Nos1+ and Nos1- excitatory neurons in the BLA regulate anxiety- and depression-related behaviors oppositely.

BACKGROUND: The basolateral amygdala (BLA) neurons are primarily glutamatergic and have been associated with emotion regulation. However, little is known about the roles of BLA neurons expressing neuronal nitric oxide synthase (nNOS, Nos1) in the regulation of emotional behaviors. METHODS: Using Nos1-cre mice and chemogenetic and optogenetic manipulations, we specifically silenced or activated Nos1+ or Nos1- neurons in the BLA, or silenced their projections to the anterdorsal bed nucleus of the stria terminalis (adBNST) and ventral hippocampus (vHPC). We measured anxiety behaviors in elevated plus maze (EPM) and open-field test (OFT), and measured depression behaviors in forced swimming test (FST) and tail suspension test (TST). RESULTS: BLA Nos1+ neurons were predominantly glutamatergic, and glutamatergic but not GABAergic Nos1+ neurons were involved in controlling anxiety- and depression-related behaviors. Interestingly, by selectively manipulating the activities of BLA Nos1+ and Nos1- excitatory neurons, we found that they had opposing effects on anxiety- and depression-related behaviors. BLA Nos1+ excitatory neurons projected to the adBNST, this BLA-adBNST circuit controlled the expression of anxiety- and depression-related behaviors, while BLA Nos1- excitatory neurons projected to vHPC, this BLA-vHPC circuit contributed to the expression of anxiety- and depression-related behaviors. Moreover, excitatory vHPC-adBNST circuit antagonized the role of BLA-adBNST circuit in regulating anxiety- and depression-related behaviors. CONCLUSIONS: BLA Nos1+ and Nos1- excitatory neuron subpopulations exert different effects on anxiety- and depression-related behaviors through distinct projection circuits, providing a new insight of BLA excitatory neurons in emotional regulation. LIMITATIONS: We did not perform retrograde labeling from adBNST and vHPC regions.

Prevention of the return of extinguished fear by disrupting the interaction of neuronal nitric oxide synthase with its carboxy-terminal PDZ ligand.

Exposure therapy based on the extinction of fear memory is first-line treatment for post-traumatic stress disorder (PTSD). However, fear extinction is relatively easy to learn but difficult to remember, extinguished fear often relapses under a number of circumstances. Here, we report that extinction learning-induced association of neuronal nitric oxide synthase (nNOS) with its carboxy-terminal PDZ ligand (CAPON) in the infralimbic (IL) subregion of medial prefrontal cortex negatively regulates extinction memory and dissociating nNOS-CAPON can prevent the return of extinguished fear in mice. Extinction training significantly increases nNOS-CAPON association in the IL. Disruptors of nNOS-CAPON increase extracellular signal-regulated kinase (ERK) phosphorylation and facilitate the retention of extinction memory in an ERK2-dependent manner. More importantly, dissociating nNOS-CAPON after extinction training enhances long-term potentiation and excitatory synaptic transmission, increases spine density in the IL, and prevents spontaneous recovery, renewal and reinstatement of remote fear of mice. Moreover, nNOS-CAPON disruptors do not affect other types of learning. Thus, nNOS-CAPON can serve as a new target for treating PTSD.

Dorsal Hippocampus to Infralimbic Cortex Circuit is Essential for the Recall of Extinction Memory.

Posttraumatic stress disorder subjects usually show impaired recall of extinction memory, leading to extinguished fear relapses. However, little is known about the neural mechanisms underlying the impaired recall of extinction memory. We show here that the activity of dorsal hippocampus (dHPC) to infralimbic (IL) cortex circuit is essential for the recall of fear extinction memory in male mice. There were functional neural projections from the dHPC to IL. Using optogenetic manipulations, we observed that silencing the activity of dHPC-IL circuit inhibited recall of extinction memory while stimulating the activity of dHPC-IL circuit facilitated recall of extinction memory. "Impairment of extinction consolidation caused by" conditional deletion of extracellular signal-regulated kinase 2 (ERK2) in the IL prevented the dHPC-IL circuit-mediated recall of extinction memory. Moreover, silencing the dHPC-IL circuit abolished the effect of intra-IL microinjection of ERK enhancer on the recall of extinction memory. Together, we identify a dHPC to IL circuit that mediates the recall of extinction memory, and our data suggest that the dysfunction of dHPC-IL circuit and/or impaired extinction consolidation may contribute to extinguished fear relapses.

Anterior Cingulate Cortex to Ventral Hippocampus Circuit Mediates Contextual Fear Generalization.

Contextual fear memory becomes less context-specific over time, a phenomenon referred to as contextual fear generalization. Overgeneralization of contextual fear memory is a core symptom of post-traumatic stress disorder (PTSD), but circuit mechanisms underlying the generalization remain unclear. We show here that neural projections from the anterior cingulate cortex (ACC) to ventral hippocampus (vHPC) mediate contextual fear generalization in male mice. Retrieval of contextual fear in a novel context at a remote time point activated cells in the ACC and vHPC, as indicated by significantly increased C-fos+ cells. Using chemogenetic or photogenetic manipulations, we observed that silencing the activity of ACC or vHPC neurons reduced contextual fear generalization at the remote time point, whereas stimulating the activity of ACC or vHPC neurons facilitated contextual fear generalization at a recent time point. We found that ACC neurons projected to the vHPC unidirectionally, and importantly, silencing the activity of projection fibers from the ACC to vHPC inhibited contextual fear generalization at the remote time point. Together, our findings reveal an ACC to vHPC circuit that controls expression of fear generalization and may offer new strategies to prevent or reverse contextual fear generalization in subjects with anxiety disorders, especially in PTSD.SIGNIFICANCE STATEMENT Overgeneralization of contextual fear memory is a cardinal feature of PTSD, but circuit mechanisms underlying it remain unclear. Our study indicates that neural projections from the anterior cingulate cortex to ventral hippocampus control the expression of contextual fear generalization. Thus, manipulating the circuit may prevent or reverse fear overgeneralization in subjects with PTSD.

Uncoupling nNOS-PSD-95 in the ACC can inhibit contextual fear generalization.

A typical feature of the contextual fear memory is increased fear generalization with time. Though much attention has been given to the neural structures that underlie the long-term consolidation of a contextual fear memory, the molecular mechanisms regulating fear generalization remain unclear. We observed that retrieval of contextual fear in a novel context at a remote time point increased coupling of neuronal nitric oxide synthase (nNOS) with postsynaptic density-95 (PSD-95) and c-Fos expression in the anterior cingulate cortex (ACC). Disrupting nNOS-PSD-95 coupling in the ACC decreased the expression of Histone deacetylase 2 (HDAC2), and inhibited contextual fear generalization at a remote time point. Together, our findings reveal nNOS-PSD-95 interaction in the ACC could be a promising target to prevent or reverse contextual fear generalization.

PSD-95-nNOS Coupling Regulates Contextual Fear Extinction in the Dorsal CA3.

Fear extinction depends on N-methyl-D-aspartate glutamate receptors (NMDARs) and brain-derived neurotrophic factor (BDNF) activation in the limbic system. However, postsynaptic density-95 (PSD-95) and neuronal nitric oxide synthase (nNOS) coupling, the downstream signaling of NMDARs activation, obstructs the BDNF signaling transduction. Thus, we wondered distinct roles of NMDAR activation and PSD-95-nNOS coupling on fear extinction. To explore the mechanisms, we detected protein-protein interaction using coimmunoprecipitation and measured protein expression by western blot. Contextual fear extinction induced a shift from PSD-95-nNOS to PSD-95-TrkB association in the dorsal hippocampus and c-Fos expression in the dorsal CA3. Disrupting PSD-95-nNOS coupling in the dorsal CA3 up-regulated phosphorylation of extracellular signal-regulates kinase (ERK) and BDNF, enhanced the association of BDNF-TrkB signaling with PSD-95, and promoted contextual fear extinction. Conversely, blocking NMDARs in the dorsal CA3 down-regulated BDNF expression and hindered contextual fear extinction. NMDARs activation and PSD-95-nNOS coupling play different roles in modulating contextual fear extinction in the hippocampus. Because inhibitors of PSD-95-nNOS interaction produce antidepressant and anxiolytic effect without NMDAR-induced side effects, PSD-95-nNOS could be a valuable target for PTSD treatment.

(+)-Borneol suppresses conditioned fear recall and anxiety-like behaviors in mice.

Fear- and anxiety-related psychiatric disorders have been one of the major chronic diseases afflicting patients for decades, and new compounds for treating such disorders remain to be developed. (+)-Borneol, a bicyclic monoterpene found in several species of Artemisia and Dipterocarpaceae, is widely used for anxiety, pain and anesthesia in Chinese medicine. Meanwhile, it can potentiate GABA (γ-aminobutyric acid) activity directly in recombinant GABAA receptors. The present study was to investigate the effects of (+)-Borneol on both contextual and cued fear recall. Interestingly, microinjection of (+)-Borneol into the dorsal hippocampus inhibited 24 h and 7 d contextual fear, whereas its infusion into ventral hippocampus only reduced 24 h cued fear responses. Moreover, microinjection of (+)-Borneol into dorsal but not ventral hippocampus suppressed anxiety-like behaviors in the open field test, light/dark exploration and the elevated plus maze test. As selective GABAA receptor antagonist bicuculline reversed the effect of (+)-Borneol on contextual fear paradigm and the drug potentiated GABA-evoked currents in acute hippocampus slices, modulation of the GABAergic neurotransmission may explain the effects of (+)-Borneol. Our findings suggest that (+)-Borneol can serve as a new therapeutic in fear- and anxiety-related disorders.