Hippocampal NNOS-PSD-95 Coupling Regulates Fear Extinction

As a normal part of human emotions,fear is a mental state induced by an actual or imaginary threat.Learning to fear allows animals to assess the risk of situations in time and organize appropriate defensive behaviors to threats.However,pathological fear memories may lead to fear-related disorders,including posttraumatic stress disorder（PTSD）,panic disorder and phobia.According to epidemiological data,lifetime prevalence rates of PTSD have been estimated at 5-10%in the community,and around 10-40%in specific trauma populations（such as veterans）.In recent years,there has been a sensible rise in the rates of PTSD along with the increased traumatic events.The cost of such disorder on the individual and society can be tremendous.Yet treatment of fear-related disorders is a considerable challenge insofar.Fear memories are rapidly acquired,temporally enduring,and can generalize across contexts.Moreover,exposure therapy based on fear extinction is often slow to develop,short lived,and context dependent.Given the high rates and therapeutic status of fear-related disorders,there is an urgent need for a deeper understanding of the pathological fear mechanisms.In the laboratory,Pavlovian fear conditioning and contextual fear conditiong are powerful animal models of fear-related disorders for understanding the underlying mechanisms of pathological fear memories.As one of components of the limbic system,the hippocampus plays an important role in mood and cognitive disorders.Previous work has shown that hippocampus is involved in the development and treatment of PTSD.The trisynaptic excitatory circuits including CA3 in hippocampus is necessary for contextual fear conditioning.Yet the precise hippocampus circuits and mechanisms involved in fear extinction remain poorly understood.Neuronal nitric oxide synthase（nNOS）,a main NO synthase in the central nervous system,is enriched in the limbic system.Pharmacological and genetic studies reveal that nNOS and NO signaling may contribute to fear acquisition and extinction.Post-synaptic density protein-95（PSD95）,a multivalent excitatory synaptic scaffolding protein and core component of the post-synaptic density,can link to nNOS via direct PDZ-PDZ domain,forming nNOS-PSD-95 complex.PSD-95 has an integral functional role within postsynaptic machinery mediating glutamatergic plasticity.Recent studies indicate that PSD-95 is a key contributor to the regulation of synaptic functions critical for fear memory formation,stability and extinction.NMDA receptor-mediated synaptic plasticity may be the core mechanism of PTSD.In addition,We find that the nNOS-PSD-95 interaction is related to NMDAR activation.Therefore,it is possible that nNOS-PSD-95 interaction may be implicated in regulation of fear extinction.Accordingly,we investigated（1）whether hippocampal nNOS-PSD-95 coupling regulates fear extinction;（2）the molecular mechanism underlying regulation of fear extinction by nNOS-PSD-95 coupling.In part one,we began by testing the contribution of nNOS-PSD-95 coupling to fear memory extinction processes.To this end,we subjected adult mice to contextual fear extinction trials.These mice was sacrificed 1h after the last extinction trial and assessed the nNOS-PSD-95 complex levels in dorsal hippocampus by Coimmunoprecipitation.We found contextual fear extinction significantly decreased nNOS-PSD-95 complex levels.To investigate whether nNOS-PSD-95 coupling regulates fear extinction,immediately after memory test,we treated mice with a small molecule ZL006（20mg/kg,i.p.）for 7 days before extinction trials and immediately after each extinction trial to block nNOS-PSD-95 binding.The data showed that systemic administration of ZL006 facilitated both contextual fear extinction and cued fear extinction.To examine the real phase of extinction regulated by nNOS-PSD-95uncoupling,conditioned mice were respectively treated with ZL006 for 7 days before extinction,or immediately after each extinction trial.We found that treatment of ZL006 before extinction facilitated contextual fear extinction,while within extinction sections had no effects.Otherwise,treatment of ZL006（20mg/kg,i.p.）for 7 days before fear conditioning had no influence on contextual-fear and cued-fear acquisition.To explore the key subregion of hippocampus involved in fear extinction,we disrupted the nNOS-PSD-95 interaction respectively in dorsal hippocampal CA1,CA3 and DG by implanting microcannula into these regions.Three agents including a small molecule named ZL006,a small peptide named Tat-nNOS1-133 and a lentiviral vector called LV-nNOS_1-133–GFP were applied in researches to block the interaction of nNOS with PSD95.Behavior results revealed that mice treated with ZL006 in CA3showed a significant reduction of freezing time in contextual fear extinction.Similar results were acquired in Tat-nNOS_1-133-treated and LV-nNOS_1-133–GFP-treated mice.But the effect of ZL006 in CA3 disappeared in nNOS gene knock-out（nNOS-/-）mice.Interestingly,no reduction of freezing behavior was observed in mice injected with Tat-nNOS_1-133-133 in CA1 or ZL006 in DG.Notably,treatment with LV-nNOS_1-133–GFP did not affect extinction of remote contextual fear memory.Otherwise,injection of ZL006 in CA3 also facilitated cued-fear extinction.In conclusion,these observations suggest that nNOS-PSD-95 coupling in hippocampal CA3 negatively regulates fear extinction via affecting acquisition of extinction.In part 2,we explored the possible mechanism by which nNOS-PSD-95uncoupling regulates contextual-fear extinction.Firstly,we divided conditioned mice equally into two groups after contextual fear conditioning,no-extinction and extinction group.Western blot was carried out to detect the protein expression in dorsal hippocampus.Compare to no-extinction group,contextual fear extinction trials led to high levels of BDNF.Next,we subjected mice to contextual fear conditioning procedure,and infused LV-nNOS1-133–GFP into hippocampal CA3 by microcannula before extinction trials.Infusion of LV-nNOS_1-133–GFP diminished freezing behavior at the first extinction trial（E1）.Then the mice were killed 1h after E1 and the expression of BDNF in CA3was measured by WB.As expect,treatment with LV-nNOS_1-133–GFP induced BDNF overexpression.Similar results were obtained when mice were injected with ZL006.As previous,conditioned mice were injected with ZL006 for four days.Freezing behavior was remarkably declined at E2.Then we killed mice 1h after E2 to assess BDNF level.Consistent with the effect of LV-nNOS_1-133–GFP,mice displayed enhanced BDNF level in CA3.Meanwhile,the expression of BDNF in nNOS-/-mice treated with ZL006 is not affected.These data indicate that BDNF may account for regulation of contextual-fear extinction by nNOS-PSD-95 uncoupling.To exclude the possibility that upregulation of BDNF contributes to the modulation of extinction by nNOS-PSD-95 uncoupling,we explored TrkB-FC,a BDNF scavenger,into CA3 within extinction trials.The enhancement of intra-CA3LV-nNOS_1-133–GFP in fear extinction was completely reversed by TrkB-FC.Moreover,the effect of ANA-12,a specific TrkB receptor antagonist,was similar to that of TrkB-FC.Thus,BDNF does be a key regulator implicating in the role of nNOS-PSD-95 coupling in contextual-fear extinction.BDNF expression may also be regulated by HDAC2 under epigenetic mechanisms.Our previous studies demonstrated that nNOS-PSD-95 disassociation led to changes in HDAC2 expression and activity.To gain further insight into the underlying mechanisms of fear extinction,we investigated the HDAC2 role in the regulation of extinction by nNOS-PSD-95 uncoupling.As previous procedure,conditioned mice were equally divided into two groups,no-extinction and extinction group.WB and immunofluorescence assay were performed to detect the HDAC2expression.Compared to no-extinction,contextual-fear extinction training downregulated HDAC2 in dorsal hippocampus.Next,we examined the effect of nNOS-PSD-95 disassociation on HDAC2expression.We subjected mice to contextual fear conditioning and microinjected ZL006 into CA3 for four days before extinction training.Behavior results showed a significant decline at E2 in ZL006-treated mice.Then we prepared hippocampal lysates at 1h after E2.The HDAC2 level in ZL006-treated mice was only 30%of controls.In addition,WB and immunofluorescence assay revealed that ZL006 was ineffective in nNOS-/-mice.To further confirm the role of HDAC2 in nNOS-PSD-95 uncoupling regulation,we used adenoviral vectors expressing HDAC2,named AD-HDAC2-flag,and inactive HDAC2 extract,named AD-HDAC2（7989-1）-flag.Conditioned mice were microinfused with AD-HDAC2-flag or AD-HDAC2（7989-1）-flag,and systemic administration with ZL006 for seven 7days before extinction.We observed the enhancement of ZL006 in fear extinction was completely reversed by AD-HDAC2-flga.Moreover,compared to vehicle controls,AD-HDAC2-flag even significantly inhibited fear extinction.Taken together,HDAC2 downregulation and BDNF upregulation in hippocampal CA3 contribute to the promotion of fear extinction by nNOS-PSD-95 disassociation.Overall,our data indicate that nNOS-PSD-95 coupling in hippocampal CA3negatively regulates fear extinction.HDAC2 downregulation and BDNF upregulation account for the promotion of fear extinction by nNOS-PSD-95 disassociation。...