Endocannabinoid (eCB) systems have been implicated in the development of stress-induced anxiety. In this study, we investigated the changes in eCB levels in the mouse brain due to restraint stress. In addition, we examined the effects of eCB-degrading enzyme inhibitors on anxiety-like behavior in restraint-stressed mice. For restraint stress, the mice were immobilized in a 50 ml syringe with holes for airflow for 30 min. After restraint stress, the contents of 2-arachidonoylglycerol (2-AG) and N-arachidonoylethanolamine (AEA) were measured via UPLC‒MS/MS. Immediately after restraint stress, 2-AG content was significantly decreased in the prefrontal cortex (PFC) and hippocampus (HC). Similarly, AEA content was decreased in the PFC and HC, although 2-AG and AEA contents did not change in the striatum, periamygdaloid cortex or medulla oblongata. On the other hand, at both 30 and 60 min after restraint stress, 2-AG content was significantly increased in the PFC and HC. However, AEA content remained reduced in the PFC for up to 30 min after stress exposure, but the significant reduction was no longer observed at 60 min. In the elevated plus-maze test, the time spent in the open arms decreased in restraint-stressed mice, which indicated the occurrence of anxiogenic behavior. This anxiogenic behavior was ameliorated by the administration of JZL184 (a monoacylglycerol lipase inhibitor) or URB597. (a fatty acid amide hydrolase inhibitor) These results suggest that eCB levels are highly responsive to time-dependent and brain region-specific changes in response to acute stress stimuli. Furthermore, restraint stress induces anxiogenic behavior, which is ameliorated by inhibitors of eCB-degrading enzymes. These findings indicate that the reduction in eCB levels in the PFC and HC may be due to the development of stress-induced anxiety.
