反复应激引起前额叶皮层上谷氨酸受体的表达

1、反复应激引起前额叶皮层上谷氨酸受体的表达第1页，共35页，2022年，5月20日，18点16分，星期二Introduction:Adrenal corticosterone, the major stress hormone, through the activation of glucocorticoid receptor (GR) and mineralocorticoid receptor (MR), can induce long-lasting inuences on cognitive and emotional processes. Mounting evidence sugge

2、sts that inappropriate stress responses act as a trigger for many mental illnesses.It has been proposed that glutamate receptor-mediated synaptic transmission that controls PFC neuronal activity is crucial for working memory In this study, we sought to determine whether repeated stress might negativ

3、ely inuence PFC-mediated cognitive processes by disturbing glutamatergic signaling in juvenile animals第2页，共35页，2022年，5月20日，18点16分，星期二Repeated 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-accessibl

4、e 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.第3页，共35页，2022年，5月20日，18点16分，星期二Results:To test the impact of stress on cognitive functions, they measured the recognition memory

5、 taskRU486: GR antagonist第4页，共35页，2022年，5月20日，18点16分，星期二To 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 plat

6、form (30 min), cage movement (30 min),lights on overnight, immobilization (RT, 1 hr), and food and water deprivation overnight.第5页，共35页，2022年，5月20日，18点16分，星期二Object location task:To test the specicity of this stress-induced memory decit, we also subjected animals to the object location task, a parad

7、igm for the PFC-independent memory第6页，共35页，2022年，5月20日，18点16分，星期二Open 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 第7页，共35页，2022年，5月20日，18点16分，星期二Temporal Order

8、Recognition Memory Task :To nd 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.第8页，共35页，2022年，5月20日，18点16分，星期二Temporal Order Recognition Memory Task :To test whether glutamatergic transmission in PFC is cr

9、iticalfor the object recognition memory, we gave animals a stereotaxic injection of the NMDAR antagonist APV and AMPAR antagonist CNQX to PFC prelimbic regions bilaterally. 第9页，共35页，2022年，5月20日，18点16分，星期二The conclusion of the Behavioral Testing:Taken together, it suggests that repeated stress has a

10、detrimental effect on recognition memory, whichmay be due to the loss of glutamatergic transmission in PFC.第10页，共35页，2022年，5月20日，18点16分，星期二The Electrophysiological Recordings:To nd out the impact of repeated stress on glutamatergictransmission, we examined the input/output curves of AMPAR-and NMDAR-

11、mediated synaptic currents (EPSC) in PFC pyra-midal neurons from stressed, young (4-week-old) male rats. 第11页，共35页，2022年，5月20日，18点16分，星期二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

12、 ratio (PPR) of AMPAR- andNMDAR-EPSC.PPR was not different in control versus stressed animals, suggesting a lack of gross change in presynaptic function.第12页，共35页，2022年，5月20日，18点16分，星期二miniature EPSC (mEPSC):To further conrm the involvement of postsynaptic glutamatereceptors, we measured miniature E

13、PSC (mEPSC), a synapticresponse resulting from quantal release of single glutamatevesicles, in PFC slices.第13页，共35页，2022年，5月20日，18点16分，星期二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 (

14、a pure postsynaptic preparation). 第14页，共35页，2022年，5月20日，18点16分，星期二Inject the GR antagonist RU486:Systemic injections of RU486Local injections of RU486 to the PFC第15页，共35页，2022年，5月20日，18点16分，星期二The ralationship with the time of stress:To understand the complex actions of stress hormones, we exposed a

15、nimals to various days of restraint stress. 第16页，共35页，2022年，5月20日，18点16分，星期二To test the different regions:To test the regional specicity of the effect of repeated stress,we also examined glutamatergic transmission in striatum and hippocampus from young male rats.第17页，共35页，2022年，5月20日，18点16分，星期二Bioch

16、emical 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.第18页，共35页，2022年，5月20日，18点16分，星期二The amount of the glutamate

17、 recptors:第19页，共35页，2022年，5月20日，18点16分，星期二Inject the GR antagonist RU486:第20页，共35页，2022年，5月20日，18点16分，星期二To test the different regions:No signicant changes were found in other brain areas, including striatum and hippocampus.第21页，共35页，2022年，5月20日，18点16分，星期二The results in vitro:We next examined whethe

18、r the effect of repeated stress in vivo may be mimicked by corticosterone (CORT) application in vitro. 第22页，共35页，2022年，5月20日，18点16分，星期二Inject the GR antagonist and MR antagonist :RU486:The GR antagonist; RU28318:The MR antagonist第23页，共35页，2022年，5月20日，18点16分，星期二Measure the cluster density :第24页，共35页，

19、2022年，5月20日，18点16分，星期二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.第25页，共35页，2022年，5月20日，18点16分，星期二Ubiquitin/

20、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.第26页，共35页，2022年，5月20日，18点16分，星期二Test other proteasome inhibitor:MG132:proteasome inhibitor

21、Lact: another proteasome inhibitorChloro: general lysosomal enzyme inhibitorLeupeptin: lysosomal protease inhibitor11R-CS:membrane-permeable calpain protease inhibitor第27页，共35页，2022年，5月20日，18点16分，星期二Test the effect of proteasome inhibitor:第28页，共35页，2022年，5月20日，18点16分，星期二ShRNA Lentiviral Knockdown:Gi

22、ven 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.第29页，共35页，2022年，5月20日，18点16分，星期二ShRNA Lentiviral Knockdown in vitro:第30页，共35页，2022年，5月20日，18点16分，星期二ShRNA Lentiviral Knockdown in vivo:第31页，共35页，2022年，5月20日，18点16分，星期二S