Search

IntroductionThe endocannabinoids (eCBs), 2-arachidonoylglycerol (2-AG) and arachidonoyl ethanolamine (AEA), are produced by separate enzymatic pathways, activate cannabinoid receptors with distinct pharmacology, and differentially regulate pathophysiological processes. The genetically encoded sensor, GRABeCB2.0, detects real-time changes in eCB levels in cells in culture and preclinical model systems; however, its activation by eCB analogues produced by cells and by phyto-cannabinoids remains uncharacterized, a current limitation when interpreting changes in its response. This information could provide additional utility for the tool in in vivo pharmacology studies of phyto-cannabinoid action.MethodsGRABeCB2.0was expressed in cultured HEK293 cells. Live cell confocal microscopy and high-throughput fluorescent signal measurements.Results2-AG increased GRABeCB2.0fluorescent signal (EC50= 85 nM), and the cannabinoid 1 receptor (CB1R) antagonist, SR141617, decreased GRABeCB2.0signal (SR1, IC50= 3.3 nM), responses that mirror their known potencies at cannabinoid 1 receptors (CB1R). GRABeCB2.0fluorescent signal also increased in response to AEA (EC50= 815 nM), the eCB analogues 2-linoleoylglycerol and 2-oleoylglycerol (2-LGand2-OG, EC50s = 1.5 and 1.0 μM, respectively), Δ9-tetrahydrocannabinol (Δ9-THC) andΔ8-THC(EC50s = 1.6 and 2.0 μM, respectively), and the artificial CB1R agonist, CP55,940 (CP, EC50= 82 nM); however their potencies were less than what has been described at CB1R. Cannabidiol (CBD) did not affect basal GRABeCB2.0fluorescent signal and yet reduced the 2-AG stimulated GRABeCB2.0responses (IC50= 8.8 nM).Conclusions2-AG and SR1 modulate the GRABeCB2.0fluorescent signal with EC50s that mirror their potencies at CB1R whereas AEA, eCB analogues, THC and CP increase GRABeCB2.0fluorescent signal with EC50s significantly lower than their potencies at CB1R. CBD reduces the 2-AG response without affecting basal signal, suggesting that GRABeCB2.0retains the negative allosteric modulator (NAM) property of CBD at CB1R. This study describes the pharmacological profile of GRABeCB2.0to improve interpretation of changes in fluorescent signal in response to a series of known eCBs and CB1R ligands.

BackgroundNo preclinical approach enables the study of voluntary oral consumption of high dose Δ9-tetrahydrocannabinol (THC) and its intoxicating effects, mainly owing to the aversive response of rodents to THC that limits intake. Here we developed a palatable THC formulation and an optimized access paradigm in mice.MethodsTHC was formulated in chocolate gelatin (THC-E-gel). Adult male and female mice were allowedad libitumaccess for 2 h. Cannabimimetic responses (hypolocomotion, analgesia, and hypothermia) were measured following access. Levels of THC and its metabolites were measured in blood and brain samples. Acoustic startle responses were measured to investigate THC-induced psychotomimetic behavior.ResultsAccess to high-dose THC-E-gel (≈30 mg/kg over 2 h) resulted in robust consumption and CB1receptor-dependent behavioral responses. High-dose THC-E-gel consumption resulted in parallel accumulation of THC and its psychoactive metabolite 11-OH-THC in brain, a profile that contrasts with the known rapid decline in brain 11-OH-THC levels following intraperitoneal THC injections. High-dose THC-E-gel consumption increased the acoustic startle response preferentially in males, and this psychotomimetic response was remarkably different from the response triggered by intraperitoneal contingent administration of THC. Comparing cannabimimetic responses elicited by intraperitoneal versus oral administration enabled us to model a “predicted dose” of THC that triggers these responses.ConclusionVoluntary consumption of high-dose THC-E-gel triggered equivalent cannabimimetic responses in male and female mice but an increased acoustic startle response preferentially in males. These findings indicate that THC-E-gel offers a robust preclinical consumption model to study cannabimimetic responses in mice, including sex-dependent psychotomimetic responses.

The most abundant endocannabinoid (eCB) in the brain, 2-arachidonoyl glycerol (2-AG), is hydrolyzed by α/β-hydrolase domain containing 6 (ABHD6); yet how ABHD6 controls stimuli-dependent increases in 2-AG production is unknown. To explore this question, we leveraged the recently developed 2-AG sensor, GRABeCB2.0, and found that stimulation of Neuro2a cells in culture with bradykinin (BK) acting at metabotropic B2K receptors and ATP acting at ionotropic P2X7 receptors led to differential increases in 2-AG levels. B2K triggered increases in 2-AG levels via diacylglycerol lipase (DAGL), and this mechanism was potentiated by increases in intracellular calcium and ABHD6 inhibition. By contrast, P2X7-triggered increases in 2-AG levels were dependent on DAGL and extracellular calcium but unaffected by ABHD6 inhibition. Thus, ABHD6 preferentially regulates metabotropic-dependent increases in 2-AG levels over ionotropic-dependent increases in 2-AG levels. Our study indicates that ABHD6 selectively controls stimuli-dependent increases in 2-AG production and emphasizes its specific role in eCB signaling.