Cannabidiol enhances morphine antinociception, diminishes NMDA-mediated seizures and reduces stroke damage via the sigma 1 receptor

Abstract

Cannabidiol (CBD), the major non-psychotomimetic compound present in the Cannabis sativa plant, exhibits therapeutic potential for various human diseases, including chronic neurodegenerative diseases, such as Alzheimer's and Parkinson's, ischemic stroke, epilepsy and other convulsive syndromes, neuropsychiatric disorders, neuropathic allodynia and certain types of cancer. CBD does not bind directly to endocannabinoid receptors 1 and 2, and despite research efforts, its specific targets remain to be fully identified. Notably, sigma 1 receptor (σ1R) antagonists inhibit glutamate N-methyl-D-aspartate acid receptor (NMDAR) activity and display positive effects on most of the aforesaid diseases. Thus, we investigated the effects of CBD on three animal models in which NMDAR overactivity plays a critical role: opioid analgesia attenuation, NMDA-induced convulsive syndrome and ischemic stroke. In an in vitro assay, CBD disrupted the regulatory association of σ1R with the NR1 subunit of NMDAR, an effect shared by σ1R antagonists, such as BD1063 and progesterone, and prevented by σ1R agonists, such as 4-IBP, PPCC and PRE084. The in vivo administration of CBD or BD1063 enhanced morphine-evoked supraspinal antinociception, alleviated NMDA-induced convulsive syndrome, and reduced the infarct size caused by permanent unilateral middle cerebral artery occlusion. These positive effects of CBD were reduced by the σ1R agonists PRE084 and PPCC, and absent in σ1R^-/- mice. Thus, CBD displays antagonist-like activity toward σ1R to reduce the negative effects of NMDAR overactivity in the abovementioned experimental situations.

Keywords: Acute pain; Cannabidiol; Cannabinoids; Epilepsy; NMDA receptor; Neuropathology; Sigma 1 receptor; Stroke.

Fig. 1

CBD disrupts the association of σ1R with the NR1 subunits of NMDA receptors. In vitro assay determining ligand activity for σ1R. NHS-activated Sepharose beads covalently coupled to a sequence of the NR1 subunit containing seven residues of the transmembrane region plus C0-C1-C2 cytosolic segments were incubated with excess σ1R (1:3). Unbound σ1R was washed out, and the NR1 C1-coupled σ1R was exposed to serial concentrations of the ligands under study. σ1R that remained attached to the NR1 subunits was then evaluated by SDS-PAGE and immunoblotting. a Inhibitory effects of the σ1R antagonists progesterone and BD106 and of CBD on the association of σ1R with the NR1 C1 subunit. The assays were performed in the presence of 50 mM Tris-HCl (pH 7.5), 0.2% CHAPS and 2.5 mM calcium. Representative blots are shown. The ED50 values were computed using the software SigmaPlot v.14. b The σ1R agonists PPCC, PRE084 and 4-IBP did not alter σ1R-NR1 associations but reduced the capacity of progesterone and CBD to disrupt such associations. PPCC reduced the capacity of CBD to inhibit the binding of σ1R to NR1 subunit in a concentration-dependent manner. The assays were performed twice, and each point was duplicated. *Significant difference with respect to the control group (DMSO or saline); φ significant difference with respect to the group receiving only CBD or progesterone; ANOVA, Dunnett’s multiple comparison, p < 0.05

Fig. 2

Effect of CBD on morphine-induced supraspinal antinociception. Mice received 10 nmol CBD icv 10 min before 6 nmol morphine, and analgesia was evaluated with the thermal warm water (52 °C) “tail-flick” test at the indicated post-opioid intervals. Each point represents the mean ± SEM of data from eight to ten mice. a CBD exhibited no significant analgesic effect in this test. The analgesia produced by the combination of CBD and morphine was significantly higher than that produced by morphine alone. The σ1R agonist PPCC did not alter morphine analgesia, but icv-injection 20 min before CBD prevented the enhancement of this effect of morphine. b The σ1R agonist BD1063 did not produce analgesia in this test but increased morphine antinociception. This potentiation was absent when PPCC was injected icv 20 min before BD1063. c Morphine promotes a higher analgesic effect in σ1R^−/− mice than in wild type control mice. In σ1R^−/− mice, BD1063 and CBD did not modify morphine analgesia. *Significantly different from the control group receiving only 6 nmol morphine, φ significantly different from the effect of morphine in wild-type mice. ANOVA, Dunnett’s multiple comparison vs control group, p < 0.05

Fig. 3

Anticonvulsant effects of CBD in a mouse model of seizures induced by NMDAR overactivation. a Behavioral alterations produced by the icv administration of 1 nmol NMDA to mice. Each bar indicates the percentage of mice showing the indicated sign and represents the mean ± SEM of 8 mice. b Effects of PPCC, CBD and BD1063 on seizures induced by NMDA. The mice received the NMDAR agonist NMDA icv (1 nmol) 30 min after the drugs (3 nmol PPCC, BD1063, CBD or 5 nmol WAY100635) and were then immediately evaluated. c Effect of the 5HT1AR antagonist WAY100635 on the convulsive syndrome evoked by 0.3 nmol NMDA. d Lack of an effect of CBD and BD1063 on seizures induced by 1 nmol NMDA in σ1R^−/− mice. e Effects of the treatments on the latency and duration of the seizure episodes induced by 1 nmol NMDA in wild type and σ1R^−/− mice. *Significant difference from the control group receiving NMDA and saline instead of the drugs. φ Significant difference from the corresponding NMDA-induced behavioral signs exhibited by the group receiving only CBD or BD1063, p < 0.05. ANOVA, Dunnett’s multiple comparison vs control group, p < 0.05

Fig. 4

CBD administration diminishes ischemic brain damage in wild-type but not σ1R^−/− mice. Upper panel, representative TTC-stained brain section images obtained from saline- and drug-treated mice (1 h after surgery) 48 h after pMCAO. White indicates infarction; red staining indicates normal tissue. Lower panel, the bar graphs quantitatively compare the infarct volume based on TTC staining from the wild type and σ1R^−/− mice treated with saline, the σ1R agonists PRE084 and PPCC (white bars), or the σ1R antagonist BD1063 and CBD (gray bars) 1 h after surgery. The groups consisted of 8 to 10 mice, and the data are presented as the mean ± SEM. *Significantly different from the saline-treated mice. φ Significantly different from mice receiving only the σ1R antagonist BD1063 or CBD; ANOVA, Dunnett’s multiple comparison vs the corresponding control group, p < 0.05