The endocannabinoid system functions as a significant neuromodulatory network, playing a central role in molecular processes associated with stress, anxiety, and fear (Lutz et al., 2015; Scheyer et al., 2023) and in neurodegenerative diseases (Wilkerson et al., 2021; Pasquarelli et al., 2015). Its primary molecular targets are the cannabinoid receptors (CB receptors). Indeed, the CB1 receptor is the most abundantly expressed G protein-coupled receptor in the brain (Busquets-Garcia et al., 2018). This receptor is activated by two principal endogenous ligands: the endocannabinoids N-arachidonyl ethanolamide (anandamide, AEA) and 2-arachidonyl glycerol (2-AG) (Hillard, 2018). Endocannabinoids are synthesized and released “on demand” in the post-synaptic neuron (Hillard, 2018; Straub et al., 2024), where they traverse the synaptic cleft retrogradely to bind to CB1 receptors, which are predominantly localized on pre-synaptic terminals. Activation of CB1 receptors by endocannabinoids triggers a negative feedback mechanism that broadly suppresses synaptic activity (Scheyer et al., 2023). Endocannabinoid breakdown is primarily mediated by two hydrolytic enzymes: monoacylglycerol lipase (MAGL), which metabolizes 2-AG and fatty acid amide hydrolase (FAAH), which degrades AEA (Di Marzo, 2006).

The endocannabinoid system plays a critical role in regulating stress response. Acute stress is typically associated with a transient reduction in brain AEA levels in regions such as the amygdala, prefrontal cortex, hippocampus, and hypothalamus (Hill et al., 2018; Lutz et al., 2015; Morena et al., 2016). This decrease facilitates rapid adaptation to stress and contributes to the regulation of stress and anxiety (Petrie et al., 2021). In humans, threat conditioning has been shown to reduce AEA levels, with this reduction correlating with increased amygdala activity during threat learning (Weisser et al., 2022). Mechanistically, this effect is thought to be mediated by AEA's role in facilitating excitatory activity in principal neurons via disinhibition of parvalbumin-positive interneurons in the lateral amygdala (Lesuis et al., 2024). The pivotal role of the endocannabinoid system in stress regulation makes its dysregulation a key factor in the development of stress-related disorders. Altered plasma endocannabinoid levels have been observed in various psychiatric conditions, including post-traumatic stress disorder (Hill et al., 2013; deRoon-Cassini et al., 2022; Marusak et al., 2024), depression (Mazurka et al., 2024; Walther et al., 2023), substance use disorders (Kroll et al., 2023, 2024) and non-suicidal self-injurious behavior (Ferger et al., 2024). Position emission tomography (PET) imaging has been particularly informative in elucidating endocannabinoid system role in specific brain regions in psychiatric disorders and identified the amygdala as a key region of interest. Increased FAAH binding in the amygdala and prefrontal cortex has been reported in individuals with borderline personality disorder (BPD), with FAAH binding in these regions positively correlating with measures of hostility in BPD patients (Kolla et al., 2020). In healthy participants, lower FAAH levels in regions such as the amygdala, medial prefrontal cortex, cingulate cortex, and hippocampus have been associated with a reduced amygdala response to threatening social cues (Green et al., 2022). Furthermore, FAAH levels in the amygdala negatively correlated with amygdala functional connectivity (Green et al., 2021).

The growing body of evidence highlighting the role of the endocannabinoid system in psychiatric disorders has spurred interest in targeting this system as a novel therapeutic approach, with the primary goal of increasing endocannabinoid concentrations, as this has demonstrated positive effects on stress and fear-related behaviors both in animals and humans (Mayo et al., 2020, 2022). Discovery of inhibitors of its main hydrolyzing enzyme FAAH, made a substantial increase in AEA possible (Tripathi, 2020). Subsequently, similar advancements were made for 2-AG by targeting its degradation via MAGL inhibition (Granchi et al., 2017; Bononi et al., 2021). In recent years, numerous FAAH and MAGL inhibitors have been developed and investigated across various therapeutic areas (Di Marzo, 2018; Hill et al., 2023; Mayo et al., 2025), with a particular focus on psychiatric disorders. One of the most widely studied FAAH inhibitors is JNJ-42165279, a highly selective and reversible FAAH inhibitor (Postnov et al., 2018), which has been evaluated in clinical trials for patients with Social Anxiety Disorder (Schmidt et al., 2021), Major Depression with Anxious Distress (NCT02498392), and Autism Spectrum Disorder (Klein et al., 2024). Although FAAH inhibition has not demonstrated efficacy in Major Depressive Disorder, clinical trials have reported significant improvements in several secondary endpoints for patients with Social Anxiety Disorder and Autism Spectrum Disorder (Schmidt et al., 2021; Klein et al., 2024). In contrast, fewer trials have been conducted with MAGL inhibitors. Among these, elcubragistat (formerly ABX-1431; Lu-AG06466) the most commonly clinical studied compound and a highly selective and irreversible MAGL inhibitor (Cisar et al., 2018) has been tested in psychiatric indications like Tourette Syndrome. An initial phase 1b trial successfully met its primary endpoint; however, a subsequent phase 2 trial failed to confirm the efficacy of MAGL inhibition for tic reduction in a larger patient cohort with Tourette Syndrome (Müller-Vahl et al., 2021).

Determining changes in the levels of 2-AG and AEA provides a crucial means of evaluating the efficacy of enzyme inhibition as a target modulation measurement for MAGL and FAAH inhibitors in the brain regions of interest. Since endocannabinoids exert their effects via release into the extracellular space, in vivo microdialysis is one of the most suitable sampling technique to assess their levels and the efficacy of enzyme inhibitors.

Here, we demonstrate the effects of MAGL and FAAH inhibitors on 2-AG and AEA levels in both extracellular microdialysate and tissue. Microdialysate samples were collected from the basolateral amygdala (BLA) and nucleus accumbens (NAcc) of adult male Wistar rats to evaluate dynamic changes in endocannabinoid levels. In a supplementary study, we investigated the effects of the same compounds on endocannabinoid levels in postmortem BLA tissue from C57BL/6 mice.