Functional deletion of neuropeptide Y receptors type 2 in local synaptic networks of anteroventral BNST facilitates recall and increases return of fear.

Return of previously extinguished fear memories presents a major hurdle in treatment of fear-related disorders. Neuropeptide Y receptors type 2 (Y2R) in the bed nucleus of stria terminalis (BNST) seem to play a crucial role in modulation of remote fear memories. Here, we targeted Cre-channelrhodopsin-2 to defined subregions of BNST or central amygdala (CeA) in floxed Y2R mice (Y2lox/lox) for functional deletion of Y2R. We combined fear training and behavioral studies in vivo with optogenetic-electrophysiological analysis of BNST synaptic network activity ex vivo, in order to identify regional and cellular specificities of Y2R influence. Deletion of Y2R in the ventral section of anterior BNST (BNSTav) did not affect fear acquisition, but increased conditioned fear during recall and extinction learning, and aggravated remote fear return. By contrast, deletion of Y2R in the dorsal section of anterior BNST (BNSTad) or CeA did not influence acquisition, extinction or return of fear memories. Ex vivo optogenetic-electrophysiological analysis revealed Y2R-expressing local GABAergic inhibitory networks in BNST, both within (intraregional) and in-between (inter-regional) BNST subregions. Stimulation of Y2R resulted in a presynaptically mediated reduction of GABAergic responses, which did not differ between intraregional but predominantly affected inter-regional connections from BNSTav to BNSTad. Moreover, deletion of Y2R decreased the excitation/inhibition balance in BNSTav neurons, suggesting a regulatory influence of endogenous NPY via intraregional GABAergic microcircuits. This study reveals Y2R within local GABAergic networks in BNST as key elements in facilitating extinction and reducing return of remote fear memories, suggesting a potential avenue for translational purposes.

Single stimulation of Y2 receptors in BNSTav facilitates extinction and dampens reinstatement of fear.

RATIONALE: Return of fear by re-exposure to an aversive event is a major obstacle in the treatment of fear-related disorders. Recently, we demonstrated that local pharmacological stimulation of neuropeptide Y type 2 receptors (Y2R) in anteroventral bed nucleus of stria terminalis (BNSTav) facilitates fear extinction and attenuates retrieval of remote fear with or without concomitant extinction training. Whether Y2R activation could also protect against re-exposure to traumatic events is still unknown. OBJECTIVE: Therefore, we investigated reinstatement of remote fear following early Y2R manipulation in BNSTav in relation to concomitant extinction training in mice. METHODS: We combined local pharmacological manipulation of Y2Rs in BNSTav with or without extinction training and tested for reinstatement of remote fear 15 days later. Furthermore, we employed immediate early gene mapping to monitor related local brain activation. RESULTS: Y2R stimulation by local injection of NPY3-36 into BNSTav facilitated extinction, reduced fear reinstatement at remote stages, and mimicked the influence of extinction in groups without prior extinction training. In contrast, Y2R antagonism (JNJ-5207787) delayed extinction and increased reinstatement. Y2R treatment immediately before remote fear tests had no effect. Concomitantly, Y2R activation at early time points reduced the number of c-Fos positive neurons in BNSTav during testing of reinstated remote fear. CONCLUSION: Local Y2R stimulation in BNSTav promotes fear extinction and stabilizes suppression of reinstated fear through a long-term influence, even without extinction training. Thus, Y2Rs in BNST are crucial pharmacological targets for extinction-based remote fear suppression.

Glucocorticoid-endocannabinoid uncoupling mediates fear suppression deficits after early - Life stress.

Early-life stress (ELS) creates life-long vulnerability to stress-related anxiety disorders through altering stress and fear systems in the brain. The endocannabinoid system has emerged as an important regulator of the stress response through a crosstalk with the glucocorticoid system, yet whether it plays a role in the persistent effects of ELS remains unanswered. By combining, behavioral, pharmacological and biochemical approaches in adult male rats, we examined the impact of ELS on the regulation of endocannabinoid function by stress and glucocorticoids. We employed a postnatal limited-nesting/bedding induced ELS between postnatal days 2-9 in rats. Exposure to postnatal ELS compromised the ability of both acute stress and glucocorticoid administration to mobilize the endocannabinoid ligand 2-arachidonoyl glycerol (2-AG) in the hippocampus of adult male rats. These findings suggest that ELS compromises the coupling of the glucocorticoid and endocannabinoid systems in the hippocampus. Since 2-AG signaling is essential in mediating glucocorticoid-induced suppression of fear recall, we further examined the impact of ELS on the ability of glucocorticoids to suppress fear memory recall. While ELS did not affect normative fear recall, it impaired the ability of glucocorticoids to dampen fear recall. Notably, bypassing glucocorticoids and directly amplifying hippocampal 2-AG signaling with a monoacyl glycerol lipase inhibitor produced a suppression of fear memory recall in animals exposed to ELS. These findings suggest that ELS results in an uncoupling of glucocorticoid-endocannabinoid signaling in the hippocampus, which, in turn, relates to alterations in stress regulation of memory recall. These data provide compelling evidence that ELS-induced deficits in the glucocorticoid-endocannabinoid coupling following stress could predispose susceptibility to stress-related psychopathology.