Background and Purpose: Women are twice as likely as men to develop posttraumatic stress disorder (PTSD) making the search for biological mechanisms underlying these gender disparities especially crucial. One of the hallmark symptoms of PTSD is an alteration in the ability to extinguish fear responses to trauma-associated cues. In male rodents, the endocannabinoid system can modulate fear extinction and has been suggested as a therapeutic target for PTSD. However, whether and how the endocannabinoid system may modulate fear expression and extinction in females remains unknown. Experimental Approach: To answer this question, we pharmacologically manipulated endocannabinoid signalling in male and female rats prior to extinction of auditory conditioned fear and measured both passive (freezing) and active (darting) conditioned responses. Key Results: Surprisingly, we found that acute systemic inhibition of the endocannabinoid anandamide (AEA) or 2-arachidonoyl glycerol (2-AG) hydrolysis did not significantly alter fear expression or extinction in males. However, the same manipulations in females produced diverging effects. Increased AEA signalling at vanilloid TRPV1 receptors impaired fear memory extinction. In contrast, inhibition of 2-AG hydrolysis promoted active over passive fear responses acutely via activation of cannabinoid 1 (CB 1) receptors. Measurement of AEA and 2-AG levels after extinction training revealed sex-and brain region-specific changes. Conclusion and Implications: We provide the first evidence that AEA and 2-AG signalling affect fear expression and extinction in females in opposite directions. These findings are relevant to future research on sex differences in mechanisms of fear extinction and may help develop sex-specific therapeutics to treat trauma-related disorders.

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Understanding sex differences in behavioral and molecular effects of stress has important implications for understanding the vulnerability to chronic psychiatric disorders associated with stress response circuitry. The amygdala is critical for emotional learning and generating behavioral responses to stressful stimuli, and preclinical studies indicate that amygdalar endocannabinoid (eCB) signaling regulates emotional states. This study measured eCB contents in the basolateral (BLA) and central (CeA) amygdala of male and female rats exposed to predator odor stress (bobcat urine) and tested for contextual avoidance 24 h later. Stressed females had lower levels of 2-arachidonoyl glycerol (2-AG) in the BLA and higher levels of anandamide (AEA) in the CeA, while exposure to bobcat urine did not affect amygdalar eCB contents in males. We previously reported that female rats exposed to bobcat urine exhibit blunted acoustic startle reactivity (ASR) 48 h after predator odor stress. Therefore, we tested the hypothesis that intra-BLA injection of a diacylglycerol lipase (DAGL) inhibitor (which would be expected to reduce 2-AG levels in BLA) and intra-CeA injection of a fatty acid amide hydrolase (FAAH) inhibitor (which would be expected to increase AEA levels in CeA) would mimic previously observed predator odor stress-induced reductions in ASR. Contrary to our hypothesis, microinjections of either the DAGL inhibitor DO34 into the BLA or the FAAH inhibitor URB597 into the CeA significantly increased ASR in females compared to vehicle-treated rats. These findings describe sex-specific effects of predator odor stress on amygdalar eCBs, and new roles for amygdalar eCBs in regulating behavior in females.

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Cannabinoids, including cannabis derived phytocannabinoids and endogenous cannabinoids (endocannabinoids), are typically considered anti-inflammatory. One such endocannabinoid is N-arachidonoylethanolamine (anandamide, AEA), which is metabolized by fatty acid amide hydrolase (FAAH). In humans, there is a loss of function single nucleotide polymorphism (SNP) in the FAAH gene (C385A, rs324420), that leads to increases in the levels of AEA. Using a mouse model with this SNP, we investigated how this SNP affects inflammation in a model of inflammatory bowel disease. We administered 2,4,6-trinitrobenzene sulfonic acid (TNBS) intracolonically, to adult male FAAH SNP mice and examined colonic macroscopic tissue damage and myeloperoxidase activity, as well as levels of plasma and amygdalar cytokines and chemokines 3 days after administration, at the peak of colitis. We found that mice possessing the loss of function alleles (AC and AA), displayed no differences in colonic damage or myeloperoxidase activity compared to mice with wild type alleles (CC). In contrast, in plasma, colitis-induced increases in interleukin (IL)-2, leukemia inhibitory factor (LIF), monocyte chemoattractant protein (MCP)-1, and tumor necrosis factor (TNF) were reduced in animals with an A allele. A similar pattern was observed in the amygdala for granulocyte colony stimulating factor (G-CSF) and MCP-1. In the amygdala, the mutant A allele led to lower levels of IL-1α, IL-9, macrophage inflammatory protein (MIP)-1β, and MIP-2 independent of colitis—providing additional understanding of how FAAH may serve as a regulator of inflammatory responses in the brain. Together, these data provide insights into how FAAH regulates inflammatory processes in disease.