Effects of cannabinoids on [35S]GTPγS binding in specific regions of the mouse brain

Imogen Dennis, Ben Whalley, Gary Stephens. University of Reading, Reading, United Kingdom.

Recent work in our group has shown that the phytocannabinoid Δ9-tetrahydrocannabivarin (Δ9-THCV) and the CB1–selective agent AM251 increase inhibitory neurotransmission in mouse cerebellum and, also, exhibit anticonvulsant activity in a piriform cortical (PC) brain slice model of epilepsy (Ma et al.; Weston et al., BPS Oxford Meeting 2006 abstracts). Possible modes of action for Δ9-THCV and AM251 include antagonism (by reducing endocannabinoid tone) or inverse agonism (at constitutively active receptors). Using whole mouse brain membranes, Δ9-THCV has recently been reported to act as a CB1 receptor antagonist (Thomas et al., 2005). Here, we investigate the effects of Δ9-THCV and synthetic cannabinoids on [35S]GTPγS binding in specific regions of the mammalian brain, including the cerebellum and PC, to determine mode of action and binding characteristics of synthetic and plant-derived cannabinoids.

Cerebellar and PC membranes were prepared from 3-5 week old male TO mice (10-20g). [35S]GTPγS binding assays were performed (minimum 3 experiments) to establish log concentration-response curves for CB1/CB2 receptor agonists WIN55212-2 and CP-55940, the antagonist/inverse agonist AM251 (all 10pM-10μM), and Δ9-THCV (10pM-100μM).

In cerebellar (Fig. 1) and PC membranes, WIN-55,212-2 and CP-55,940 showed a concentration-dependent agonist action. AM251 depressed basal [35S]GTPγS binding at all concentrations; whilst Δ9-THCV had no clear effects at sub-μM concentrations, but did reduce [35S]GTPγS binding at higher concentrations (Fig. 1). AM251 (100pM-10nM) and Δ9-THCV (100nM-5μM; Fig. 1) also produced rightward shifts in log concentration-response curves for WIN-55,212-2. Schild analyses gave KB values of 28pM (AM251; n=4) and 7nM (Δ9-THCV; n=3). In addition, basal [35S]GTPγS binding was consistently higher in PC compared to cerebellar membranes.

These results suggest that AM251 and Δ9-THCV can act as CB1 receptors antagonists in the cerebellum, with AM251 having a higher potency than Δ9-THCV. AM251 and Δ9-THCV (at higher concentrations only) also show inverse agonist effects. These findings on isolated membranes complement recent electrophysiological studies.

Thomas et al. (2005) Br J Pharmacol 146: 917-926