CB1R blockade unmasks TRPV1-mediated contextual fear generalization in female, but not male rats.

Increasing evidence suggests that the neurobiological processes that govern learning and memory can be different in males and females, but many of the specific mechanisms underlying these sex differences have not been fully defined. Here we investigated potential sex differences in endocannabinoid (eCB) modulation of Pavlovian fear conditioning and extinction, examining multiple defensive behaviors, including shock responsivity, conditioned freezing, and conditioned darting. We found that while systemic administration of drugs acting on eCB receptors did not influence the occurrence of darting, females that were classified as Darters responded differently to the drug administration than those classified as Non-darters. Most notably, CB1R antagonist AM251 produced an increase in cue-elicited freezing and context generalization selectively in female Non-darters that persisted across extinction and extinction retrieval tests but was prevented by co-administration of TRPV1R antagonist Capsazepine. To identify a potential synaptic mechanism for these sex differences, we next employed biochemical and neuroanatomical tracing techniques to quantify anandamide (AEA), TRPV1R, and perisomatic CB1R expression, focusing on the ventral hippocampus (vHip) given its known role in mediating contextual fear generalization. These assays identified sex-specific effects of both fear conditioning-elicited AEA release and vHip-BLA circuit structure. Together, our data support a model in which sexual dimorphism in vHip-BLA circuitry promotes a female-specific dependence on CB1Rs for context processing that is sensitive to TRPV1-mediated disruption when CB1Rs are blocked.

CB1R blockade unmasks TRPV1-mediated contextual fear generalization in female, but not male rats.

Increasing evidence suggests that the neurobiological processes that govern learning and memory can be different in males and females, and here we asked specifically whether the endocannabinoid (eCB) system could modulate Pavlovian fear conditioning in a sex-dependent manner. Systemic (i.p.) injection of CB1R antagonist AM251 in adult male and female Sprague Dawley rats prior to auditory cued fear conditioning produced a female-specific increase in freezing that persisted across extinction and extinction retrieval tests but was prevented by co-administration of TRPV1R antagonist Capsazepine. Notably, AM251 also produced robust freezing in a novel context prior to auditory cue presentation the day following drug administration, but not the day of, suggesting that CB1R blockade elicited contextual fear generalization in females. To identify a potential synaptic mechanism for these sex differences, we next used liquid chromatography/tandem mass spectrometry, Western Blot, and confocal-assisted immunofluorescence techniques to quantify anandamide (AEA), TRPV1R, and perisomatic CB1R expression, respectively, focusing on the ventral hippocampus (vHip). Fear conditioning elicited increased vHip AEA levels in females only, and in both sexes, CB1R expression around vHip efferents targeting the basolateral amygdala (BLA) was twice that at neighboring vHip neurons. Finally, quantification of the vHip-BLA projections themselves revealed that females have over twice the number of neurons in this pathway that males do. Together, our data support a model in which sexual dimorphism in vHip-BLA circuitry promotes a female-specific dependence on CB1Rs for context processing that is sensitive to TRPV1-mediated disruption when CB1Rs are blocked.

Sex and stressor modality influence acute stress-induced dynamic changes in corticolimbic endocannabinoid levels in adult Sprague Dawley rats.

Research over the past few decades has established a role for the endocannabinoid system in contributing to the neural and endocrine responses to stress exposure. The two endocannabinoid ligands, anandamide (AEA) and 2-arachidonoyl glycerol (2-AG), both play roles in regulating the stress response and both exhibit dynamic changes in response to stress exposure. Most of this previous research, however, was conducted in male rodents. Given that, especially in rodents, the stress response is influenced by sex, an understanding of how these dynamic responses of endocannabinoids in response to stress is influenced by sex could provide insight into sex differences of the acute stress response. We exposed adult, Sprague Dawley rats to different commonly utilized acute stress modalities, specifically restraint, swim and foot shock stress. Thirty minutes following stress onset, we excised the amygdala, hippocampus and medial prefrontal cortex, corticolimbic brain regions involved in the stress response, to measure endocannabinoid levels. When AEA levels were altered in response to restraint and swim stress, they were reduced, whereas exposure to foot shock stress led to an increase in the amygdala. 2-AG levels, when they were altered by stress exposure were only increased, specifically in males in the amygdala following swim stress, and in the hippocampus and medial prefrontal cortex overall following foot shock stress. This increase in 2-AG levels following stress only in males was the only sex difference found in stress-induced changes in endocannabinoid levels. There were no consistent sex differences observed. Collectively, these data contribute to our further understanding of the interactions between stress and endocannabinoid function.

Genetic Variants of Fatty Acid Amide Hydrolase Modulate Acute Inflammatory Responses to Colitis in Adult Male Mice.

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)-1ß, 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.

Amygdalar endocannabinoids are affected by predator odor stress in a sex-specific manner and modulate acoustic startle reactivity in female rats.

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.

Endocannabinoids, cannabinoids and the regulation of anxiety.

Cannabis has been used for hundreds of years, with its ability to dampen feelings of anxiety often reported as a primary reason for use. Only recently has the specific role cannabinoids play in anxiety been thoroughly investigated. Here we discuss the body of evidence describing how endocannabinoids and exogenous cannabinoids are capable of regulating the generation and termination of anxiety states. Disruption of the endogenous cannabinoid (eCB) system following genetic manipulation, pharmacological intervention or stress exposure reliably leads to the generation of an anxiety state. On the other hand, upregulation of eCB signaling is capable of alleviating anxiety-like behaviors in multiple paradigms. When considering exogenous cannabinoid administration, cannabinoid receptor 1 (CB1) agonists have a biphasic, dose-dependent effect on anxiety such that low doses are anxiolytic while high doses are anxiogenic, a phenomenon that is evident in both rodent models and humans. Translational studies investigating a loss of function mutation in the gene for fatty acid amide hydrolase, the enzyme responsible for metabolizing AEA, have also shown that AEA signaling regulates anxiety in humans. Taken together, evidence reviewed here has outlined a convincing argument for cannabinoids being powerful regulators of both the manifestation and amelioration of anxiety symptoms, and highlights the therapeutic potential of targeting the eCB system for the development of novel classes of anxiolytics. This article is part of the special issue on 'Cannabinoids'.

Sex-dependent effects of endocannabinoid modulation of conditioned fear extinction in rats.

BACKGROUND AND PURPOSE: Women are twice as likely as men to develop post-traumatic 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 cannabinoid1 (CB1 ) 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.