Analgesia by intrathecal delta-9-tetrahydrocannabinol is dependent on Cav3.2 calcium channels
Molecular Brain volume 16, Article number: 47 (2023)
Delta-9-tetrahydrocannabinol (Δ9-THC) is known to produce systemic analgesia that involves CB1 and CB2 cannabinoid receptors. However, there is compelling evidence that Δ9-THC can potently inhibit Cav3.2T-type calcium channels which are highly expressed in dorsal root ganglion neurons and in the dorsal horn of the spinal cord. Here, we investigated whether spinal analgesia produced by Δ9-THC involves Cav3.2 channels vis a vis cannabinoid receptors. We show that spinally delivered Δ9-THC produced dose-dependent and long-lasting mechanical anti-hyperalgesia in neuropathic mice, and showed potent analgesic effects in models of inflammatory pain induced by formalin or Complete Freund’s Adjuvant (CFA) injection into the hind paw, with the latter showing no overt sex differences. The Δ9-THC mediated reversal of thermal hyperalgesia in the CFA model was abolished in Cav3.2 null mice, but was unaltered in CB1 and CB2 null animals. Hence, the analgesic effects of spinally delivered Δ9-THC are due to an action on T-type calcium channels, rather than activation of spinal cannabinoid receptors.
T-type Ca2+ channels are known to be important regulators of pain transmission in primary afferent sensory neurons and the spinal cord . Among the three isoforms of T-type Ca2+ channels that are expressed in the mammalian genome, the Cav3.2 channel isoform appears to be the predominant T-type channel subtype involved in this process . It is expressed in a subpopulation of primary afferent fibers and the spinal dorsal horn , and its expression is enhanced in these tissues in a wide range of chronic pain conditions in rodents . Consequently, systemic or intrathecal delivery of T-type channel inhibitors mediates analgesia (for review see [1, 4]. T-type channels can be inhibited by different types of endocannabinoids , terpenes  and phytocannabinoids such as cannabidiol and Δ9-THC [7, 8]. In particular, Δ9-THC mediates strongly state dependent inhibition of Cav3.2 channels with a preference for binding to inactivated channels [7, 8]. It is known that spinally delivered Δ9-THC inhibits mechanical and cold allodynia in models of neuropathic pain , and analgesia exerted by Δ9-THC delivered to the brain involves modulation of both CB1 and CB2 receptors . However, it is unclear whether the spinal actions of Δ9-THC involve T-type channels, cannabinoid receptors, or a combination thereof. Thus, the present study was designed to investigate contributions of spinal CB receptor subtypes and Cav3.2 channels on the antihyperalgesic effect of spinally delivered Δ9-THC. All experiments were carried out with approval of an animal protocol by the Institutional Animal Care and Use Committee, and all efforts were made to minimize animal suffering according to the policies and recommendations of the International Association for the Study of Pain. Δ9-THC was delivered by intrathecal injection as described before [6, 8] into male and female C57BL/6J (wild-type), or male CB1 null, CB2 null, or Cav3.2 null mice (20 − 25 g, 8–10 weeks; Jackson Laboratories). We first assessed the analgesic action of spinally delivered Δ9-THC in the acute nociceptive (phase 1) and inflammatory pain (phase 2) phases of a standard formalin test . Intrathecally delivered Δ9-THC, 20 min before testing, significantly and dose-dependently reduced the duration of nocifensive responses in the first (Fig. 1a) and second (Fig. 1b) phases of the formalin test. Next, we verified whether spinal Δ9-THC was also able to inhibit mechanical hyperalgesia caused by chronic neuropathy 21 days after partial sciatic nerve injury which was performed as described by us before . Mechanical hyperalgesia was measured using a Dynamic Plantar Aesthesiometer (Ugo Basile, Varese, Italy). When compared to the neuropathic control group, treatment of mice with Δ9-THC (10.0 µg/i.t.), but not vehicle (PBS, 10 µl/i.t.) produced marked anti-hyperalgesia when evaluated 45 min after treatment (Fig. 1c). These data show that Δ9-THC mediates robust analgesia in wild type mice. Next, we investigated the effect of Δ9-THC in a model of persistent inflammatory pain. 20 μl of Complete Freund's Adjuvant (CFA) were given intraplantarly (i.pl.) in the ventral surface of the right hindpaw, whereas sham groups received 20 μl of PBS. Thermal hyperalgesia was examined by measuring the latency to withdrawal of ipsilateral hind paws in response to a focused beam of radiant heat (IR = 30) using a plantar test apparatus (UgoBasile, Varese, Italy). Two days after CFA injection, intrathecal treatment with Δ9-THC (10 µg/i.t) but not with vehicle (10 µl/i.t.) resulted in anti-hyperalgesia that remained significant up to 3 h (Fig. 1d). Δ9-THC was also effective in increasing paw withdrawal latencies when delivered to female mice (tested 45 min after its spinal delivery, Fig. 1e). To determine whether the analgesic effects observed for spinally delivered Δ9-THC were mediated by cannabinoid CB1 or CB2 receptors, we repeated the CFA model using male CB1 (Fig. 1f) and male CB2 (Fig. 1g) null mice in comparison with male wild-type mice that were simultaneously tested. For this purpose, mice were injected intrathecally with either vehicle (control) or Δ9-THC (10.0 µg/i.t.) and tested 45 min later. Similar to wild type animals, Δ9-THC produced significant analgesic effects, indicating that neither of these two receptors are essential for the observed analgesia even though this compound is an agonist of both receptor types .
We then tested the analgesic effect of spinal Δ9-THC in Cav3.2 null mice. These mice develop CFA-induced hypersensitivity despite the absence of Cav3.2 channels , most likely due to compensatory mechanisms that are not fully understood. As shown in Fig. 1h, Δ9-THC lost its analgesic effects when delivered to Cav3.2 null mice, indicating that the key biological target for spinally delivered Δ9-THC are T-type channels.
There is considerable evidence that CB1 receptor activation mediates analgesia , however there are also reports that the analgesic activity of Δ9-THC is lost in CB receptor null mice [10, 14]. We do not challenge a possible involvement of these receptors when Δ9-THC is delivered systemically. Our focus was to specifically isolate a spinal effect, and this can be cleanly accomplished by the intrathecal route of delivery used in our study (Additional file 1: Fig S1). What we do not know is the overall contribution of the spinal action to the overall analgesic properties of Δ9-THC. We attempted testing the effect of systemically delivered Δ9-THC in Cav3.2 null mice, however, we found that these mice became lethargic, thus confounding the types of pain behavioral measurements that we typically perform. Finally, our laboratory has previously reported that the analgesic effect of intrathecally delivered mixed CB receptor/Cav3.2 ligands are abolished in Cav3.2 null mice, but they retain activity upon blocking CB1 receptors with AM-281 . Interestingly, inhibition of CB2 receptors with AM-630 did attenuate the analgesic effects of these compounds and we concluded that although CB2 receptors may be involved in their actions, this may be due to CB2 receptor modulation of Cav3.2 channel activity. Hence, we cannot rule out the possibility that Δ9-THC might activate spinal CB2 receptors which may in turn inhibit Cav3.2 in addition to the direct inhibitory actions of Δ9-THC on these channels. We note that CB1 receptors do not functionally inhibit Cav3.2 in heterologous systems  but we could at that time not explore such coupling for CB2 receptors for technical reasons. Nonetheless, even if CB2 receptors augment direct inhibition of Cav3.2 channels, our results clearly implicate Cav3.2 channels as an essential target of Δ9-THC in the actions of spinal Δ9-THC as an analgesic, whereas CB receptors are not required.
Availability of data and materials
All data generated or analysed during this study are included in this published article.
Complete Freund’s adjuvant
Partial sciatic nerve injury
Harding EK, Zamponi GW. Central and peripheral contributions of T-type calcium channels in pain. Mol Brain. 2022;15:39.
Bourinet E, Alloui A, Monteil A, Barrère C, Couette B, Poirot O, et al. Silencing of the Cav3.2 T-type calcium channel gene in sensory neurons demonstrates its major role in nociception. EMBO J. 2005;24:315–24.
François A, Schüetter N, Laffray S, Sanguesa J, Pizzoccaro A, Dubel S, et al. The low-threshold calcium channel Cav3.2 determines low-threshold mechanoreceptor function. Cell Rep. 2015;10:370–82.
Snutch T, Zamponi GW. Recent advances in the development of T-type calcium channel blockers for pain intervention. Br J Pharmacol. 2018;175:2375–83.
Chemin J, Monteil A, Perez-Reyes E, Nargeot J, Lory P. Direct inhibition of T-type calcium channels by the endogenous cannabinoid anandamide. EMBO J. 2001;20:7033–40.
Gadotti VM, Huang S, Zamponi GW. The terpenes camphene and alpha-bisabolol inhibit inflammatory and neuropathic pain via Cav3.2 T-type calcium channels. Mol Brain. 2021;14:166.
Ross HR, Napier I, Connor M. Inhibition of recombinant human T-type calcium channels by Delta9-tetrahydrocannabinol and cannabidiol. J Biol Chem. 2008;283:16124–34.
Harding EK, Souza IA, Gandini MA, Gadotti VM, Ali MY, Huang S, et al. Differential regulation of Cav 3.2 and Cav 2.2 calcium channels by CB1 receptors and cannabidiol. Br J Pharmacol. 2023. https://doi.org/10.1111/bph.16035.
Casey SL, Mitchell VA, Sokolaj EE, Winters BL, Vaughan CW. Intrathecal actions of the cannabis constituents Δ(9)-Tetrahydrocannabinol and cannabidiol in a mouse neuropathic pain model. Int J Mol Sci. 2022;23(15):8649.
Wang XF, Galaj E, Bi GH, Zhang C, He Y, Zhan J, et al. Different receptor mechanisms underlying phytocannabinoid- versus synthetic cannabinoid-induced tetrad effects: Opposite roles of CB1 /CB2 versus GPR55 receptors. Br J Pharmacol. 2020;177:1865–80.
García-Caballero A, Gadotti VM, Stemkowski P, Weiss N, Souza IA, Hodgkinson V, et al. The deubiquitinating enzyme USP5 modulates neuropathic and inflammatory pain by enhancing Cav3.2 channel activity. Neuron. 2014;83(5):1144–58.
Pertwee RG. The diverse CB1 and CB2 receptor pharmacology of three plant cannabinoids: Δ9-tetrahydrocannabinol, cannabidiol and Δ9-tetrahydrocannabivarin. Brit J Pharmacol. 2008;153:199–215.
Elikottil J, Guopota P, Gupta K. The analgesic potential of cannabinoids. J Opioid Manag. 2009;5:341–57.
Zimmer A, Zimmer AM, Hohmann AG, Herkenham M, Bonner TI. Increased mortality, hypoactivity, and hypoalgesia in cannabinoid CB1 receptor knockout mice. Proc Natl Acad Sci U S A. 1999;96:5780–5.
Gadotti VM, You H, Petrov RR, Berger ND, Diaz P, Zamponi GW. Analgesic effect of a mixed T-type channel inhibitor/CB2 receptor agonist. Mol Pain. 2013;9:32.
This work was supported by grants to GWZ from Alberta Innovates and the Canadian Institutes of Health Research. GWZ holds a Canada Research Chair. FTTA holds an Eyes High Fellowship from the University of Calgary.
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Figure S1: Graphical representation of the primary afferent pain pathway. Intrathecal injection of Δ9-THC induces analgesia in mice lacking either the CB1 or the CB2 receptor, but not in Cav3.2 null mice.
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Gadotti, V.d., Antunes, F.T.T. & Zamponi, G.W. Analgesia by intrathecal delta-9-tetrahydrocannabinol is dependent on Cav3.2 calcium channels. Mol Brain 16, 47 (2023). https://doi.org/10.1186/s13041-023-01036-8
- Cannabinoid receptors
- Cav3.2 channel