Human-Specific Neuropeptide S Receptor Variants Regulate Fear Extinction in the Basal Amygdala of Male and Female Mice Depending on Threat Salience.

BACKGROUND: A nonsynonymous single nucleotide polymorphism in the neuropeptide S receptor 1 (NPSR1) gene (rs324981) results in isoleucine-to-asparagine substitution at amino acid 107. In humans, the ancestral variant (NPSR1 I107) is associated with increased anxiety sensitivity and risk of panic disorder, while the human-specific variant (NPSR1 N107) is considered protective against excessive anxiety. In rodents, neurobiological constituents of the NPS system have been analyzed in detail and their anxiolytic-like effects have been endorsed. However, their implication for anxiety and related disorders in humans remains unclear, as rodents carry only the ancestral NPSR1 I107 variant. METHODS: We hypothesized that phenotypic correlates of NPSR1 variants manifest in fear-related circuits in the amygdala. We used CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/Cas9)-mediated gene editing to generate a "humanized" mouse strain, in which individuals express either NPSR1 I107 or NPSR1 N107. RESULTS: Stimulation of NPSR1 evoked excitatory responses in principal neurons of the anterior basal amygdala with significant differences in magnitude between genotypes, resulting in synaptic disinhibition of putative extinction neurons in the posterior basal amygdala in mice expressing the human-specific hypofunctional N107 but not the ancestral I107 variant. N107 mice displayed improved extinction of conditioned fear, which was phenocopied after pharmacological antagonism of NPSR1 in the anterior basal amygdala of I107 mice. Differences in fear extinction between male and female mice were related to an interaction of Npsr1 genotype and salience of fear training. CONCLUSIONS: The NPS system regulates extinction circuits in the amygdala depending on the Npsr1 genotype, contributing to sex-specific differences in fear extinction and high anxiety sensitivity of individuals bearing the ancestral NPSR1 I107 variant.

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.

Impact of Life History on Fear Memory and Extinction.

Behavioral profiles are strongly shaped by an individual's whole life experience. The accumulation of negative experiences over lifetime is thought to promote anxiety-like behavior in adulthood ("allostatic load hypothesis"). In contrast, the "mismatch hypothesis" of psychiatric disease suggests that high levels of anxiety-like behavior are the result of a discrepancy between early and late environment. The aim of the present study was to investigate how different life histories shape the expression of anxiety-like behavior and modulate fear memory. In addition, we aimed to clarify which of the two hypotheses can better explain the modulation of anxiety and fear. For this purpose, male mice grew up under either adverse or beneficial conditions during early phase of life. In adulthood they were further subdivided in groups that either matched or mismatched the condition experienced before, resulting in four different life histories. The main results were: (i) Early life benefit followed by late life adversity caused decreased levels of anxiety-like behavior. (ii) Accumulation of adversity throughout life history led to impaired fear extinction learning. Late life adversity as compared to late life benefit mainly affected extinction training, while early life adversity as compared to early life benefit interfered with extinction recall. Concerning anxiety-like behavior, the results do neither support the allostatic load nor the mismatch hypothesis, but rather indicate an anxiolytic effect of a mismatched early beneficial and later adverse life history. In contrast, fear memory was strongly affected by the accumulation of adverse experiences over the lifetime, therefore supporting allostatic load hypothesis. In summary, this study highlights that anxiety-like behavior and fear memory are differently affected by specific combinations of adverse or beneficial events experienced throughout life.

Distinct state anxiety after predictable and unpredictable fear training in mice.

Sustained fear paradigms in rodents have been developed to monitor states of anxious apprehension and to model situations in patients suffering from long-lasting anxiety disorders. A recent report describes a fear conditioning paradigm, allowing distinction between phasic and sustained states of conditioned fear in non-restrained mice. However, so far no prospective studies have yet been conducted to elucidate whether induction of phasic or sustained fear can affect states of anxiety. Here, we used CS (conditioned stimulus) and US (unconditioned stimulus) pairing with predictable and unpredictable timing to induce phasic and sustained fear in mice. State anxiety during various fear response components was assessed using the elevated plus-maze test. Training with unpredictable CS-US timing resulted in CS-evoked sustained components of fear (freezing), while predictable CS-US timing resulted in rapid decline. Data suggested the influence of training procedure on state anxiety which is dependent on progression of conditioned fear during fear memory retrieval. Animals trained with unpredictable CS-US timing showed an unchanged high anxiety state throughout behavioral observation. In contrast, mice trained with predictable CS-US timing showed anxiolytic-like behavior 3 min after CS onset, which was accompanied by a fast decline of the fear conditioned response (freezing). Further systematic studies are needed to validate the phasic/sustained fear model in rodents as translational model for anxiety disorders in humans.