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Repeated Stress Causes Cognitive Impairment by Suppressing Glutamate Receptor Expression and Function in Prefrontal CortexIntroduction:1)Adrenal corticosterone,the major stress hormone,through the activation of glucocorticoid receptor(GR)and mineralocorticoid receptor(MR),can induce long-lasting influences on cognitive and emotional processes.2)Mounting evidence suggests that inappropriate stress responses act as a trigger for many mental illnesses.3)It has been proposed that glutamate receptor-mediated synaptic transmission that controls PFC neuronal activity is crucial for working memory 4)In this study,we sought to determine whether repeated stress might negatively influence PFC-mediated cognitive processes by disturbing glutamatergic signaling in juvenile animalsRepeated restraint stress:Juvenile(3-to 4-week-old)Sprague Dawley male rats were used in this study.For repeated restraint stress,rats were placed in air-accessible cylinders for2 hr daily(10:00 a.m.to 12:00 p.m.)for 57 days.The container size was similar to the animal size,which made the animal almost immobile in the container.Results:To test the impact of stress on cognitive functions,they measured the recognition memory taskRU486:GR antagonistTo examin whether other stressors could produce a similar effectRepeated unpredictable stress Rats were subjected each day to twostressors that were randomly chosen from six different stressors,including forced swim(RT,30 min),elevated platform(30 min),cage movement(30 min),lights on overnight,immobilization(RT,1 hr),and food and water deprivation overnight.Object location task:To test the specificity of this stress-induced memory deficit,we also subjected animals to the object location task,a paradigm for the PFC-independent memoryOpen field task:In contrast to the impaired temporal order recognitionmemory,rats exposed to repeated restraint stress showed no changes in anxiety-related behavior or locomotive activity Temporal Order Recognition Memory Task:To find out the onset of the detrimental effects of stress oncognition,we exposed young male rats to various days(1,3,5 and 7)of restraint stress.Temporal Order Recognition Memory Task:To test whether glutamatergic transmission in PFC is criticalfor the object recognition memory,we gave animals a stereotaxic injection of the NMDAR antagonist APV and AMPAR antagonist CNQX to PFC prelimbic regions bilaterally.The conclusion of the Behavioral Testing:Taken together,it suggests that repeated stress has a detrimental effect on recognition memory,whichmay be due to the loss of glutamatergic transmission in PFC.The Electrophysiological Recordings:To find out the impact of repeated stress on glutamatergictransmission,we examined the input/output curves of AMPAR-and NMDAR-mediated synaptic currents(EPSC)in PFC pyra-midal neurons from stressed,young(4-week-old)male rats.paired pulse ratio(PPR):To test whether the reduced synaptic transmission by re-peated stress may result from a presynaptic mechanism,wemeasured the paired pulse ratio(PPR)of AMPAR-andNMDAR-EPSC.PPR was not different in control versus stressed animals,suggesting a lack of gross change in presynaptic function.miniature EPSC(mEPSC):To further confirm the involvement of postsynaptic glutamatereceptors,we measured miniature EPSC(mEPSC),a synapticresponse resulting from quantal release of single glutamatevesicles,in PFC slices.Whole-cell ionic current:Moreover,we measured whole-cell ionic current elicited byAMPA(100 mM)or NMDA(100 mM)application in acutely dissociated PFC neurons(a pure postsynaptic preparation).Inject the GR antagonist RU486:Systemic injections of RU486Local injections of RU486 to the PFCThe ralationship with the time of stress:To understand the complex actions of stress hormones,we exposed animals to various days of restraint stress.To test the different regions:To test the regional specificity of the effect of repeated stress,we also examined glutamatergic transmission in striatum and hippocampus from young male rats.Biochemical Measurement of Surface and Total Proteins:We performed western blotting and surface biotinylationexperiments to detect the total and surface levels of AMPAR and NMDAR subunits in PFC slices from stressed,young(4-week-old)male rats.The amount of the glutamate recptors:Inject the GR antagonist RU486:To test the different regions:No significant changes were found in other brain areas,including striatum and hippocampus.The results in vitro:We next examined whether the effect of repeated stress in vivo may be mimicked by corticosterone(CORT)application in vitro.Inject the GR antagonist and MR antagonist:RU486:The GR antagonist;RU28318:The MR antagonistMeasure the cluster density:Ubiquitin/Proteasome-dependent Degradation:Since the total level of NR1 andGluR1 was reduced in repeatedly stressed animals,we examined whether it could be due to the decreased synthesis or increased degradation of glutamate receptors.Ubiquitin/Proteasome-dependent Degradation:The level of ubiquitinated GluR2,NR2A,or NR2B subunitsremained unchanged.Repeated stress also failed to alter the ubiquitination of SAP97(a GluR1 binding protein)and PSD-95.Test other proteasome inhibitor:MG132:proteasome inhibitorLact:another proteasome inhibitorChloro:general lysosomal enzyme inhibitorLeupeptin:lysosomal protease inhibitor11R-CS:membrane-permeable calpain protease inhibitorTest the effect of proteasome inhibitor:ShRNA Lentiviral Knockdown:Given the role of proteasome-dependent degradation of gluta-mate receptors in the detrimental effects of repeated stress,we would like to know which E3 ubiquitin ligases are potentiallyinvolved in the stress-induced ubiquitination of GluR1 and NR1subunits in PFC.Nedd4-1:an E3 ligase necessary for GluR1 ubiquitination in response to the agonist AMPA.Fbx2:an E3 ligase in the ER that ubiquitinates NR1 subunits.ShRNA Lentiviral Knockdown in vitro:ShRNA Lentiviral Knockdown in vivo:ShRNA Lentiviral Knockdown in vivo:Test other region:To understand the potential mechanism underlying the re-gion specificity of the effects of repeated stress on glutamatereceptor expression and function,we examined the level ofNedd4-1 and Fbx2 in PFC,striatum,and hippocampus fromcontrol versus stressed young male rats.Summary:Repeated stressCORTNedd4-1Fbx2GluR1 ubiquitinationNR1 ubiquitinationAMPANMDARBehavioral TestQuestion:The biochemical signaling cascades underlying the GR-induced changes in the activity and expression of Nedd4-1 and Fbx2.Question:Whether the EC cortex has the similar effect?Thanks for your suggestions!
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