| Fear,a kind of basic emotion related to adaptive ability,is defined as a learning capacity motivated by threatening or potenticlly threatening stimuli.It helps animals to organize appropriate defensive behaviors in response to threat.Reasonable fear allows animals to assess the risk of situations in time,avoide potencial danger and enhance survivability in the novel environment.However,pathological fear memories,such as fear generalization,can also lead to post-traumatic stress disorder(PTSD),simple phobia,social anxiety disorder,panic disorder in humans.PTSD has been the fourth most common psychiatric diagnosis.Estimates are that more than 90%of all people will be exposed to at least one severe traumatic event during their lifetime,and experiencing one extremely traumatic event can lead to PTSD.According to epidemiological data,the prevalence of PTSD is approximately 5-10%of the general population,however,closer to 20-30%in highly exposed trauma populations.The characteristics of PTSD are rapidly acquired,temporally enduring,and broadly generalized across both familiar and novel contexts.Our therapeutic method is still limited.Exposure therapy is widely used to treat fear disorders,but it is often slow to develop,short lived,and context dependent.PTSD has brought burden to individual and society.Thus,there is an urgent need for a deeper study of fear extinction which will provide a new target for treating fear-related disorders.In recent years,more and more attention has been devoted to the relationship between neurogenesis and PTSD,especially the hippocampal dentate gyrus(DG)neurogenesis.The DG subregion of the hippocampus is a substrate for both cognitionand mood regulation.The trisynaptic excitatory circuits including DG in hippocampus is necessary for contextual fear memory.Recently,it has been reported that hippcampal neurogenesis correlates with PTSD closely.For example,stimulating adult hippocampal neurogenesis can promote pattern separation.Therefore,stimulating adult hippocampal neurogenesis may be a novel therapeutic strategy for treating fear-related disorders,such as PTSD.Neuronal nitric oxide synthase(nNOS)is mainly expressed in neurons,to some extent in astrocytes and neuronal stem cells.nNOS can link to PSD-95 via direct PDZ-PDZ domain,forming nNOS-PSD-95 complex.Postsynaptic density 95(PSD-95)is a synaptic protein regulating glutamatergic plasticity,synaptic stability and formation of dendritic spine.nNOS-PSD-95 complex mediates synapse formation and is implicated in N-methyl-D-aspartic acid(NMDA)-induced neuronal death.Pharmacological studies reveal that nNOS,enriched in the hippocampus,is an endogenous repressor of neurogenesis.Under physiological and pathological conditions,nNOS-derived nitric oxide(NO)is implicated in synapse loss-mediated early cognitive and motor deficitsin and negatively regulates neurogenesis.According to our experiments,decreasing nNOS-PSD-95 complex level facilitates the proliferation of newborn cells and dendritic spine formation of mature neurons in the ischemic brain of middle cerebral artery occlusion(MCAO)rats.Taken together,it remains in question whether nNOS-PSD-95 couplex regulates extinction of fear memory by regulating hippocampal neurogenesis,and whether nNOS-PSD-95 couplex could be a potential drug target for PTSD.Accoding to the experiments,we find that disrupting the interaction between nNOS and PSD-95 facilitates DG neurogenesis,and it is possible that the mechanism underlying the change of neurogenesis may promote the retrieval of extinction memory.Accordingly,we investigated(1)whether disrupting hippocampal nNOS-PSD-95 coupling facilitates adult neurogenesis;(2)whether disrupting hippocampal nNOS-PSD-95 coupling promotes the retrieval of extinction memory;(3)whether ehanced adult neurogenesis induced by disrupting hippocampal nNOS-PSD-95 coupling is implicated in the retrieval of extinction memory.1.To investigate whether disrupting hippocampal nNOS-PSD-95 coupling facilitates adult neurogenesis,we firstly tested the contextual remote fear conditioning procedure,and the behavioral experiment confirmed its reliability.Next,we implanted microcannula into the brain of mice and subjected mice to contextual remote fear conditioning procedure.After 24 h,we microinjected a small molecule named ZL006 or a lentiviral vector called LV-nNOS-N1-133-GFP into the hippocampus DG region through microcannula to disrupt nNOS-PSD-95 binding.Then,to examine the effect of disrupting nNOS-PSD-95 complex on neurogenesis,we treated mice with 5-Bromodeoxyuridine(BrdU)solution.We found that decreasing the nNOS-PSD-95 complex level can significantly facilitate neuronal proliferation and survival.To explore the effect of disrupting nNOS-PSD-95 complex on synaptic plasticity,as previous procedure,mice were treated with LV-nNOS-N1-133-GFP for 2 days and sacrificed 4 weeks later to detect the dendritic spine density with Golgi-staining kit.The data showed that disrupting nNOS-PSD-95 compling can significantly increase spine density.In conclusion,these observations suggest that disrupting nNOS-PSD-95 coupling in hippocampal DG promotes adult neurogenesis and improve synaptic plasticity.2.To investigate whether disrupting hippocampal nNOS-PSD-95 coupling enhances the extinction memory,we began by subjecting mice to contextual remote fear conditioning procedure after microcannul implantation.Then,Three agents including a small molecule named ZL006,a small peptide named Tat-nNOS 1-133 and a lentiviral vector called LV-nNOS-N1-133-GFP were were respectively microinjected into the hippocampus DG to disrupt nNOS-PSD-95 binding.Behavior results revealed that mice treated with ZL006 in DG showed a significant reduction of freezing time in the retrieval of extinction memory.Similar results were acquired in Tat-nNOS 1-133-treated and LV-nNOS-Ni-133-GFP-treated mice.To futher explore the effect of delayed administration of LV-nNOS-N1-133-GFP,we microinjected LV-nNOS-N1-133-GFP into the hippocampus DG 4 weeks after contextual remote fear conditioning.Behavior results shown that delayed administration of LV-nNOS-N1-133-GFP significantly reduce the freezing time in the retrieval of extinction memory.In conclusion,our study indicated that disrupting hippocampal nNOS-PSD-95 coupling enhances the extinction memory,and the underlying mechanism may be related to enhanced adult neurogenesis.3.To further confirm the neuronal mechanism underlying the promotion of extinxtion memory retrival induced by disrupting nNOS-PSD-95 binding,firstly,we microinjected LV-nNOS-N1-133-GFP into hippocampual DG of nNOS-/-mice region after contextual remote fear conditioning,and found that no reduction of freezing behavior was observed in nNOS-/-mice injected with LV-nNOS-N1-133-GFP in DG.Our observation suggest that,disrupting nNOS-PSD-95 coupling in DG contribute to the promotion of fear memory retrival.Secondly,to demonstrate the key role of adult neurohenesis in the promotion of extinction memory retrieval,we randomly divided conditioned mice equally into three groups after contextual remote fear conditioning,control group,treatment group and radiation group.Treatment group and radiation group were both treated with LV-nNOS-N1-133-GFP to disrupt nNOS-PSD-95 coupling in DG.Then we suppress adult neurogenesis induced by disrupting nNOS-PSD-95 couplex by exposing radiation group to Xray.We observed that treatment group significantly reduced the freezing time in extinction memory retrieval trial,but no reduction of freezing behavior was observed in radiation group.Behaviour tests indicated that enhanced adult neurogenesis induced by disrupting hippocampal nNOS-PSD-95 coupling is implicated in the retrieval of extinction memory.In conclusion,these observation suggest that ZL006,Tat-nNOS1-133 and LV-nNOS-Ni1-33-GFP can significantly enhance the extinction memory by facilitating DG adult neurogenesis and synaptic plasticity due to block of nNOS-PSD-95 coupling. |
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