Effect of Forskolin on CBD-Mediated TRPV1 Desensitization
The present study shows that CBD activated calcium influx in DRG neurons at 10 and 50 µMol/L, but between 1 nMol/L to 1 µMol/L concentrations, CBD inhibited capsaicin responses by blocking the adenylyl cyclase – cAMP signaling pathway, which is essential for maintaining TRPV1 sensitization. Increased cAMP levels in neurons are generally associated with increased nociception, whereas agents that decrease cAMP synthesis have analgesic effects. Forskolin stimulation leads to activation of adenylyl cyclase with the formation of cAMP, which plays an important role in maintaining TRPV1 sensitization, as TRPV1 was shown to be sensitized when phosphorylated by PKA, and desensitized when dephosphorylated. 35 , 41 Similarly, cAMP generation in response to inflammatory mediators such as prostaglandins, and the direct activation of PKA with cAMP analogues, is known to cause behavioural hypersensitivity. 45 , 46
In contrast to Δ 9 -THC, CBD has the advantage of not being psychoactive 5 , 6 but is reported to potentiate the beneficial effects of Δ 9 -THC, to enhance its tolerability and to widen its therapeutic window. 16 The presence of CBD in cannabis preparations containing a high ratio of CBD:Δ 9 -THC may protect against the development of psychotic symptoms, compared with preparations with low CBD:Δ 9 -THC ratios. 17 The beneficial effects of CBD have been described in several experimental animal models of neurological disorders, especially epilepsy, via neuronal inhibition. In a model of paclitaxel-induced neuropathy in mice, treatment with CBD alleviated the development of allodynia. 18
In each experiment, for each neuron, the second response to capsaicin was expressed as a percentage of the first, with or without added drugs. Percent responses for each experiment were normalized to the vehicle-treated control. Averages were calculated for each group, and the non-parametric Mann–Whitney test was used to compare between groups, using Graphpad Prism software. Data are presented as mean ± s.e.m., *P<0.05 was considered statistically significant, **P<0.01, and ***P<0.001. “n” indicates the number of animals used for each group.
CBD mediates calcium influx in DRG neurons. Sample traces demonstrating calcium influx in individual adult rat DRG neurons, with 1 µMol/L CBD (A), 10 µMol/L CBD (B) and 50 µMol/L CBD (C) indicated by increase in 340/380 ratio (y-axis), and duration in seconds (x-axis). Calcium responses to 50 µMol/L CBD (D), were abolished in the presence of 10 µMol/L TRPV1 antagonist SB705498 (E), and partly restored after washout and reapplication of 50 µMol/L CBD (F). Graph showing summary of CBD dose-related calcium influx (G); arrow on y-axis indicates threshold of detection of positive responses i.e. increase of 0.02 from baseline. EC50 is estimated between 1 and 10 µMol/L CBD (*P<0.05). Dose-related increase in the proportion of neurons responding to CBD application with calcium influx (H), shows that maximum 6% neurons responded to the highest concentration (50 µMol/L) of CBD tested.
Note: Data showing the desensitization effects of CBD on capsaicin responses are reversed in the presence of forskolin.
Bilateral DRG from all levels were micro-dissected from freshly euthanized adult female Wistar rats (Charles River UK Ltd, Margate, Kent, UK) (with approvals from the Animal Welfare Ethical Review Body, Imperial College, following UK Home Office approved procedures, and in keeping with the 3Rs ARRIVE guidelines). DRG were collected in Ham’s F12 medium and enzyme digested in Ham’s F12 nutrient medium containing 0.2% collagenase and 0.5% dispase for 3 hours at 37°C, as previously described. 32 , 33 Enzyme digested tissue was triturated in modified BSF2 medium [containing 2% HIFCS, 0.1 mg/mL transferrin, 60 ng/mL progesterone, 0.16 μg/mL sodium selenite, 3 mg/mL bovine serum albumin (BSA), penicillin/streptomycin 100 μg/mL each, 16 μg/mL putrescine, 10 μg/mL insulin], soybean trypsin inhibitor and DNAse to obtain a neuronal suspension. One rat was used for each experiment, and the neuron suspension was plated on several poly-l-lysine and laminin (20 μg/mL each) coated glass-bottom MatTek dishes (MatTek Corp, USA), at 5000 neurons per dish for calcium imaging studies. Two-milliliter BSF2 medium supplemented with 100 ng/mL of NGF and 50 ng/mL GDNF were added to all culture dishes and incubated at 37°C in a humidified environment of 5% CO2 in air, for at least 48 hours before further study.
Results: There was a significant interaction between genotype and disease (F(6,154)=24.35; P<0.0001) and main effects of genotype (F(1,154)=169.6; P<0.0001) and treatment (F(6,154)=113.4; P<0.001) upon CC50 which arose from significantly attenuated responses in TRPV1−/− mice to CBD (50 & 100 mg/kg; each P<0.0001), diazepam (P<0.001) and capsazepine (P<0.05) when compared to WT. No significant difference in brain CBD concentrations was noted between WT and TRPV1−/− mice (P>0.05).
GW Research Ltd
Objective: Evaluate effect of CBD upon seizure threshold in wild-type (WT) and TRPV1 knockout (TRPV1−/−) mice.
Design/Methods: Mice received intraperitoneal CBD (10–200 mg/kg; GW Pharmaceuticals), CBD vehicle (ethanol:kolliphorEL:saline, 1:1:18 ratio) 60 minutes before testing, the TRPV1 antagonist capsazepine (10 mg/kg in 2% DMSO), or diazepam (2.5 mg/kg in saline) 30 minutes before testing (n=12/group). A constant current stimulus (1–300 mA; 0.1 s duration) was delivered via corneal electrodes and mice assessed for tonic hind limb extension. Stimulus intensity was varied by a shock titration method and the current required to produce maximal seizures in 50% of animals tested (CC50) values calculated. Statistical analysis was performed using two-way ANOVA with post-hoc Sidak’s tests. Brain and plasma CBD concentrations were assessed by liquid chromatography/tandem mass spectrometry.
Conclusions: CBD dose-dependently increased seizure threshold in WT mice, an effect markedly reduced in TRPV1−/− mice. Brain CBD concentrations were consistent with those required for TRPV1 activation and desensitization, suggesting that CBD’s anticonvulsant effects in acute, generalized seizures are partially mediated via TRPV1 receptor interaction.
Study Supported by:
Background: TRPV1 receptor expression is increased in rodent epilepsy models and temporal lobe epilepsy patients. CBD is a TRPV1 agonist that rapidly desensitizes TRPV1. A pharmaceutical formulation of purified CBD has been shown to reduce seizure frequency in Lennox-Gastaut and Dravet syndrome patients.