Inhibition of Diacylglycerol Lipase Impairs Fear Extinction in Mice

Abstract

Elucidating the underlying molecular mechanisms regulating fear and extinction learning may offer insights that can lead to novel treatments for debilitating anxiety and trauma-related disorders including posttraumatic stress disorder. The endocannabinoid (eCB) system is a retrograde inhibitory signaling pathway involved in regulating central responses to stress. The eCB 2-arachidonoylglycerol (2-AG) has recently been proposed to serve as a homeostatic signal mitigating adverse effects of stress exposure, however, less well understood is 2-AG's role in fear learning and fear extinction. In this study, we have sought to explore 2-AG's role in fear conditioning and fear extinction by disrupting 2-AG synthesis utilizing the DAGL inhibitor (DO34) and DAGLα knock-out mice (DAGLα^-/-). We found that DAGLα^-/- mice, and male and female C57B6/J mice treated with DO34, exhibited impairment in extinction learning in an auditory cue fear-conditioning paradigm. DO34 did not increase unconditioned freezing. Interestingly, inhibition of fatty-acid amide hydrolase was not able to restore normal fear extinction in DO34-treated mice suggesting increased Anandamide cannot compensate for deficient 2-AG signaling in the regulation of fear extinction. Moreover, augmentation of CB1R signaling with tetrahydrocannabinol also failed to restore normal fear extinction in DO34-treated mice. Overall, these data support the hypothesis that DAGLα plays an important role in fear extinction learning. Although genetic and pharmacological disruption of DAGL activity causes widespread lipidomic remodeling, these data combined with previous studies putatively suggest that deficient 2-AG signaling could be a susceptibility endophenotype for the development of trauma-related psychiatric disorders.

Keywords: 2-arachidonoylglycerol; FAAH; cannabinoid; endocannabinoid; extinction; fear; stress.

FIGURE 1

Male and female DAGLα^−/− mice have impaired fear extinction. (A) Schematic diagram of the experimental paradigm. (B) (Far left panel) % freezing by male DAGLα^−/− and WT mice during acquisition of cue-conditioned fear. (Middle panels) % freezing during auditory cue during extinction training days 2 and 3. (Far right panel) % freezing during extinction recall (n = 5 WT, n = 4 KO mice). (C) (Far left panel) % freezing by female DAGLα^−/− and WT mice during acquisition of cue-conditioned fear. (Middle panels) % freezing during auditory cue during extinction training days 2 and 3. (Far right panel) % freezing during extinction recall (n = 5 WT, n = 4 KO mice). F- and P-values and η² for genotype effect shown in each panel. All values are presented as mean ± SEM.

FIGURE 2

Pharmacological DAGL inhibition does not affect acquisition of conditioned-fear. (A) Schematic diagram of the experimental paradigm. (B) (Far left panel) % freezing by C57BL/6J male mice during acquisition of cue-conditioned fear when DO34 (50 mg kg⁻¹) was injected IP 2 h prior to trial. (Middle panels) % freezing during auditory cue during extinction training days 2 and 3. (Far right panel) % freezing during extinction recall (n = 20 male mice per condition). F- and P-values and η² for main effect of drug treatment show in each panel. All values are presented as mean ± SEM.

FIGURE 3

Pharmacological DAGL inhibition impairs fear extinction. (A) Schematic diagram of the experimental paradigm. (B) (Far left panel) % freezing by C57BL/6J male mice during acquisition of cue-conditioned fear. (Middle panels) % freezing during auditory cue by mice during extinction training days 2 and 3 when D034 (50 mg kg⁻¹) was injected IP 2 h prior to trial. (Far right panel) % freezing during extinction recall (n = 17 DO34-treated male mice, n = 18 vehicle-treated male mice). (C) (Far left panel) % freezing by C57BL/6J female mice during acquisition of cue-conditioned fear when DO34 (50 mg kg⁻¹) was injected IP 2 h prior to trial. (Middle panels) % freezing during auditory cue during extinction training days 2 and 3. (Far right panel) % freezing during extinction recall (n = 18 DO34-treated female mice n = 20 vehicle-treated female mice). F- and P-values and η² for main effects of drug treatment shown in each panel. All values are presented as mean ± SEM.

FIGURE 4

Pharmacological DAGL inhibition does not affect unconditioned freezing. (A) Schematic diagram of the experimental paradigm. (B) (Far left panel) % freezing by C57BL/6J male mice during sham-conditioning assay-no shock was administered after tone. (Middle panels) % freezing during auditory cue during extinction training days 2 and 3. (Far right panel) % freezing during extinction recall (n = 15 male mice per condition). F- and P-values and η² for main effect of drug treatment shown in each panel. All values are presented as mean ± SEM.

FIGURE 5

AEA augmentation does not reverse impaired extinction after DAGL inhibition. (A) Schematic diagram of the experimental paradigm. (B) (Far left panel) % freezing by C57BL/6J male mice during acquisition of cue-conditioned fear. (Middle panels) % freezing during auditory cue by mice during extinction training days 2 and 3 when DO34 (50 mg kg⁻¹) was injected alone or in addition to PF-3845 (FAHH inhibitor 1 mg kg⁻¹) IP 2 h prior to trial. (Far right panel) % freezing during extinction recall (n = 18 male mice per condition). F- and P-values and η² for main effect of drug shown in each panel. All values are presented as mean ± SEM.