Genetic deletion of NAPE-PLD alters stress responsiveness and HPA-axis functionality in a context-dependent manner in mice.

The endocannabinoid (eCB) system regulates stress responsiveness and hypothalamic-pituitary-adrenal (HPA) axis activity. The enzyme N-acyl phosphatidylethanolamine phospholipase-D (NAPE-PLD) is primarily responsible for the synthesis of the endocannabinoid signaling molecule anandamide (AEA) and other structurally related lipid signaling molecules known as N-acylethanolamines (NAEs). However, little is known about how activity of this enzyme affects behavior. As AEA plays a regulatory role in stress adaptation, we hypothesized that reducing synthesis of AEA and other NAEs would dysregulate stress reactivity. To test this hypothesis, we evaluated wild type (WT) and NAPE-PLD knockout (KO) mice in behavioral assays that assess stress responsiveness and anxiety-like behavior. NAPE-PLD KO mice exhibited anxiety-like behaviors in the open field test after a period of single housing. NAPE-PLD KO mice exhibited an exaggerated freezing response at baseline but blunted response 2,3,5-trimethyl-3-thiazoline (TMT) predator odor when compared to WT mice. NAPE-PLD KO mice also exhibited a context-dependent dysregulation of HPA axis in response to TMT in the paraventricular hypothalamic nucleus at a neuronal level, as measured by c-Fos immunohistochemstry. Male, but not female, NAPE-PLD knockout mice showed higher levels of circulating corticosterone relative to same-sex wildtype mice in response to TMT exposure, suggesting a sexually dimorphic dysregulation of the HPA axis at the hormonal level. Sex specific findings observed here mirror the sexually dimorphic drug response we recently identified in NAPE-PLD KO mice (Woodward et al., 2025). Together, these findings suggest that the enzymatic activity of NAPE-PLD regulates emotional resilience and recovery from both acute and sustained stress.