Upregulation of Anandamide Hydrolysis in the Basolateral Complex of Amygdala Reduces Fear Memory Expression and Indices of Stress and Anxiety.

Increased anandamide (AEA) signaling through inhibition of its catabolic enzyme fatty acid amide hydrolase (FAAH) in the basolateral complex of amygdala (BLA) is thought to buffer against the effects of stress and reduces behavioral signs of anxiety and fear. However, examining the role of AEA signaling in stress, anxiety, and fear through pharmacological depletion has been challenging due to the redundant complexity of its biosynthesis and the lack of a pharmacological synthesis inhibitor. We developed a herpes simplex viral vector to rapidly yet transiently overexpress FAAH specifically within the BLA to assess the impact of suppressing AEA signaling on stress, fear, and anxiety in male rats. Surprisingly, FAAH overexpression in BLA dampened stress-induced corticosterone release, reduced anxiety-like behaviors, and decreased conditioned fear expression. Interestingly, depleting AEA signaling in the BLA did not prevent fear conditioning itself or fear reinstatement. These effects were specific to the overexpression of FAAH because they were reversed by intra-BLA administration of an FAAH inhibitor. Moreover, the fear-suppressive effects of FAAH overexpression were also mitigated by intra-BLA administration of a low dose of a GABAA receptor antagonist, but not an NMDA/AMPA/kainate receptor antagonist, suggesting that they were mediated by an increase in GABAergic neurotransmission. Our data suggest that a permissive AEA tone within the BLA might gate GABA release and that loss of this tone through elevated AEA hydrolysis increases inhibition in the BLA, which in turn reduces stress, anxiety, and fear. These data provide new insights on the mechanisms by which amygdalar endocannabinoid signaling regulates emotional behavior.SIGNIFICANCE STATEMENT Amygdala endocannabinoid signaling is involved in the regulation of stress, anxiety, and fear. Our data indicate that viral-mediated augmentation of anandamide hydrolysis within the basolateral amygdala reduces behavioral indices of stress, anxiety, and conditioned fear expression. These same effects have been previously documented with inhibition of anandamide hydrolysis in the same brain region. Our results indicate that the ability of anandamide signaling to regulate emotional behavior is nonlinear and may involve actions at distinct neuronal populations, which could be influenced by the basal level of anandamide. Modulation of anandamide signaling is a current clinical therapeutic target for stress-related psychiatric illnesses, so these data underscore the importance of fully understanding the mechanisms by which anandamide signaling regulates amygdala-dependent changes in emotionality.

Characterizing ATP processing by the AAA+ protein p97 at the atomic level.

The human enzyme p97 regulates various cellular pathways by unfolding hundreds of protein substrates in an ATP-dependent manner, making it an essential component of protein homeostasis and an impactful pharmacological target. The hexameric complex undergoes substantial conformational changes throughout its catalytic cycle. Here we elucidate the molecular motions that occur at the active site in the temporal window immediately before and after ATP hydrolysis by merging cryo-EM, NMR spectroscopy and molecular dynamics simulations. p97 populates a metastable reaction intermediate, the ADP·Pi state, which is poised between hydrolysis and product release. Detailed snapshots reveal that the active site is finely tuned to trap and eventually discharge the cleaved phosphate. Signalling pathways originating at the active site coordinate the action of the hexamer subunits and couple hydrolysis with allosteric conformational changes. Our multidisciplinary approach enables a glimpse into the sophisticated spatial and temporal orchestration of ATP handling by a prototype AAA+ protein.

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.

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum.

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca2+ in the ambient buffer. Subsequent depletion of Ca2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation.

Emotional arousal state influences the ability of amygdalar endocannabinoid signaling to modulate anxiety.

Systemic activation of cannabinoid receptors often induces biphasic effects on emotional memory and anxiety depending on the levels of emotional arousal associated to the experimental context. The basolateral nucleus of the amygdala (BLA) represents a crucial structure for the ability of endocannabinoid (eCB) signaling to modulate emotional behaviour, and receives dense projections from brainstem arousal system nuclei. We examined whether changes in emotional arousal state would influence the ability of acute eCB manipulations within the BLA to modulate anxiety. Rats were tested in an elevated plus maze (EPM) under low or high arousal conditions. The low emotional arousal group was extensively handled and habituated to the experimental room and tested under red light condition, the high emotional arousal group was not handled or habituated and tested under high light condition. We examined amygdalar eCB anandamide (AEA) and 2-arachidonoylglycerol (2-AG) levels immediately after the EPM and the effects of intra-BLA administration of the AEA hydrolysis inhibitor URB597 or the 2-AG hydrolysis inhibitor KML29 on anxiety behaviour. The modulation of anxiety-like behaviour by eCBs in the BLA was strictly dependent on the environmental-associated emotional arousal. Pharmacologically-induced elevations of AEA or 2-AG in the BLA decreased anxiety under conditions of low emotional arousal. Conversely, when the level of emotional arousal increased, local eCB manipulation was ineffective in the modulation of the emotional arousal-induced anxiety response. These findings suggest that, depending on the emotional arousal state, eCB system is differentially activated to regulate the anxiety response in the amygdala and help to understand the state-dependency of many interventions on anxiety.