Abstract
<jats:p> The endocannabinoid system modulates diverse physiological processes via two endogenous lipid ligands, 2-arachidonoylglycerol (2-AG) and anandamide (AEA); however, their specific spatiotemporal dynamics remain poorly understood owing to the lack of selective probes. Here, we developed GRAB <jats:sub>2-AG2.0</jats:sub> and GRAB <jats:sub>AEA2.0</jats:sub> , two genetically encoded fluorescent sensors that selectively detect 2-AG and AEA, respectively. Both sensors exhibited high apparent affinity and molecular specificity for their respective ligands, enabling the real-time detection of 2-AG and AEA release evoked by electrical stimulation in cultured neurons and acute brain slices. In freely behaving mice, these sensors revealed ligand- and context-specific eCB dynamics: aversive stimulation preferentially evoked 2-AG, whereas psychoactive drugs produced distinct 2-AG and AEA responses. Notably, Δ9-THC elicited a sustained 2-AG signal in the nucleus accumbens shell, and local deletion of Dagla markedly attenuated both this signal and Δ9-THC-induced hypolocomotion. These sensors therefore enable detecting 2-AG and AEA signaling seperately and reveal an endogenous 2-AG component of the behavioral response to Δ9-THC. </jats:p>