Discovery of a NAPE-PLD inhibitor that modulates emotional behavior in mice.

N-acylethanolamines (NAEs), which include the endocannabinoid anandamide, represent an important family of signaling lipids in the brain. The lack of chemical probes that modulate NAE biosynthesis in living systems hamper the understanding of the biological role of these lipids. Using a high-throughput screen, chemical proteomics and targeted lipidomics, we report here the discovery and characterization of LEI-401 as a CNS-active N-acylphosphatidylethanolamine phospholipase D (NAPE-PLD) inhibitor. LEI-401 reduced NAE levels in neuroblastoma cells and in the brain of freely moving mice, but not in NAPE-PLD KO cells and mice, respectively. LEI-401 activated the hypothalamus-pituitary-adrenal axis and impaired fear extinction, thereby emulating the effect of a cannabinoid CB1 receptor antagonist, which could be reversed by a fatty acid amide hydrolase inhibitor. Our findings highlight the distinctive role of NAPE-PLD in NAE biosynthesis in the brain and suggest the presence of an endogenous NAE tone controlling emotional behavior.

A cortico-amygdala neural substrate for endocannabinoid modulation of fear extinction.

Preclinical and clinical studies implicate endocannabinoids (eCBs) in fear extinction, but the underlying neural circuit basis of these actions is unclear. Here, we employed in vivo optogenetics, eCB biosensor imaging, ex vivo electrophysiology, and CRISPR-Cas9 gene editing in mice to examine whether basolateral amygdala (BLA)-projecting medial prefrontal cortex (mPFC) neurons represent a neural substrate for the effects of eCBs on extinction. We found that photoexcitation of mPFC axons in BLA during extinction mobilizes BLA eCBs. eCB biosensor imaging showed that eCBs exhibit a dynamic stimulus-specific pattern of activity at mPFC→BLA neurons that tracks extinction learning. Furthermore, using CRISPR-Cas9-mediated gene editing, we demonstrated that extinction memory formation involves eCB activity at cannabinoid CB1 receptors expressed at vmPFC→BLA synapses. Our findings reveal the temporal characteristics and a neural circuit basis of eCBs' effects on fear extinction and inform efforts to target the eCB system as a therapeutic approach in extinction-deficient neuropsychiatric disorders.

Selective sub-nucleus effects of intra-amygdala oxytocin on fear extinction.

There is growing evidence that the neuropeptide oxytocin (OT) modulates fear and extinction in humans and rodents through actions in corticolimbic circuits including the central amygdala (CeA). Prior studies have, however, been limited to subjects that exhibit intact basal extinction, rather than extinction-impaired populations that could potentially therapeutically benefit from viable OT-targeting treatments. Here, we assessed the effects of pre-extinction training infusion of OT into the CeA, or basolateral amygdala (BLA), on extinction in an inbred mouse strain (S1) model of impaired extinction. We found that intra-CeA OT, at a dose of 0.01 µg, enabled extinction memory formation, as evidenced by lesser freezing as compared to vehicle-infused controls on a drug-free retrieval test. Conversely, infusion of a higher, 1.0 µg OT dose, markedly reduced freezing and increased grooming during extinction training and produced elevated freezing on drug-free retrieval. Infusion of the 0.01 µg dose into the BLA was without behavioral effects. Together, our data show that OT acts in a dose-dependent manner within the CeA to promote extinction in otherwise extinction-deficient mice. These findings provide further support for the potential utility of OT as an adjunctive treatment to extinction-based therapies for trauma and anxiety disorders.