When tested on a range of mechanical stimuli, Tx3-3 exhibited higher potency to inhibit dorsal horn neuronal responses in SNL than naive rats. Tx3-3 inhibited to the same extent neuronal responses evoked by noxious punctate mechanical stimulation (von Frey 26 and 60 g) in SNL and naive rats (Fig. 3A, B), but the same level of inhibition was achieved by a 100-fold lower dose of Tx3-3 in SNL rats (inhibition of 46.7 ± 10.8% was achieved by Tx3-3 at 0.3 pmol/site; F(4,33) = 9.781, P < 0.0001) in comparison to naive rats (42.2 ± 3.9% was achieved by Tx3-3 at 30 pmol/site; F(3,26) = 4.547, P = 0.0109) (Fig. 3C). A leftward shift in the dose–response curve of Tx3-3 in SNL rats in comparison to naive rats was also seen in neuronal response evoked by nonnoxious mechanical stimuli (von Frey 8 g). In SNL rats, a significant inhibitory effect on neuronal response evoked by nonnoxious punctate mechanical stimulus (von Frey 8 g) was reached by application of 0.3 pmol/site of Tx3-3 (inhibition of 54.5 ± 8.6%; F(4,33) = 4.294, P = 0.0066), while 30 pmol/site of Tx3-3 was needed to inhibit neuronal response in naive rats (inhibition of 41.7 ± 13.7%; F(3,26) = 3.493, P = 0.0297) (Fig. 3C). Moreover, Tx3-3 exhibited inhibitory effect on nonnoxious dynamic mechanical stimulation (brush) in SNL rats (inhibition of 52.2 ± 8.7; F(4,33) = 2.840, P = 0.0404), but not in naive rats (F(3,26) = 2.739, P = 0.0637) (Fig. 3A, B).
Neuronal responses to thermal stimulation (40, 45, and 48°C) were also inhibited by spinal application of Tx3-3 in naive and SNL rats. Tx3-3 inhibited neuronal responses evoked by noxious thermal stimulus (48°C) in both naive (inhibition of 41 ± 15%; F(3,23) = 3.324, P = 0.0375) and SNL rats (inhibition of 38.7% ± 7.6; F(3,25) = 6.912, P = 0.00015) (Fig. 3D, E). In SNL rats, Tx3-3 mediated a broader inhibition of neuronal response to thermal stimulus, inhibiting also the responses evoked by 40°C (inhibition of 62.5% ± 11.6; F(3,25) = 4.532; P = 0.0118). Moreover, lower doses of Tx3-3 were required to achieve significant inhibition in SNL rats than in naive rats (Fig. 3D, E).
Overall, the maximum inhibitions produced by Tx3-3 were established around 10 to 20 minutes, and the inhibitory effect lasted approximately 60 minutes (Appendix). No difference was found in timecourse effect of Tx3-3 between naive and SNL rats (data not shown).
In this study, we addressed the effect of the peptide toxin Tx3-3, a P/Q- and R-type VGCC blocker, in in vivo electrophysiological measurements of dorsal horn neuronal responses under physiological and neuropathic conditions. We showed that Tx3-3 mediated differential inhibitory effect on deep dorsal horn neuronal responses under physiological and neuropathic conditions, exhibiting greater potency after nerve injury.
When tested on neuronal responses evoked by electrical stimulation, Tx3-3 exhibited a greater inhibitory profile in SNL than in naive rats. Under neuropathic conditions, Tx3-3 inhibited Aδ, C-fibre, and input responses as well as postdischarge response, while only postdischarge was inhibited in normal animals. Moreover, Tx3-3 produced a greater inhibition of neuronal wind-up in SNL than naive rats. The VGCCs targeted by Tx3-3 are differentially expressed in dorsal horn spinal neurones. P/Q-type VGCCs are expressed on nerve terminals of nonpeptidergic IB4-positive C-fibres, while R-type VGCCs are predominantly localized to somatodendritic compartments51 but are also expressed on nerve terminals of peptidergic C-fibres and Aδ-fibres.17 Subcellular localization functionally link VGCC subtypes with specific neuronal processes. P/Q-type VGCCs are involved in initiating the release of neurotransmitters at presynaptic sites,7,9 while R-type VGCCs play minor role in basal neurotransmitter release, being involved in presynaptic mechanisms of plasticity.5,14,53 The inhibition of synaptic transmission through inhibition of neurotransmitter release may account for the effect of Tx3-3 on postdischarge and wind-up responses evoked by electrical stimulation in physiological conditions. In agreement, the blockade of spinal P/Q-type VGCCs by application of ω-agatoxin-IVA in rats attenuated both postdischarge and wind-up neuronal responses.30 However, the inhibitory profile of ω-agatoxin-IVA remained unchanged after SNL.30 Keeping this in mind, the differential effect mediated by Tx3-3 in neuropathic conditions is thus unlikely to be mediated by its action on P/Q-type VGCCs. Furthermore, classically, P/Q-type VGCC blockers present modest effects against neuropathic pain.10,50,55 Interestingly, the inhibitory profile of Tx3-3 on electrically evoked neuronal responses resemble those elicited by the R-type VGCC blocker SNX-482.29 Both Tx3-3 and SNX-482, a peptide isolated from the venom of the spider Hysterocrates gigas,34 inhibited postdischarge and wind-up responses under control conditions, but exhibited a greater inhibitory profile in neuropathic conditions induced by SNL, inhibiting also nociceptive C-fibre and Aδ-fibre responses.29 Moreover, like SNX-482, the input and wind-up responses were rather affected by Tx3-3 after neuropathy.
Thus the effect of Tx3-3 on electrically evoked dorsal horn neuronal responses in neuropathic conditions are consistent with a major blockade of R-type VGCCs. In the SNL animals, the wind-up profile differed from that in the control group as the initial response, an index of presynaptic mechanisms delivering the input onto the neurones, was markedly increased, perhaps indicative of enhanced presynaptic spinal mechanisms in keeping with the augmented effects of the toxin after neuropathy.
The antinociceptive properties of Tx3-3 were previously shown in neuropathic pain models of sciatic nerve ligation and diabetic neuropathy.13 In the present study, we have addressed the effect of Tx3-3 in in vivo electrophysiology recordings of dorsal horn neurones after the SNL model of chronic neuropathic pain.23 In line to the earlier behavioural study, in which Tx3-3 attenuated the tactile allodynia induced by traumatic neuropathy,13 here we show that Tx3-3 inhibits dorsal horn neuronal response evoked by innocuous mechanical stimulation in SNL rats. Despite the effect on innocuous mechanical response, Tx3-3 did not affect Aβ-fibres responses. However, electrically evoked responses are suprathreshold and syncronious; by contrast, natural stimuli evoked by 8 g von Frey stimulation applied over 10 seconds are more amenable to inhibition and are likely to activate also Aδ-fibres, which could explain this effect. Importantly, we show that mechanically evoked dorsal horn responses were more sensitive to Tx3-3 in conditions of neuropathy. The higher potency of Tx3-3 in SNL rats suggests a higher affinity of Tx3-3 to VGCC after neuropathy. It has been shown that in neuropathic states the high frequency of fiber activation produces a dynamic change in the VGCC conformational state, switching between resting, activated, and inactivated state.1 The access to the inactivated channel is greater during high-frequency neuronal firing conditions, improving the affinity of certain VGCC blockers, as occurring with some ω-conotoxins that exhibit higher potency when binding to the inactivated state of VGCC.3,47,52,58 Such state-dependent blockade property could account for the more potent blockade of Tx3-3 during the higher frequency of spontaneous neuronal firing present after SNL.11 The higher magnitude of inhibition and dose-related effect of Tx3-3 on wind-up responses in neuropathic rats further support the notion that the reorganization of VGCCs kinetics properties following nerve damage favors the Tx3-3 binding. In this regard, it is noteworthy that neuronal responses evoked by nonnoxious thermal and mechanical stimuli were sensitive to Tx3-3 inhibition preferentially in neuropathic conditions, indicating an enhanced excitability of spinal transmission from afferents that expressed Tx3-3-sensitive VGCCs after neuropathy. In agreement, dorsal horn neuronal response evoked by nonnoxious punctate mechanical stimulus is more sensitive to the R-type VGCC blocker SNX-482 in neuropathic conditions.29 Furthermore, P/Q-type VGCCs are also found in the deeper laminae of the dorsal horn,51 suggesting its presence on touch-sensitive large fibres, and there are reports of increased levels of P/Q-type in DRG, especially in medium and large myelinated afferent fibers, in neuropathic pain models.49,56 Thus, nerve injury may establish a functional reorganization of neuronal phenotypes within the spinal cord, by changing the pattern of expression or activity of some ion channels, including the VGCCs target by Tx3-3, which explains the outcome effect of Tx3-3 on neuronal response evoked by nonnoxious stimuli in neuropathic rats.
It is also worth mentioning the possible role of the auxiliary α2δ subunit of VGCCs in our current results. The α2δ subunits of VGCCs (comprises α2δ-1, α2δ-2, α2δ-3, and α2δ-4) regulate VGCC biophysical properties, trafficking, and membrane expression2,15,22 and are upregulated in the dorsal horn spinal cord, mediating spinal hyperexcitability, under neuropathic conditions.4,25,26 Recently it was shown that the disruption of the α2δ1 gene expression leads to a delay in development of mechanical hypersensitivity in neuropathic mice, which correlates with a reduction in the response of deep dorsal horn neurones to a range of mechanical stimuli.37 Which subtype of Cav2 family is mainly affected by α2δ regulation in neuropathic conditions remains to be clearly defined. However, most aspects of the R-type pore-forming α1 subunit (alpha 1E) are affected by α2δ regulation, including the kinetics of activation-inactivation-deactivation of R-type VGCCs,40 and the trafficking of Cav2.1 (which conducts P/Q-type currents) to cell surface is prevented by mutation in the α2δ2 subunits.20,21 Therefore, it is possible that the adaptations on VGCC function and expression carried out by α2δ subunits in neuropathic conditions underlie the gain of effect of Tx3-3.
The Tx3-3 affected the neuronal responses evoked by thermal stimulation in naive and SNL rats. These results are in accordance with the previously reported inhibitory effect of Tx3-3 in heat-induced nociceptive pain.13 Notably, the doses of Tx3-3 used here are comparable to those used in the above-mentioned behavioral study. In the present study, however, the lower dose of Tx3-3 showed a tendency to increase the thermally evoked neuronal response in naive rats. This may result from the blockade of P/Q-type VGCCs presented on inhibitory interneurones.48,51 Likewise, spinal application of the P/Q-type VGCC blocker ω-agatoxin IVA tended to facilitate neuronal responses at lower doses.30 However, this phenomenon was not seen in SNL rats, possibly because in this neuropathic condition, R-type VGCCs come into play, as demonstrated by the increased sensitivity of thermally evoked neuronal response to the R-type VGCC blocker SNX-482 in SNL rats.29 Therefore, possibly, the neuropathy may cause a pathophysiological upregulation of R-type VGCC activity (or promote a given conformational state) that favors the action of Tx3-3 on it, explained by the enhanced sensitivity of SNL rats to Tx3-3 inhibitory effect on thermally evoked neuronal responses (as lower doses were needed to achieve inhibitory effect in SNL rats compared to control rats).
This is the first electrophysiological study addressing the effects of Tx3-3 on sensory transmission in spinal cord of rats. The present data extend results from previous behavioural studies, showing a prevalent antinociceptive effect of Tx3-3 on neuropathic states and suggest the R-type VGCC as the main target of such action.
A significant functional role of VGCCs in neuropathic pain mechanisms has been substantiated by studies using toxins isolated from animal venoms.38,54 Here, we showed that the toxin Tx3-3, a P/Q- and R-type VGCC blocker isolated from the venom of the spider P. nigriventer, mediated differential inhibitory effect on deep dorsal horn neuronal responses under physiological and neuropathic pain conditions, exhibiting greater potency after nerve damage. The profile of action of Tx3-3 highlights the role of VGCCs, drawing attention to R-type VGCC, in pathological pain and may provide insight into the development of specific analgesics for the treatment of neuropathic pain.
The authors have no conflict of interest to declare.
Supported by the Wellcome Trust London Pain Consortium strategic award. G. D. Dalmolin is supported by Capes/Toxinologia (process 8849-11-0). There are not any financial or other relationships that might lead to a conflict of interest in this study.
Timecourse of cumulative doses of toxin Tx3-3 in mechanical evoked neuronal response (n = 3).
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