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Chronic pain patients often have anxiety disorders, and some of them suffer from anxiety even after analgesic administration. In this study, we investigated the role of AMPAR-mediated synaptic transmission in the ventromedial prefrontal cortex (vmPFC) in chronic pain-induced persistent anxiety in mice and explored potential drug targets. Chronic inflammatory pain was induced in mice by bilateral injection of complete Freund's adjuvant (CFA) into the planta of the hind paws; anxiety-like behaviours were assessed with behavioural tests; S-nitrosylation and AMPAR-mediated synaptic transmission were examined using biochemical assays and electrophysiological recordings, respectively. We found that CFA induced persistent upregulation of AMPAR membrane expression and function in the vmPFC of anxious mice but not in the vmPFC of non-anxious mice. The anxious mice exhibited higher S-nitrosylation of stargazin (an AMPAR-interacting protein) in the vmPFC. Inhibition of S-nitrosylation by bilaterally infusing an exogenous stargazin (C302S) mutant into the vmPFC rescued the surface expression of GluA1 and AMPAR-mediated synaptic transmission as well as the anxiety-like behaviours in CFA-injected mice, even after ibuprofen treatment. Moreover, administration of ZL006, a small molecular inhibitor disrupting the interaction of nNOS and PSD-95 (20 mg·kg

Stress-induced depression is common worldwide. NAc, a “reward” center, is recently reported to be critical to confer the susceptibility to chronic social defeat stress (CSDS) and the depression-related outcome. However, the underlying molecular mechanisms have not been well characterized. In this study, we induced depression-like behaviors with CSDS and chronic mild stress in male mice to mimic social and environmental factors, respectively, and observed animal behaviors with social interaction test, tail suspension test, and sucrose preference test. To determine the role of neuronal nitric oxide synthase (nNOS) and its product nitric oxide (NO), we used brain region-specifically nNOS overexpression and stereotaxic injection of NO inhibitor or donor. Moreover, the downstream molecular cyclin-dependent kinase 5 (CDK5) was explored by conditional KO and gene mutation. We demonstrate that nNOS-implicated mechanisms in NAc shell (NAcSh), including increased cell number, increased protein expression levels, and increased specific enzyme activity, contribute the susceptibility to social defeat and the following depression-like behaviors. NAcSh nNOS does not directly respond to chronic mild stress but facilitates the depression-like behaviors. The increased NAcSh nNOS expression after CSDS leads to the social avoidance and depression-like behaviors in defeated mice, which is dependent on the nNOS enzyme activity and NO production. Moreover, we identify the downstream signal in NAcSh. S-nitrosylation of CDK5 by NO contributes to enhanced CDK5 activity, leading to depression-related behaviors in susceptible mice. Therefore, NAcSh nNOS mediates susceptibility to social defeat stress and the depression-like behaviors through CDK5.SIGNIFICANCE STATEMENTStress-induced depression is common worldwide, and chronic exposure to social and psychological stressors is important cause of human depression. Our study conducted with chronic social defeat stress mice models demonstrates that nNOS in NAcSh is crucial to regulate the susceptibility to social defeat stress and the following depression-like behaviors, indicating NAcSh nNOS as the responding molecule to social factors of depression. Moreover, we discover the downstream mechanism of NAcSh nNOS in mediating the susceptibility is NO and S-nitrosylation of CDK5. Thus, NAcSh nNOS mediates susceptibility to social defeat stress through CDK5 is a potential mechanism for depression, which may interpret how the brain transduces social stress exposure into depression.

Overactivation of N-methyl-D-aspartate receptor (NMDAR) in the spinal cord dorsal horn (SDH) in the setting of injury represents a key mechanism of neuropathic pain. However, directly blocking NMDAR or its downstream signaling, interaction between postsynaptic density-95 (PSD-95) and neuronal nitric oxide synthase (nNOS), causes analgesic tolerance, mainly due to GABAergic disinhibition. The aim of this study is to explore the possibility of preventing analgesic tolerance through co-targeting NMDAR downstream signaling and γ-aminobutyric acid type A receptors (GABAARs). Methods: Mechanical/thermal hyperalgesia were quantified to assess analgesic effects. Miniature postsynaptic currents were tested by patch-clamp recording to evaluate synaptic transmission in the SDH. GABA-evoked currents were tested on HEK293 cells expressing different subtypes of recombinant GABAARs to assess the selectivity of (+)-borneol and ZL006-05. The expression of α2 and α3 subunits of GABAARs and BDNF, and nNOS-PSD-95 complex levels were analyzed by western blotting and coimmunoprecipitation respectively. Open field test, rotarod test and Morris water maze task were conducted to evaluate the side-effect of ZL006-05. Results: (+)-Borneol selectively potentiated α2- and α3-containing GABAARs and prevented the disinhibition of laminae I excitatory neurons in the SDH and analgesic tolerance caused by chronic use of ZL006, a nNOS-PSD-95 blocker. A dual-target compound ZL006-05 produced by linking ZL006 and (+)-borneol through an ester bond blocked nNOS-PSD-95 interaction and potentiated α2-containing GABAAR selectively. Chronic use of ZL006-05 did not produce analgesic tolerance and unwanted side effects. Conclusion: By targeting nNOS-PSD-95 interaction and α2-containing GABAAR simultaneously, chronic use of ZL006-05 can avoid analgesic tolerance and unwanted side effects. Therefore, we offer a novel candidate drug without analgesic tolerance for treating neuropathic pain.

Rationale: Stroke is a leading cause of adult disability worldwide, but no drug provides functional recovery during the repair phase. Accumulating evidence demonstrates that environmental enrichment (EE) promotes stroke recovery by enhancing network excitability. However, the complexities of utilizing EE in a clinical setting limit its translation. Methods: We used multifaceted approaches combining electrophysiology, chemogenetics, optogenetics, and floxed mice in a mouse photothrombotic stroke model to reveal the key target of EE-mediated stroke recovery. Results: EE reduced tonic gamma-aminobutyric acid (GABA) inhibition and facilitated phasic GABA inhibition in the peri-infarct cortex, thereby promoting network excitability and stroke recovery. These beneficial effects depended on GAT-1, a GABA transporter regulating both tonic and phasic GABA signaling, as EE positively regulated GAT-1 expression, trafficking, and function. Furthermore, GAT-1 was necessary for EE-induced network plasticity, including structural neuroplasticity, input synaptic strengthening in the peri-infarct cortex, output synaptic strengthening in the corticospinal tract, and sprouting of uninjured corticospinal axons across the midline into the territory of denervated spinal cord, and functional recovery from stroke. Moreover, restoration of GAT-1 function in the peri-infarct cortex by its overexpression showed similar beneficial effects on stroke recovery as EE exposure. Conclusion: GAT-1 is a key molecular substrate of the effects of EE on network excitability and consequent stroke recovery and can serve as a novel therapeutic target for stroke treatment during the repair phase.

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.

The paraventricular nucleus of the thalamus (PVT), which serves as a hub, receives dense projections from the medial prefrontal cortex (mPFC) and projects to the lateral division of central amygdala (CeL). The infralimbic (IL) cortex plays a crucial role in encoding and recalling fear extinction memory. Here, we found that neurons in the PVT and IL were strongly activated during fear extinction retrieval. Silencing PVT neurons inhibited extinction retrieval at recent time point (24 h after extinction), while activating them promoted extinction retrieval at remote time point (7 d after extinction), suggesting a critical role of the PVT in extinction retrieval. In the mPFC-PVT circuit, projections from IL rather than prelimbic cortex to the PVT were dominant, and disrupting the IL-PVT projection suppressed extinction retrieval. Moreover, the axons of PVT neurons preferentially projected to the CeL. Silencing the PVT-CeL circuit also suppressed extinction retrieval. Together, our findings reveal a new neural circuit for fear extinction retrieval outside the classical IL-amygdala circuit. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12264-020-00603-6) contains supplementary material, which is available to authorized users.

Anxiety is common in patients suffering from chronic pain. Here, we report anxiety-like behaviors in mouse models of chronic pain and reveal that nNOS-expressing neurons in ventromedial prefrontal cortex (vmPFC) are essential for pain-induced anxiety but not algesia, using optogenetic and chemogenetic strategies. Additionally, we determined that excitatory projections from the posterior subregion of paraventricular thalamic nucleus (pPVT) provide a neuronal input that drives the activation of vmPFC nNOS-expressing neurons in our chronic pain models. Our results suggest that the pain signal becomes an anxiety signal after activation of vmPFC nNOS-expressing neurons, which causes subsequent release of nitric oxide (NO). Finally, we show that the downstream molecular mechanisms of NO likely involve enhanced glutamate transmission in vmPFC CaMKIIα-expressing neurons through S-nitrosylation-induced AMPAR trafficking. Overall, our data suggest that pPVT excitatory neurons drive chronic pain-induced anxiety through activation of vmPFC nNOS-expressing neurons, resulting in NO-mediated AMPAR trafficking in vmPFC pyramidal neurons., Chronic pain usually induces anxiety. Here, the authors report that vmPFC nNOS-expressing neurons are activated by excitatory inputs from pPVT during chronic pain and subsequently induce anxiety-like behaviors in mice through promoting AMPAR trafficking.

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.

Drug abuse is a dramatic challenge for the whole society because of high relapse rate. Environmental cues are crucial for the preference memory of drug abuse. Extinction therapy has been developed to inhibit the motivational effect of drug cues to prevent the reinstatement of morphine abuse. However, extinction therapy alone only forms a new kind of unstable inhibitory memory. We found that morphine conditioned place preference (CPP) extinction training increased the association of nitric oxide synthase (nNOS) with its carboxy-terminal PDZ ligand (CAPON) in the dorsal hippocampus (dHPC) significantly and blocking the morphine-induced nNOS–CAPON association using Tat-CAPON-12C during and after extinction training reversed morphine-induced hippocampal neuroplasticity defect and prevented the reinstatement and spontaneous recovery of morphine CPP. Moreover, in the hippocampal selective ERK2 knock-out or nNOS knockout mice, the effect of Tat-CAPON-12C on the reinstatement of morphine CPP and hippocampal neuroplasticity disappeared, suggesting ERK2 is necessary for the effects of Tat-CAPON-12C. Together, our findings suggest that nNOS–CAPON interaction in the dHPC may affect the consolidation of morphine CPP extinction and dissociating nNOS–CAPON prevents the reinstatement and spontaneous recovery of morphine CPP, possibly through ERK2-mediated neuroplasticity and extinction memory consolidation, offering a new target to prevent the reinstatement of drug abuse.

Neuropathic pain is usually persistent due to maladaptive neuroplasticity-induced central sensitization and, therefore, necessitates long-term treatment. N-methyl-D-aspartate receptor (NMDAR)-mediated hypersensitivity in the spinal dorsal horn represents key mechanisms of central sensitization. Short-term use of NMDAR antagonists produces antinociceptive efficacy in animal pain models and in clinical practice by reducing central sensitization. However, how prolonged use of NMDAR antagonists affects central sensitization remains unknown. Surprisingly, we find that prolonged blockage of NMDARs does not prevent but aggravate nerve injury-induced central sensitization and produce analgesic tolerance, mainly due to reduced synaptic inhibition. The disinhibition that results from the continuous decrease in the production of nitric oxide from neuronal nitric oxide synthase, downstream signal of NMDARs, leads to the reduction of GABAergic inhibitory synaptic transmission by upregulating brain-derived neurotrophic factor expression and inhibiting the expression and function of potassium-chloride cotransporter. Together, our findings suggest that chronic blockage of NMDARs develops analgesic tolerance through the neuronal nitric oxide synthase–brain-derived neurotrophic factor–potassium-chloride cotransporter pathway. Thus, preventing the GABAergic disinhibition induced by nitric oxide reduction may be necessary for the long-term maintenance of the analgesic effect of NMDAR antagonists. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13311-020-00883-w) contains supplementary material, which is available to authorized users.

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.

Environmental enrichment (EE) is a generally accepted strategy to promote stroke recovery and its beneficial effect is positively correlated with neuroplasticity. However, the mechanisms underlying it remain elusive. Histone deacetylase 2 (HDAC2), a negative regulator of neuroplasticity, is up-regulated after stroke. Thus, we hypothesized that HDAC2 may participate in EE-mediated stroke recovery. In this study, focal stroke was induced by photothrombosis in male mice exposing to EE or standard housing (SH) conditions. Recombinant virus vectors, including Ad-HDAC2-Flag, AAV-CAG-EGFP-Cre, LV-shHDAC2, or their controls were microinjected into the motor cortex at 3 days before stroke. Grid-walking and cylinder tasks were conducted to assess motor function. Western blot and immunostaining were used to uncover the mechanisms underlying EE-mediated stroke recovery. We found that EE exposure reversed stroke-induced HDAC2 up-regulation, implicating HDAC2 in EE-mediated functional recovery. Importantly, EE-dependent stroke recovery was counteracted by over-expressing HDAC2, and HDAC2 knockdown promoted functional recovery from stroke to the similar extent as EE exposure. Moreover, the knockdown of HDAC2 epigenetically enhanced expressions of neurotrophins and neuroplasticity-related proteins, with similar effects as EE, and consequently, whole brain and corticospinal tract (CST) rewiring. Together, our findings indicate that HDAC2 is critical for EE-dependent functional restoration. Precisely targeting HDAC2 may mimic EE and serve as a novel therapeutic strategy for stroke recovery.

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.

Anhedonia is the inability to experience pleasure from rewarding or enjoyable activities and is a core symptom of depression in humans. Here, we describe a protocol for the measurement of anhedonia in mice, in which anhedonia is measured by a sucrose preference test (SPT) based on a two-bottle choice paradigm. A reduction in the sucrose preference ratio in experimental relative to control mice is indicative of anhedonia. To date, inconsistent and variable results have been reported following the use of the SPT by different groups, probably due to the use of different protocols and equipment. In this protocol, we describe how to set up a clearly defined apparatus for SPT and provide a detailed protocol to ensure greater consistency when carrying out SPT. This optimized protocol is highly sensitive, reliable, and adaptable for evaluation of chronic stress-related anhedonia, as well as morphine-induced dependence. The whole SPT, including adaptation, baseline measurement, and testing, takes 8 d.

Mechanisms underlying functional recovery after stroke are little known, and effective drug intervention during the delayed stage is desirable. One potential drug target, the protein-protein interaction between neuronal nitric oxide synthase (nNOS) and postsynaptic density protein 95 (PSD-95), is critical to acute ischaemic damage and neurogenesis. We show that nNOS-PSD-95 dissociation induced by microinjection of a recombinant fusion protein, Tat-nNOS-N1-133 , or systemic administration of a small-molecule, ZL006, from day 4 to day 10 after photothrombotic ischaemia in mice reduced excessive tonic inhibition in the peri-infarct cortex and ameliorated motor functional outcome. We also demonstrated improved neuroplasticity including increased dendrite spine density and synaptogenesis after reducing excessive tonic inhibition by nNOS-PSD-95 dissociation. Levels of gamma-aminobutyric acid (GABA) and GABA transporter-3/4 (GAT-3/4) are increased in the reactive astrocytes in the peri-infarct cortex. The GAT-3/4-selective antagonist SNAP-5114 reduced tonic inhibition and promoted function recovery, suggesting that increased tonic inhibition in the peri-infarct cortex was due to GABA release from reversed GAT-3/4 in reactive astrocytes. Treatments with Tat-nNOS-N1-133 or ZL006 after ischaemia inhibited astrocyte activation and GABA production, prevented the reversal of GAT-3/4, and consequently decreased excessive tonic inhibition and ameliorated functional outcome. The underlying molecular mechanisms were associated with epigenetic inhibition of glutamic acid decarboxylase 67 and monoamine oxidase B expression through reduced NO production. The nNOS-PSD-95 interaction is thus a potential target for functional restoration after stroke and ZL006, a small molecule inhibitor of this interaction, is a promising pharmacological lead compound. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Neuronal nitric oxide synthase (nNOS) 1 , mainly responsible for NO release in central nervous system (CNS) 2 , plays a significant role in multiple physiological functions. However, the function of nNOS + interneurons in fear learning has not been much explored. Here we focused on the medial ganglionic eminences (MGE) 3 -derived nNOS + interneurons in fear learning. To determine the origin of nNOS + interneurons, we cultured neurons in vitro from MGE, cortex, lateral ganglionic eminence (LGE) 4 , caudal ganglionic eminences (CGE) 5 and preoptic area (POA) 6 . The results showed that MGE contained the most abundant precursors of nNOS + interneurons. Moreover, donor cells from E12.5 embryos demonstrated the highest positive rate of nNOS + interneurons compared with other embryonic periods (E11.5, E12, E13, E13.5 and E14). Additionally, these cells from E12.5 embryos showed long axonal and abundant dendritic arbors after 10 days culture, indicating the capability to disperse and integrate in host neural circuits after transplantation. To investigate the role of MGE-derived nNOS + interneurons in fear learning, donor MGE cells were transplanted into dentate gyrus (DG) 7 of nNOS knock-out (nNOS −/− ) or wild-type mice. Results showed that the transplantation of MGE cells promoted the acquisition of nNOS −/− but not the wild-type mice, suggesting the importance of nNOS + neurons in fear acquisition. Moreover, we transplanted MGE cells from nNOS −/− mice or wild-type mice into DG of the nNOS −/- mice and found that only MGE cells from wild-type mice but not the nNOS −/− mice rescued the deficit in acquisition of the nNOS −/− mice, further confirming the positive role of nNOS + neurons in fear learning.

Granule cells in the dentate gyrus regenerate constantly in adult hippocampus and then integrate into neural circuits in the hippocampus thereby providing the neural basis for learning and memory. Promoting the neurogenesis in the hippocampus facilitates learning and memory such as spatial learning, object identification, and extinction learning. The interaction between neuronal nitric oxide synthase (nNOS) and postsynaptic density protein-95 (PSD-95) is reported to negatively regulate neurogenesis in brain, so we hypothesized that disrupting this interaction might facilitate the neurogenesis in the dentate gyrus (DG) and thus enhance the extinction memory retrieval of fear learning. We found that uncoupling the nNOS-PSD-95 complex in remote contextual fear condition promoted both neuronal proliferation and survival in the DG, contributing to an enhanced retrieval of the extinction memory. Moreover, the nNOS-PSD-95 uncoupling-induced neurogenesis may be mediated by the extracellular signal-regulated kinase (ERK) as the phosphorylation level of ERK1/2 was increased after uncoupling. These findings suggest that the nNOS-PSD-95 complex may serve as a novel target for the treatment of post-traumatic stress disorder (PTSD).

Stroke is one of the leading causes of disability and death globally. It occurs when a major artery is occluded in the brain and leads to death of cells within the injured tissue. (+)-Borneol, a simple bicyclic monoterpene extracted from traditional Chinese medicine, is widely used in various types of diseases. However, no study has proved the effects of (+)-borneol on functional recovery from permanent ischemic stroke and the mechanism is still unknown. Here, we report that in the rat model of permanent cerebral ischemia, we found that (+)-borneol (1.0 mg/kg) significantly ameliorated infarct size and neurological scoresvia reducing the expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-α) in a dose dependent manner. Notably, (+)-borneol showed long-term effects on the improvement of sensorimotor functions in the photothrombotic model of stroke, which decreased the number of foot faults in the grid-walking task and forelimb asymmetry scores in the cylinder task, at least in part through reducing loss of dendritic spines in the length, brunch number and density. These findings suggest that (+)-borneol could serve as a therapeutic target for ischemic stroke.

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.

Background and Purpose— Stroke is a major public health concern worldwide. Although clinical treatments have improved in the acute period after stroke, long-term therapeutics remain limited to physical rehabilitation in the delayed phase. This study is aimed to determine whether nNOS (neuronal NO synthase)-CAPON (carboxy-terminal postsynaptic density-95/discs large/zona occludens-1 ligand of nNOS) interaction may serve as a new therapeutic target in the delayed phase for stroke recovery. Methods— Photothrombotic stroke and transient middle cerebral artery occlusion were induced in mice. Adeno-associated virus (AAV)-cytomegalovirus (CMV)-CAPON-125C-GFP (green fluorescent protein)-3Flag and the other 2 drugs (Tat-CAPON-12C and ZLc-002) were microinjected into the peri-infarct cortex immediately and 4 to 10 days after photothrombotic stroke, respectively. ZLc-002 was also systemically injected 4 to 10 days after transient middle cerebral artery occlusion. Grid-walking task and cylinder task were conducted to assess motor function. Western blotting, immunohistochemistry, Golgi staining, and electrophysiology recordings were performed to uncover the mechanisms. Results— Stroke increased nNOS-CAPON association in the peri-infarct cortex in the delayed period. Inhibiting the ischemia-induced nNOS-CAPON association substantially decreased the number of foot faults in the grid-walking task and forelimb asymmetry in the cylinder task, suggesting the promotion of functional recovery from stroke. Moreover, dissociating nNOS-CAPON significantly facilitated dendritic remodeling and synaptic transmission, indicated by increased dendritic spine density, dendritic branching, and length and miniature excitatory postsynaptic current frequency but did not affect stroke-elicited neuronal loss, infarct size, or cerebral edema, suggesting that nNOS-CAPON interaction may function via regulating structural neuroplasticity, rather than neuroprotection. Furthermore, ZLc-002 reversed the transient middle cerebral artery occlusion–induced impairment of motor function. Conclusions— Our results reveal that nNOS-CAPON coupling can serve as a novel pharmacological target for functional restoration after stroke.

It is well established that taurine shows potent protection against glutamate-induced injury to neurons in stroke. The neuroprotection may result from multiple mechanisms. Increasing evidences suggest that NADPH oxidases (Nox), the primary source of superoxide induced by N-methyl-d-aspartate (NMDA) receptor activation, are involved in the process of oxidative stress. We found that 100μM NMDA induced oxidative stress by increasing the reactive oxygen species level, which contributed to the cell death, in vitro. Neuron cultures pretreated with 25mM taurine showed lower percentage of death cells and declined reactive oxygen species level. Moreover, taurine attenuated Nox2/Nox4 protein expression and enzyme activity and declined intracellular calcium intensity during NMDA-induced neuron injury. Additionally, taurine also showed neuroprotection against H2O2-induced injury, accompanying with Nox inhibition. So, we suppose that protection of taurine against reactive oxygen species during NMDA-induced neuron injury is associated with Nox inhibition, probably in a calcium-dependent manner.

Summary In neurons, increased protein–protein interactions between neuronal nitric oxide synthase (nNOS) and its carboxy‐terminal PDZ ligand (CAPON) contribute to excitotoxicity and abnormal dendritic spine development, both of which are involved in the development of Alzheimer's disease. In models of Alzheimer's disease, increased nNOS–CAPON interaction was detected after treatment with amyloid‐β in vitro, and a similar change was found in the hippocampus of APP/PS1 mice (a transgenic mouse model of Alzheimer's disease), compared with age‐matched background mice in vivo. After blocking the nNOS–CAPON interaction, memory was rescued in 4‐month‐old APP/PS1 mice, and dendritic impairments were ameliorated both in vivo and in vitro. Furthermore, we demonstrated that S‐nitrosylation of Dexras1 and inhibition of the ERK–CREB–BDNF pathway might be downstream of the nNOS–CAPON interaction.

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.

Background Stroke is a leading cause of long‐term disability worldwide. However, current therapies that promote functional recovery from stroke are limited to physical rehabilitation. No pharmacological therapy is available. Thus, understanding the role of histone deacetylase 2 (HDAC2) in the pathophysiological process of stroke‐induced functional loss may provide a novel strategy for stroke recovery. Methods and Results Focal stroke was induced by photothrombosis. LV‐HDAC2‐shRNA‐GFP, LV‐GFP, Ad‐HDAC2‐Flag, or Ad‐inactive‐HDAC2‐Flag was microinjected into the peri‐infarct area immediately after stroke. HDAC inhibitors were microinjected into the peri‐infarct area 4 to 10 days after stroke. Grid‐walking task and cylinder task were conducted to assess motor function. Golgi‐Cox staining, chromatin immunoprecipitation, and electrophysiology were used to reveal the mechanisms underlying stroke recovery. Knockdown or knockout of HDAC2 promoted stroke recovery, whereas overexpression of HDAC2 worsened stroke‐induced functional impairment. More importantly, trichostatin A, a pan‐HDAC inhibitor, promoted functional recovery from stroke in WT mice when used in the delayed phase, but it was ineffective in Hdac2 conditional knockout ( Hdac2 CKO) mice. Treatment with suberoylanilide hydroxamic acid, a selective HDAC1 and HDAC2 inhibitor, in the delayed phase of stroke produced sustained functional recovery in mice via epigenetically enhancing neuroplasticity of surviving neurons in the peri‐infarct zone. Conclusions Our novel findings provide evidence that HDAC2 is a crucial target for functional recovery from stroke. As there are clinically available HDAC inhibitors, our findings could be directly translated into clinical research of stroke.

Abtract Chronic pain is a major public health problem categorized as inflammatory or neuropathic, each involving impaired GABAergic control in the spinal cord of mammals. (+)-Borneol, a bicyclic monoterpene present in the essential oil of plants, is used for analgesia and anesthesia in traditional Chinese medicine. It has been reported that (+)-borneol directly potentiates GABA activity at recombinant human GABA A receptors. Although borneol has antinociceptive effect on acute pain models, little is known about its effect on chronic pain and its mechanism. Here we report that (+)-borneol has remarkable anti-hyperalgesic effects on neuropathic and inflammatory pain in animal models. Neuropathic hypersensitivity was induced by segmental spinal nerve ligation (SNL), and inflammatory hypersensitivity was induced by intraplantar (i.pl.) injection of complete Freund׳s adjuvant (CFA). Both oral administration (125, 250 or 500 mg/kg) and intrathecal injection (i.t.) (15, 30 and 60 μg) of (+)-borneol reduced mechanical hypersensitivity dose-dependently in SNL and CFA models. The anti-hyperalgesic effects of (+)-borneol were abolished by a selective GABA A receptor (GABA A R) antagonist bicuculline (i.t., at 30 min after (+)-borneol injection). Furthermore, (+)-borneol (500 mg/kg, p.o. or 60 μg, i.t.) did not influence motor function. These findings suggest that (+)-borneol may ameliorate mechanical hyperalgesia by enhancing GABA A R-mediated GABAergic transmission in the spinal cord, and could serve as a therapeutic for chronic pain.

Stroke is a major public health concern. The lack of effective therapies heightens the need for new therapeutic targets. Mammalian brain has the ability to rewire itself to restore lost functionalities. Promoting regenerative repair, including neurogenesis and dendritic remodeling, may offer a new therapeutic strategy for the treatment of stroke. Here, we report that interaction of neuronal nitric oxide synthase (nNOS) with the protein postsynaptic density-95 (PSD-95) negatively controls regenerative repair after stroke in rats. Dissociating nNOS–PSD-95 coupling in neurons promotes neuronal differentiation of neural stem cells (NSCs), facilitates the migration of newborn cells into the injured area, and enhances neurite growth of newborn neurons and dendritic spine formation of mature neurons in the ischemic brain of rats. More importantly, blocking nNOS–PSD-95 binding during the recovery stage improves stroke outcome via the promotion of regenerative repair in rats. Histone deacetylase 2 in NSCs may mediate the role of nNOS–PSD-95 association. Thus, nNOS–PSD-95 can serve as a target for regenerative repair after stroke.

Neuronal nitric oxide synthase (nNOS)

Our previous study found that serotonin 1A receptor (5-HT1aR) is an endogenous suppressor of nNOS expression in the hippocampus, which accounts for anxiolytic effect of fluoxetine. However, the precise molecular mechanism remains unknown. By using 8-OH-DPAT, a selective 5-HT1aR agonist, NAN-190, a selective 5-HT1aR antagonist, and U0126, an Extracellular Regulated Protein Kinases (ERK) phosphorylation inhibitor, we investigated the role of ERK in 5-HT1aR-nNOS pathway. Western blots analysis demonstrated that 5-HT1aR activation up-regulated the level of phosphorylated ERK (P-ERK) beginning at 5 min and down-regulated the expression of nNOS beginning at 20 min. Meanwhile, blockage of 5-HT1aR resulted in a decrease in P-ERK beginning at 20 min and caused an increase in nNOS expression beginning at 6 h. Although U0126 itself did not alter nNOS expression and activity, NO level, and anxiety-related behaviors, the treatment totally reversed 8-OH-DPAT-induced reduction in nNOS expression and function, and anxiolytic effect. Besides, our data showed that ERK phosphorylation was essential for 5-HT1aR activation-induced cAMP responsive element binding protein (CREB) phosphorylation, hippocampal neurogenesis and synaptogenesis of newborn neuron. Our study suggests a crucial role of ERK phosphorylation in the regulation of nNOS expression by 5-HT1aR, which is helpful for understanding the mechanism of 5-HT1aR-based anxiolytic treatment.

Mechanisms underlying functional recovery after stroke are little known, and effective drug intervention during the delayed stage is desirable. One potential drug target, the protein-protein interaction between neuronal nitric oxide synthase (nNOS) and postsynaptic density protein 95 (PSD-95), is critical to acute ischaemic damage and neurogenesis. We show that nNOS-PSD-95 dissociation induced by microinjection of a recombinant fusion protein, Tat-nNOS-N

Consciousness can be defined by two major attributes: awareness of environment and self, and arousal, which reflects the level of awareness. The return of arousal after general anesthesia presents an experimental tool for probing the neural mechanisms that control consciousness. Here we have identified that systemic or intracerebral injection of the cannabinoid CB1 receptor (CB1R) antagonist AM281 into the dorsomedial nucleus of the hypothalamus (DMH) - but not the adjacent perifornical area (Pef) or the ventrolateral preoptic nucleus of the hypothalamus (VLPO) - accelerates arousal in mice recovering from general anesthesia. Anesthetics selectively activated endocannabinoid (eCB) signaling at DMH glutamatergic but not GABAergic synapses, leading to suppression of both glutamatergic DMH-Pef and GABAergic DMH-VLPO projections. Deletion of CB1R from widespread cerebral cortical or prefrontal cortical (PFC) glutamatergic neurons, including those innervating the DMH, mimicked the arousal-accelerating effects of AM281. In contrast, CB1R deletion from brain GABAergic neurons or hypothalamic glutamatergic neurons did not affect recovery time from anesthesia. Inactivation of PFC-DMH, DMH-VLPO, or DMH-Pef projections blocked AM281-accelerated arousal, whereas activation of these projections mimicked the effects of AM281. We propose that decreased eCB signaling at glutamatergic terminals of the PFC-DMH projection accelerates arousal from general anesthesia through enhancement of the excitatory DMH-Pef projection, the inhibitory DMH-VLPO projection, or both.

Serotonin 1a-receptor (5-HT1aR) has been specifically implicated in the pathogenesis of anxiety. However, the mechanism underlying the role of 5-HT1aR in anxiety remains poorly understood. Here we show in mice that the transcription factor cAMP response element binding protein (CREB) in the hippocampus functions as an effector of 5-HT1aR in modulating anxiety-related behaviors. We generated recombinant lentivirus LV-CREB133-GFP expressing a dominant negative CREB which could not be phosphorylated at Ser133 to specifically reduce CREB activity, and LV-VP16-CREB-GFP expressing a constitutively active fusion protein VP16-CREB which could be phosphorylated by itself to specifically enhance CREB activity. LV-CREB133-GFP neutralized 5-HT1aR agonist-induced up-regulation of synapse density, spine density, dendrite complexity, neurogenesis, and the expression of synapsin and spinophilin, two well-characterized synaptic proteins, and abolished the anxiolytic effect of 5-HT1aR agonist; whereas LV-VP16-CREB-GFP rescued the 5-HT1aR antagonist-induced down-regulation of synapse density, spine density, dendrite complexity, neurogenesis and synapsin and spinophilin expression, and reversed the anxiogenic effect of 5-HT1aR antagonist. The deletion of neurogenesis by irradiation or the diminution of synaptogenesis by knockdown of synapsin expression abolished the anxiolytic effects of both CREB and 5-HT1aR activation. These findings suggest that CREB-mediated hippoacampus structural plasticity is crucial for the role of 5-HT1aR in modulating anxiety-related behaviors.

In order to provide new information on the antioxidant activities before and after in vitro digestion of selected fruits for consumers, nutritionists, and food policy makers, total phenolic contents and antioxidant capacities of 33 fruits were evaluated by Folin–Ciocalteu method, 2,2-diphenyl-1-picrylhydrazyl radical scavenging activity assay (DPPH), Ferric reducing antioxidant power assay (FRAP), and ABTS•+ radical cation scavenging activity (ABTS) assay, respectively. The correlations between DPPH, FRAP, ABTS values and total phenolic contents were also evaluated. The results showed that different fruits had diverse antioxidant capacities and the variation was very large. Before the gastric and duodenal phases of in vitro digestion, there was a high positive correlation between the DPPH, FRAP, ABTS values, and total phenolic contents. After the digestion, however, the correlations between the results and the total phenolic contents were poor. Four fruits, plum (sanhua) (Prunus salicina Lindl), red bayberry (Myrica rubra (Lour.)), plum (green) (P. salicina Lindl), and mango (shuixian) (Mangifera indica Linn) showed the strongest antioxidant activities they were before or after the digestion, which implied that these fruits were important natural sources for preventing oxidative stress diseases.

New strategies must be developed to resolve the problems of stroke treatment. In recent years, stem cell-based therapy after stroke has come into the public and academic lens. Previously we have shown that uncoupling neuronal nitric oxide synthase (nNOS) from the postsynaptic density protein-95 (PSD-95) by ZL006, a small molecular compound, can ameliorate ischemic damage and promote neuronal differentiation of endogenous neural stem cells (NSCs) in focal cerebral ischemic male rats. In this study, we transplanted exogenous NSCs into the ipsilateral hemisphere of male rats in combination with ZL006 treatment after ischemic stroke. We show that ZL006 treatment facilitates the migration of transplanted NSCs into the ischemia-injured area and promotes neuronal differentiation of these cells, which is not due to a direct effect of ZL006 on exogenous NSCs but is associated with increased phosphorylation of cAMP response element-binding protein (CREB) in neurons and favorable microenvironment. Moreover, improved functional outcome in the ZL006-treated group was also found. Taken together, our data indicate that ZL006, uncoupling nNOS-PSD-95 in neurons, positively regulates the fate of transplanted NSCs and benefits the functional outcome after stroke in male rats.

Serotonin 1a-receptor (5-HT1aR) has been specifically implicated in the pathogenesis of anxiety. However, the mechanism underlying the role of 5-HT1aR in anxiety remains poorly understood. Here we show in mice that the transcription factor cAMP response element binding protein (CREB) in the hippocampus functions as an effector of 5-HT1aR in modulating anxiety-related behaviors. We generated recombinant lentivirus LV-CREB133-GFP expressing a dominant negative CREB which could not be phosphorylated at Ser133 to specifically reduce CREB activity, and LV-VP16-CREB-GFP expressing a constitutively active fusion protein VP16-CREB which could be phosphorylated by itself to specifically enhance CREB activity. LV-CREB133-GFP neutralized 5-HT1aR agonist-induced up-regulation of synapse density, spine density, dendrite complexity, neurogenesis, and the expression of synapsin and spinophilin, two well-characterized synaptic proteins, and abolished the anxiolytic effect of 5-HT1aR agonist; whereas LV-VP16-CREB-GFP rescued the 5-HT1aR antagonist-induced down-regulation of synapse density, spine density, dendrite complexity, neurogenesis and synapsin and spinophilin expression, and reversed the anxiogenic effect of 5-HT1aR antagonist. The deletion of neurogenesis by irradiation or the diminution of synaptogenesis by knockdown of synapsin expression abolished the anxiolytic effects of both CREB and 5-HT1aR activation. These findings suggest that CREB-mediated hippoacampus structural plasticity is crucial for the role of 5-HT1aR in modulating anxiety-related behaviors.

Phosphofructokinase-1 (PFK-1), a major regulatory glycolytic enzyme, has been implicated in the functions of astrocytes and neurons. Here, we report that PFK-1 negatively regulates neurogenesis from neural stem cells (NSCs) by targeting pro-neural transcriptional factors. Using in vitro assays, we found that PFK-1 knockdown enhanced, and PFK-1 overexpression inhibited the neuronal differentiation of NSCs, which was consistent with the findings from NSCs subjected to 5 h of hypoxia. Meanwhile, the neurogenesis induced by PFK-1 knockdown was attributed to the increased proliferation of neural progenitors and the commitment of NSCs to the neuronal lineage. Similarly, in vivo knockdown of PFK-1 also increased neurogenesis in the dentate gyrus of the hippocampus. Finally, we demonstrated that the neurogenesis mediated by PFK-1 was likely achieved by targeting mammalian achaete-scute homologue-1 (Mash 1), neuronal differentiation factor (NeuroD), and sex-determining region Y (SRY)-related HMG box 2 (Sox2). All together, our results reveal PFK-1 as an important regulator of neurogenesis.

Telomere and telomerase alterations have been reported in mood disorders. However, the role of telomerase in depression remains unclear. Here we show that chronic mild stress (CMS) led to a significant decrease in telomerase reverse transcriptase (TERT) level and telomerase activity in the hippocampus. Treatment with antidepressant fluoxetine reversed the CMS-induced TERT and telomerase activity changes. Inhibiting telomerase by systemic administration (100 mg · kg−1· d−1, i.p., for 14 d), intrahippocampal microinjection (0.7 μmol, 2 μl), or infusion (using an osmotic minipump, 0.134 μg/μl, 0.25 μl/h) of 3′-azido-deoxythymidine (AZT) resulted in depression-like behaviors and impaired hippocampal neurogenesis in mice. In contrast, overexpressing telomerase by intrahippocampal infusion of recombinant adenovirus vector expressing mouse TERT (Ad-mTERT-GFP) led to neurogenesis upregulation, produced antidepressant-like behaviors, and prevented the CMS-induced behavioral modifications. Disrupting neurogenesis in the dentate gyrus by X-irradiation (15 Gy) of a restricted region of mouse brain containing the hippocampus abolished the antidepressant-like effect of Ad-mTERT-GFP. Additionally, AZT had no effect on DNA polymerase activity and did not cause cell damagein vitroandin vivo. Microinjection of AZT into the subventricular zone of lateral ventricle (0.7 μmol, 2 μl) inhibited local neurogenesis but had no behavioral effect. These results suggest that hippocampal telomerase is involved in the modulation of depression-related behaviors, possibly by regulating adult neurogenesis.

The molecular mechanisms underlying the behavioral effects of glucocorticoids are poorly understood. We report here that hippocampal neuronal nitric oxide synthase (nNOS) is a crucial mediator. Chronic mild stress and glucocorticoids exposures caused hippocampal nNOS overexpression via activating mineralocorticoid receptor. In turn, hippocampal nNOS-derived nitric oxide (NO) significantly downregulated local glucocorticoid receptor expression through both soluble guanylate cyclase (sGC)/cGMP and peroxynitrite (ONOO−)/extracellular signal-regulated kinase signal pathways, and therefore elevated hypothalamic corticotrophin-releasing factor, a peptide that governs the hypothalamic-pituitary-adrenal axis. More importantly, nNOS deletion or intrahippocampal nNOS inhibition and NO-cGMP signaling blockade (using NO scavenger or sGC inhibitor) prevented the corticosterone-induced behavioral modifications, suggesting that hippocampal nNOS is necessary for the role of glucocorticoids in mediating depressive behaviors. In addition, directly delivering ONOO−donor into hippocampus caused depressive-like behaviors. Our findings reveal a role of hippocampal nNOS in regulating the behavioral effects of glucocorticoids.

Stroke is a major public health problem leading to high rates of death and disability in adults. Excessive stimulation of N-methyl-D-aspartate receptors (NMDARs) and the resulting neuronal nitric oxide synthase (nNOS) activation are crucial for neuronal injury after stroke insult. However, directly inhibiting NMDARs or nNOS can cause severe side effects because they have key physiological functions in the CNS. Here we show that cerebral ischemia induces the interaction of nNOS with postsynaptic density protein-95 (PSD-95). Disrupting nNOS-PSD-95 interaction via overexpressing the N-terminal amino acid residues 1-133 of nNOS (nNOS-N(1-133)) prevented glutamate-induced excitotoxicity and cerebral ischemic damage. Given the mechanism of nNOS-PSD-95 interaction, we developed a series of compounds and discovered a small-molecular inhibitor of the nNOS-PSD-95 interaction, ZL006. This drug blocked the ischemia-induced nNOS-PSD-95 association selectively, had potent neuroprotective activity in vitro and ameliorated focal cerebral ischemic damage in mice and rats subjected to middle cerebral artery occlusion (MCAO) and reperfusion. Moreover, it readily crossed the blood-brain barrier, did not inhibit NMDAR function, catalytic activity of nNOS or spatial memory, and had no effect on aggressive behaviors. Thus, this new drug may serve as a treatment for stroke, perhaps without major side effects.

It has been demonstrated that neuronal nitric oxide synthase (nNOS) negatively regulates adult neurogenesis. However, the cellular and molecular mechanisms underlying are poorly understood. Here, we show that nNOS from neural stem cells (NSCs) and from neurons play opposite role in regulating neurogenesis. The NSCs treated with nNOS inhibitor N5-(1-imino-3-butenyl)-L- ornithine (L-VNIO) or nNOS gene deletion exhibited significantly decreased proliferation and neuronal differentiation, indicating that NSCs-derived nNOS is essential for neurogenesis. The NSCs cocultured with neurons displayed a significantly decreased proliferation, and deleting nNOS gene in neurons or scavenging extracellular nitric oxide (NO) abolished the effects of coculture, suggesting that neurons-derived nNOS, a source of exogenous NO for NSCs, exerts a negative control on neurogenesis. Indeed, the NSCs exposed to NO donor DETA/NONOate displayed decreased proliferation and neuronal differentiation. The bidirectional regulation of neurogenesis by NSCs- and neurons-derived nNOS is probably related to their distinct subcellular localizations, mainly in nuclei for NSCs and in cytoplasm for neurons. Both L-VNIO and DETA/NONOate inhibited telomerase activity and proliferation in wild-type (WT) but not in nNOS−/− NSCs, suggesting a nNOS-telomerase signaling in neurogenesis. The NSCs exposed to DETA/NONOate exhibited reduced cAMP response element binding protein (CREB) phosphorylation, nNOS expression, and proliferation. The effects of DETA/NONOate were reversed by forskolin, an activator of CREB signaling. Moreover, disrupting CREB phosphorylation by H-89 or LV-CREB133-GFP simulated the effects of DETA/NONOate, and inhibited telomerase activity. Thus, we conclude that NSCs-derived nNOS stimulates neurogenesis via activating telomerase, whereas neurons-derived nNOS represses neurogenesis by supplying exogenous NO that hinders CREB activation, in turn, reduces nNOS expression in NSCs.

Increasing evidence suggests that 5-HT1Areceptor (5-HT1AR) is implicated in anxiety disorders. However, the mechanism underlying the role of 5-HT1AR in these diseases remains unknown. Here, we show that 5-HT1AR-selective agonist 8-OH-DPAT and selective serotonin reuptake inhibitor (SSRI) fluoxetine downregulated hippocampal neuronal nitric oxide synthase (nNOS) expression, whereas 5-HT1AR-selective antagonist NAN-190 upregulated hippocampal nNOS expression. By assessing anxiety-related behaviors using the novelty suppressed feeding, open-field, and elevated plus maze tests, we show that mice lacking nNOS gene [knock-out (KO)] or treated with nNOS-selective inhibitor 7-nitroindazole (7-NI; i.p., 30 mg/kg/d for 28 d; or intrahippocampal microinjection, 16.31 μg/1.0 μl) displayed an anxiolytic-like phenotype, implicating nNOS in anxiety. We also show that, in wild-type (WT) mice, administrations of 8-OH-DPAT (i.p., 0.1 mg/kg/d) or fluoxetine (i.p., 10 mg/kg/d) for 28 d caused anxiolytic-like effects, whereas NAN-190 (i.p., 0.3 mg/kg/d for 28 d) caused anxiogenic-like effects. In KO mice, however, these drugs were ineffective. Moreover, intrahippocampal infusion of 8-OH-DPAT (45.963 μg/100 μl) using 14 d osmotic minipump produced anxiolytic effects. Intrahippocampal microinjection of 7-NI (16.31 μg/1.0 μl) abolished the anxiogenic-like effects of intrahippocampal NAN-190 (4.74 μg/1.0 μl). Additionally, NAN-190 decreased and 8-OH-DPAT increased phosphorylated cAMP response element-binding protein (CREB) levels in WT mice but not in KO mice. Blockade of hippocampal CREB phosphorylation by microinjection of H89 (5.19 μg/1.0 μl), a PKA (protein kinase A) inhibitor, abolished the anxiolytic-like effects of 7-NI (i.p., 30 mg/kg/d for 21 d). These findings indicate that both hippocampal nNOS and CREB activity mediate the anxiolytic effects of 5-HT1AR agonists and SSRIs.