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).
. Baccei ML, Kocsis JD. Voltage-gated calcium currents in axotomized adult rat cutaneous afferent neurons. J Neurophysiol 2000;83:2227–38.
. Bauer CS, Nieto-Rostro M, Rahman W, Tran-Van-Minh A, Ferron L, Douglas L, Kadurin I, Sri Ranjan Y, Fernandez-Alacid L, Millar NS, Dickenson AH, Lujan R, Dolphin AC. The increased trafficking of the calcium channel subunit alpha2delta-1 to presynaptic terminals in neuropathic pain is inhibited by the alpha2delta ligand pregabalin. J Neurosci 2009;29:4076–88.
. Berecki G, Motin L, Haythornthwaite A, Vink S, Bansal P, Drinkwater R, Wang CI, Moretta M, Lewis RJ, Alewood PF, Christie MJ, Adams DJ. Analgesic (omega)-conotoxins CVIE and CVIF selectively and voltage-dependently block recombinant and native N-type calcium channels. Mol Pharmacol 2010;77:139–48.
. Boroujerdi A, Kim HK, Lyu YS, Kim DS, Figueroa KW, Chung JM, Luo ZD. Injury discharges regulate calcium channel alpha-2-delta-1 subunit upregulation in the dorsal horn that contributes to initiation of neuropathic pain. PAIN 2008;139:358–66.
. Breustedt J, Vogt KE, Miller RJ, Nicoll RA, Schmitz D. Alpha1E-containing Ca2+ channels are involved in synapticplasticity. Proc Natl Acad Sci USA 2003;100:12450–5.
. Cardoso FC, Pacífico LG, Carvalho DC, Victória JM, Neves AL, Chávez-Olórtegui C, Gomez MV, Kalapothakis E. Molecular cloning and characterization of Phoneutria nigriventer
toxins active on calcium channels. Toxicon 2003;41:755–63.
. Catterall WA, Few AP. Calcium channel regulation and presynaptic plasticity. Neuron 2008;59:882–901.
. Catterall WA, Leal K, Nanou E. Calcium channels and short-term synaptic plasticity. J Biol Chem 2013;288:10742–9.
. Catterall WA. Interactions of presynaptic Ca2+ channels and snare proteins in neurotransmitter release. Ann N Y Acad Sci 1999;868:144–59.
. Chaplan SR, Pogrel JW, Yaksh TL. Role of voltage-dependent calcium channel subtypes in experimental tactile allodynia. J Pharmacol Exp Ther 1994;269:1117–23.
. Chapman V, Suzuki R, Dickenson AH. Electrophysiological characterization of spinal neuronal response properties in anaesthetized rats after ligation of spinal nerves L5-L6. J Physiol 1998;507:881–94.
. Cordeiro Mdo N, de Figueiredo SG, Valentim Ado C, Diniz CR, von Eickstedt VR, Gilroy J, Richardson M. Purification and amino acid sequences of six TX3 type neurotoxins from the venom of the Brazilian “armed” spider Phoneutria nigriventer
. Toxicon 1993;31:35–42.
. Dalmolin GD, Silva CR, Rigo FK, Gomes GM, Cordeiro Mdo N, Richardson M, Silva MA, Prado MA, Gomez MV, Ferreira J.Antinociceptive effect of Brazilian armed spider venom toxin Tx3-3 in animal models of neuropathic pain. PAIN 2011;152:2224–32.
. Dietrich D, Kirschstein T, Kukley M, Pereverzev A, von der Brelie C, Schneider T, Beck H. Functional specialization of presynaptic Cav2.3 Ca2+ channels. Neuron 2003;39:483–96.
. Dolphin AC. The α2δ subunits of voltage-gated calcium channels. Biochim Biophys Acta 2013;1828:1541–9.
. Ertel EA, Campbell KP, Harpold MM, Hofmann F, Mori Y, Perez-Reyes E, Schwartz A, Snutch TP, Tanabe T, Birnbaumer L, Tsien RW, Catterall WA. Nomenclature of voltage-gated calcium channels. Neuron 2000;25:533–5.
. Fang Z, Park CK, Li HY, Kim HY, Park SH, Jung SJ, Kim JS, Monteil A, Oh SB, Miller RJ. Molecular basis of Ca(v)2.3 calcium channels in rat nociceptive neurons. J Biol Chem 2007;282:4757–64.
. Feng ZP, Doering CJ, Winkfein RJ, Beedle AM, Spafford JD, Zamponi GW. Determinants of inhibition of transiently expressed voltage-gated calcium channels by omega-conotoxins GVIA and MVIIA. J Biol Chem 2003;278:20171–8.
. Gonçalves L, Dickenson AH. Asymmetric time-dependent activation of right central amygdala neurones in rats with peripheral neuropathy and pregabalin modulation. Eur J Neurosci 2012; 36:3204–13.
. Heblich F, Tran Van Minh A, Hendrich J, Watschinger K, Dolphin AC. Time course and specificity of the pharmacological disruption of the trafficking of voltage-gated calcium channels by gabapentin. Channels (Austin) 2008;2:4–9.
. Hendrich J, Van Minh AT, Heblich F, Nieto-Rostro M, Watschinger K, Striessnig J, Wratten J, Davies A, Dolphin AC. Pharmacological disruption of calcium channel trafficking by the alpha2delta ligand gabapentin. Proc Natl Acad Sci USA 2008;105:3628–33.
. Jarvis SE, Zamponi GW. Trafficking and regulation of neuronal voltage-gated calcium channels. Curr Opin Cell Biol 2007;19:474–82.
. Kim SH, Chung JM. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. PAIN 1992;50:355–63.
. Leão RM, Cruz JS, Diniz CR, Cordeiro MN, Beirão PSL. Inhibition of neuronal high-voltage activated calcium channels by the omega-Phoneutria nigriventer
Tx3-3 peptide toxin. Neuropharmacology 2000;39:1756–67.
. Li CY, Song YH, Higuera ES, Luo ZD. Spinal dorsal horn calcium channel alpha2delta-1 subunit upregulation contributes to peripheral nerve injury-induced tactile allodynia. J Neurosci 2004;24:8494–9.
. Li CY, Zhang XL, Matthews EA, Li KW, Kurwa A, Boroujerdi A, Gross J, Gold MS, Dickenson AH, Feng G, Luo ZD. Calcium channel alpha2delta1 subunit mediates spinal hyperexcitability in pain modulation. PAIN 2006;125:20–34.
. Luvisetto S, Marinelli S, Panasiti MS, D'Amato FR, Fletcher CF, Pavone F, Pietrobon D. Pain sensitivity in mice lacking the Ca(v)2.1alpha1 subunit of P/Q-type Ca2+
channels. Neuroscience 2006;142:823–32.
. Malmberg AB, Yaksh TL. Voltage-sensitive calcium channels in spinal nociceptive processing: blockade of N- and P-type channels inhibits formalin-induced nociception. J Neurosci 1994;14:4882–90.
. Matthews EA, Bee LA, Stephens GJ, Dickenson AH. The Cav
2.3 calcium channel antagonist SNX-482 reduces dorsal horn neuronal responses in a rat model of chronic neuropathic pain. Eur J Neurosc 2007;25:3561–69.
. Matthews EA, Dickenson AH. Effects of spinally delivered N- and P-type voltage-dependent calcium channel antagonists on dorsal horn neuronal responses in a rat model of neuropathy. PAIN 2001;92:235–46.
. Molinski TF, Dalisay DS, Lievens SL, Saludes JP. Drug development from marine natural products. Nat Rev Drug Discov 2009;8:69–85.
. Nebe J, Ebersberger A, Vanegas H, Schaible HG. Effects of omega-agatoxin IVA, a P-type calcium channel antagonist, on the development of spinal neuronal hyperexcitability caused by knee inflammation in rats. J Neurophysiol 1999;81:2620–26.
. Nebe J, Vanegas H, Neugebauer V, Schaible HG. Omega-agatoxin IVA, a P-type calcium channel antagonist, reduces nociceptive processing in spinal cord neurons with input from the inflamed but not from the normal knee joint–an electrophysiological study in the rat in vivo. Eur J Neurosci 1997;9:2193–201.
. Newcomb R, Szoke B, Palma A, Wang G, Chen XH, Hopkins W, Cong R, Miller J, Urge L, Tarczy-Hornoch K, Loo JA, Dooley DJ, Nadasdi L, Tsien RW, Lemos J, Miljanich G. Selective peptide antagonist of the class E calcium channel from the venom of the tarantula Hysterocrates gigas. Biochemistry 1998;37:15353–62.
. Olivera BM, Cruz LJ, de Santos V, LeCheminant GW, Griffin D, Zeikus R, McIntosh JM, Galyean R, Varga J, Gray WR. Neuronal calcium channel antagonists. Discrimination between calcium channel subtypes using omega-conotoxin from Conus magus
venom. Biochemistry 1987;26:2086–90.
. Olivera BM, Miljanich GP, Ramachandran J, Adams ME. Calcium channel diversity and neurotransmitter release: the omega-conotoxins and omega-agatoxins. Annu Rev Biochem 1994;63:823–67.
. Patel R, Bauer CS, Nieto-Rostro M, Margas W, Ferron L, Chaggar K, Crews K, Ramirez JD, Bennett DL, Schwartz A, Dickenson AH, Dolphin AC. α2δ-1 gene deletion affects somatosensory neuron function and delays mechanical hypersensitivity in response to peripheral nerve damage. J Neurosci 2013;33:16412–26.
. Pexton T, Moeller-Bertram T, Schilling JM, Wallace MS. Targeting voltage-gated calcium channels for the treatment of neuropathic pain: a review of drug development. Expert Opin Investig Drugs 2011;20:1277–84.
. Pope JE, Deer TR. Ziconotide: a clinical update and pharmacologic review. Expert Opin Pharmacother 2013;14:957–66.
. Qin N, Olcese R, Stefani E, Birnbaumer L. Modulation of human neuronal alpha 1E-type calcium channel by alpha 2 delta-subunit. Am J Physiol 1998;274:1324–31.
. Randall A, Tsien RW. Pharmacological dissection of multiple types of Ca2+ channel currents in rat cerebellar granule neurons. J Neurosci 1995;15:2995–3012.
. Saegusa H, Kurihara T, Zong S, Minowa O, Kazuno A, Han W, Matsuda Y, Yamanaka H, Osanai M, Noda T, Tanabe T. Altered pain responses in mice lacking alpha 1E subunit of the voltage-dependent Ca2+ channel. Proc Natl Acad Sci USA 2000;97:6132–7.
. Saegusa H, Matsuda Y, Tanabe T. Effects of ablation of N- and R-type Ca(2+) channels on pain transmission. Neurosci Res 2002;43:1–7.
. Schmidtko A, Lötsch J, Freynhagen R, Geisslinger G. Ziconotide for treatment of severe chronic pain. Lancet 2010;375:1569–77.
. Sluka KA. Blockade of calcium channels can prevent the onset of secondary hyperalgesia and allodynia induced by intradermal injection of capsaicin in rats. PAIN 1997;71:157–64.
. Staats PS, Yearwood T, Charapata SG, Presley RW, Wallace MS, Byas-Smith M, Fisher R, Bryce DA, Mangieri EA, Luther RR, Mayo M, McGuire D, Ellis D. Intrathecal ziconotide in the treatment of refractory pain in patients with cancer or AIDS: a randomized controlled trial. JAMA 2004;291:63–70.
. Stocker JW, Nadasdi L, Aldrich RW, Tsien RW. Preferential interaction of omega-conotoxins with inactivated N-type Ca2+ channels. Neurosci 1997;17:3002–13.
. Takahashi T, Momiyama A. Different types of calcium channels mediate central synaptic transmission. Nature 1993;366:156–8.
. Umeda M, Ohkubo T, Ono J, Fukuizumi T, Kitamura K. Molecular and immunohistochemical studies in expression of voltage-dependent Ca2+ channels in dorsal root ganglia from streptozotocin-induced diabetic mice. Life Sci 2006;79:1995–2006.
. Vanegas H, Schaible H. Effects of antagonists to high-threshold calcium channels upon spinal mechanisms of pain, hyperalgesia and allodynia. PAIN 2000;85:9–18.
. Westenbroek RE, Hoskins L, Catterall WA. Localization of Ca2+ channelsubtypes on rat spinal motor neurons, interneurons, and nerve terminals. J Neurosci 1998;18:6319–30.
. Winquist RJ, Pan JQ, Gribkoff VK. Use-dependent blockade of Cav2.2 voltage-gated calcium channels for neuropathic pain. Biochem Pharmacol 2005;70:489–99.
. Wu LG, Westenbroek RE, Borst JG, Catterall WA, Sakmann B. Calcium channel types with distinct presynaptic localization couple differentially to transmitter release in single calyx-type synapses. J Neurosci 1999;19:726–36.
. Yaksh TL. Calcium channels as therapeutic targets in neuropathic pain. J Pain 2006;7:13–30.
. Yamamoto T, Sakashita Y. Differential effects of intrathecally administered N- and P-type voltage-sensitive calcium channel blockers upon two models of experimental mononeuropathy in the rat. Brain Res 1998;794:329–32.
. Yusaf SH, Goodman J, Gonzalez IM, Bramwell S, Pinnock PD, Dixon AK, Lee K. Streptozocin-induced neuropathy is associated with altered expression of voltage-gated calcium channel subunit mRNAs in rat dorsal root ganglion neurons. Biochem Biophys Res Commun 2001;289:402–6.
. Zamponi GW, Striessnig J, Koschak A, Dolphin AC. The physiology, pathology, and pharmacology of voltage-gated calcium channels and their future therapeutic potential. Pharmacol Rev 2015;67:821–70.
. Zhang JF, Ellinor PT, Aldrich RW, Tsien RW. Molecular determinants of voltage-dependent inactivation in calcium channels. Nature 1994;372:97–100.
. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals. PAIN 1983;16:109–10.