could be responsible for their modulation of primary hyperpathia and large afferent mediated allodynia. Recent work has indeed shown that BoNT/A1 decreases vesicular release of glutamate from satellite cells [96]. A recent study importantly suggests that BoNT/A1 may interfere with intra-ganglionic transmitter release and cross-excitation of neighboring neurons to the development and maintenance of chronic pain states [218]. This transported action might thus provide an important mechanism for altering the hypothesized crosstalk between populations of sensory ganglion cells (Figure 3, #6, #7). Further studies in this area will be of considerable interest.

As reviewed above, SNAREs play a ubiquitous role in the trafficking of several ionotropic and metabotropic receptors that contribute to regulating neuronal excitability and synaptic transmission in the dorsal horn. The protein receptors are transported to the cell surface in the vesicle membrane that fuses via a SNARE mediated process. SNAREs thus play a major role in the induction and maintenance of spinal sensitization. SNARE proteins are involved in the mobilization of receptor subunits of AMPA [219] and NMDA [220], which are actively involved in the process of dorsal horn sensitization [221,222,223]. Interventions by BoNT in glutamate ionophore trafficking would influence facilitated processing, thereby having profound effects upon the generation of facilitated states. In cerebellar slices, BoNTs reduce trafficking and block long-term potentiation [161]. The potential that BoNTs may affect the processing of pain information by altering receptor trafficking in addition to direct effects resulting from a block in exocytosis is an interesting future area of study.

Consistent with these spinal actions outlined above, not surprisingly, intrathecal delivery of BoNT/B1 has been shown to result in cleavage of spinal SNAREs, blockade of evoked release of afferent transmitters and prevention of evoked activation of spinal neurons as indicated by the incidence of c-Fos (+) neurons [67,224]. Intrathecal delivery of BoNT/A1 and /B1 has been shown to block the second phase of IPLT formalin-evoked pain behavior [67,73,225] to reduce visceral-evoked inflammatory states (BoNT/A1) [226], and to attenuate the allodynia otherwise observed in mononeuropathies (nerve ligation) and polyneuropathies (as with chemotherapeutics) (BoNT/B1) [84]. The specificity of these effects on the role played by the cleavage of SNAREs was provided by two observations: i) intrathecal delivery of BoNT/B1 treated with dithiotreitol to cleave the disulfide linkages had no activity, and ii) intrathecal BoNT/B1 serotype blocked formalin-evoked flinching in the mouse but not in the rat, an observation consistent with the fact that the rat VAMP1 has a mutation at the BoNT/B1 targeted cleavage site rendering the rat insensitive to BoNT/B1 activity [227].

While several lines of evidence indicate that intrathecal BoNT/A1 and /B1 can affect afferent terminal function, as reviewed above, they may also be taken up by a variety of neural types including inhibitory interneurons (Figure 3, #11). Cell culture studies have shown that several BoNTs can block depolarization-evoked release of inhibitory amino acids [92,228,229,230]. A block of spinal inhibitory interneurons would lead to excitation, and such has been reported after accidental intrathecal delivery of BoNT/A1 in humans [231]. This is a very important point of consideration when evaluating BoNTs as pain pharmaceuticals.