大鼠下丘脑及杏仁核中促肾上腺皮质素释放激素神经元的激活状态

大鼠下丘脑及杏仁核中促肾上腺皮质素释放激素神经元的激活状态

1crh-containing+kraftingdepera-depercoltic-u型微标签（crh）；（2）反演率培养计划（crh）；（3）以超可压状位的特征为特征的微微产品。微丝无意义。区域一级的大多数运动运动（crh-a）的数量是固定的。微丝无意义。区域一级的大多数运动（crh-a）的数量是固定的。微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝无意义的运动，微丝。2杏仁杏仁2.1检察官办公室2.2CUMSapplication2.4sucoseprocence测试2.5非织造性件lact/溶液phsque/．bachimsiphengeTheprimaryantibodyspecificityhadbeenidentifiedearlier.ThespecificityofCRHantibodywastestedbypreadsorbingprimaryantibodywiththeCRHpeptide(10μmol/L,Bachem)overnight.Thelabelingonthetissuewaseliminatedbyincubationwithpreadsorbedprimaryantiboday.Inaddition,negativecontroltissuewasincubatedwithsalineinsteadoftheprimaryantibody.2.6通过转色织物和rectshrafts方法,主要分为3.4.5和4.5.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.5.3.5.3.5.3.3.5.3.3.5和5.5.3.5.3.3.5.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.4和4.5.5.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.TheCRH-positivecellsinPVNandCeAwerecountedusingacomputerizedimagesystemImageProPlusversion5.0.ThesystemwasconnectedtoaJVC3CCDcameraequippedwithaZeissAixoskopmicroscope(Plan-Neofluar).Areaselectionwasperformedasfollows:ineachsectiontobeanalyzed,theareascoveringPVNorCeAwereloadedintothesystemindividually,andweredisplayedontheimageanalysismonitor.PVNandCeAwereoutlinedmanuallyat2.5×objectiveofthemicroscope.Subsequently,theimaginganalysissystemoverlaidagridofrectangularfieldswithintheoutlinedarea.Eachfieldwasequalinsizetotheareadisplayedbythecameraat40×objective.Foranalysis,alloftherectangularfieldswereselected.Inordertopreventdoublecounting,onlycellscontaininganucleoluswerecounted,andanucleoluswasconsideredtobeaprofile.Celldensitywascalculatedbydividingthetotalnumberofnucleolicountedbythesampledvolume.Theunitofdensitywasprofiles/mm3.2.7内脏器官3产品系统3.1体育用品3.2sucoseprocence测试3.4ldensitysofteritysofter规模ExposuretochronicstressresultedinanincreaseinthedensityofCRHneuronsinPVN.Forinstance,themedianofCRHcelldensitywas53410profiles/mm3inCUMSgroup(n=6),approximately20timeshigherthanthatinthecontrol(2673profiles/mm3,n=6,z=-3.0,P=0.001)(Fig.1).Inaddition,asignificantincreaseinthedensityofCRHneuronswasalsofoundinCeAafterchronicmildstress(12751profiles/mm3,n=6),comparedwiththatinthecontrol(245profiles/mm3,n=6,z=-2.88,P=0.002)(Fig.2).4关于crh-非价格主义的strs和strsThepresentstudyrevealeddepression-likebehavioralchangesandaregionalincreaseofCRH-neurondensityinPVNofthehypothalamusandinCeAinCUMSrats.BothPVNandCeAplayimportantrolesinstressresponse.Thechronicstressprocedure,asintroducedbyWillneretal.,isavalidatedwaytoobtainananimalmodelofdepressionbyapplyingmildstressors.Theprotocolisregardedtocloselymimicthehumansituation,consistingofmoredailyhasslesthantraumaticevents.Theattenuatedpreferenceforsolutionservesasamarkerofgeneralizeddecreaseofsensitivitytoreward,thatis,anhedonia.Indeed,ourdataareconsistentwiththepreviousfindingsthatratsexposedtostressorsconsumelesssucrosethanthecontrols.Thisindicatesthatanhedonia,thecoresymptomofdepression,canbeinducedbyCUMSinrats.OtherobservationsintheCUMSmodel,suchasdecreasesinbodyweightgainandlocomotoractivity,alsoparallelthesymptomsofdepression.Moreover,herewefindastrongincreaseinCRH-positivecelldensityinPVNofCUMSrats.IthasbeenrevealedthatCRHmRNAlevelisincreasedinPVNafterchronicmildstress.OurfindingisconsistentwiththepreviousreportsthatdemonstratehyperactivationofCRHneuronsinPVNbothinstressedratsandinthebrainofdepressivepatients.Besides,wehavefoundinthesamemodelthatCRHmRNAexpressionisincreasedafterCUMS.OurstudyalsoshowsthatthenumberofCRH-positivecellsinCeAissignificantlyincreasedinCUMSrats.AnumberofsyndromesproducedbystressfollowingcentraladministrationofCRHcanalsobeelicitedbyelectricstimulationtotheamygdala,particularlytoCeAinrats.ItisofinterestthatHPA-axisbecomesactivatedduringstimulationofratamygdala,alongwithanincreaseinthelevelofcorticosteroneinserumandsymptomsthataresimilartothoseobservedduringstressresponse,suchasfreezing.CRHadministrationinachemicallesionoftheamygdalafailedtoinducethebehavioraleffects.Furthermore,applicationofaCRHantagonistintheamygdalacouldsignificantlyreducetheresponsetorepeatedstress.Thesefindingssuggestthatstress-inducedchangesintheamygdalaplayapivotalroleintheresponsestostress,suchashighlevelsofstresshormonesinserum,adecreaseinsensitivitytorewardandfreezing.Fear-elicitingproceduresinduceanincreaseinCRHmRNAexpressionlevelinCeAthatparticipatesinmediatingacutestresseffectsonmemoryconsolidation.Similarly,wefoundanincreaseinthenumberofCRHneuronsinCeAafterchronicstress,whichindicatesthatCRHintheamygdalamaybeinvolvedinthememoriesofaversiveexperiences.Consequently,itregulatestheactivationoftheHPA-axisviaprojectionstothehypothalamus,whichmightunderlietheincreaseofdepressivebehaviorsandmoodalterations.SomereportshaveindicatedthatneuronsinCeAhavedirectprojectionstomanyareasinbrain,includingPVNinthehypothalamus.TransmitterreleasefromtheamygdalaontoPVNhasbeenidentifiedbyGray.ThemainoutputneuronsoftheamygdalahavebeendemonstratedtocontainCRHpeptide.TherearealsosynapsesonCRHneuronsintheratPVN.TheaboveevidencecombinedwithourfindingssuggeststhatthedirectCRHneuron-involvingconnectionbetweenPVNandCeAplaysanimportantroleinthestressresponse.ItwouldbeinterestingtoexplorethemarkersofsynapsesbetweenCRHneuronsinPVNandCeA.CRHR1hasbeenobservedinCRH-positiveneuronsanditsexpressionisshowntoincreaseafteracutestressinrats.Inourpreviouspostmortemstudy,wealsofoundanincreaseofCRHR1mRNAexpressioninPVNofdepressedpatients.CRHR1maybeessentialfortheactivationofpostsynapticCRHcellsinPVNduringstress-induceddepression.Inconclusion,ourstudiesshowthatCRHimmunoreactivityinPVNandCeAaresignificantlyincreasedduringchronicmildstress,whichaddsfurtherevidencetothepropositionthatCRHneuronsinbothPVNandCeAareinvolvedinchronicmildstressresponse.TwentyadultmaleSprague-Dawleyrats[obtainedfromAnhuiExperimentalAnimalCenter,weighting(250±10)g],werehoused(6animalspercage)toallowaccustomizationtotheenvironmentoneweekbeforethestressprocedure.Animalswerekeptat21-22°Cundera12:12hlight/darkcycle,withfreeaccesstofoodandwater.Alltheexperimentswereconductedinaccordancewithallrelevantlocalguidelinesandlegislationstominimizepainandsufferingoftheanimals.Ratswiththescoreofhorizontalactivity>100or<30wereexcludedfromfurtherexperiments.Thescoreswerecalculatedbymeasuringthenumberofsquarescrossedintheopenfieldtest.Theremaininganimals(n=12)werethenrandomizedintocontrolandCUMSgroups(n=6ineachgroup).Stresswasappliedconsecutivelyfor21dwithdifferentmildstressors,accordingtotheprocedurespresentedbyDuncko,GronliandWu.Theanimalswererandomlyexposedtooneofthefollowingstressorseachday:hotstressintheovenat45°Cfor5min,swimmingin8°Cwaterfor5min,shakingfrequently(100/min)for5min,60inescapableelectricfootshocksata1.5mAintensityand2sdurationwith1sintervals,tailclampingfor1min,waterandfooddeprivationfor24h,andplacementincagesthatweretilted30°fromthehorizontal.Controlanimalswerekeptwithoutanystressapplicationinthesameconditionsoflightandtemperature,withfreeaccesstowaterandfoodexceptfortheperiodofsucrosepreferencetest.2.3通过转色剂和风价值的remowelloffixationforade,remowelloffixationoffixationremowelloffixation.roOpenfieldtestwasperformed12hafterCUMSprocedure.Theapparatusconsistedofan81cm×81cmarenawithfour28-cmhighblackwalls.Theareawasdividedinto16squares.Theratwasplacedinthecenterofthefieldandwasobservedfor4min.Thenumberofcrossedsquares(horizontalactivity),frequencyofrearing(verticalactivity),andthenumberoftimesofgroomingwererecorded.ThistestwasconductedbeforeCUMS,onday7ofCUMS,andonday21ofCUMS,respectively.BeforeCUMSprocedure,alltheratsweregivenonebottleof1%sucroseandonebottleofwatertoadaptthetaste.Foodandwaterwerethenremoved.Afterwaterandfooddeprivationfor23h,ratsweregivenabottleof1%sucroseandtapwater.Thentheconsumptionamountof1%sucroseandtotalliquidwasmeasuredinthenexthour.Theamountofconsumedsucrosewasdividedbytotalliquidconsumption.ThetestwasperformedbeforeCUMS,onday7ofCUMS,andonday21ofCUMS,respectively.Alltheanimalsweredeeplyanesthetizedwithchloralhydrateanddecapitatedaftertheopenfieldtest.Thebrainwasrapidlyremoved.Thewholerightsideofbrainwasdissectedonice,followedby48-hfixationin4%paraformaldehydeat4°C.Theleftsideofbrainwasfrozeninliquidnitrogenandstoredat-80°C.Sectionswerecutbyacryostatatthethicknessof15μm,andwashedin0.01mol/Lphosphatebuffersaline(PBS;pH7.4).EndogenousperoxidaseactivitywasinhibitedbywashinginPBScontaining1%hydrogenperoxideand1%tritonX-100for30min.Sectionswerewashed3timesinPBSandblockedwith5%goatnormalseruminPBSfor30min.Thenthesectionswereincubatedwithpolyclonalanti-CRHantibody(1:1500,Bachem,T-4037)at37°Cfor1handat4°Cfor24h.Afterthat,thesectionswerewashedinPBS(3×10min)andincubatedwithbiotinylatedgoatanti-rabbitsecondaryantibody(1:200,VectorLaboratories)at37°Cfor1h.ThesectionswerewashedagaininPBS(3×10min),andthenincubatedwithavidinbiotincomplex(ABC)reagent(1:500,VectorLaboratories)for1h.AfterbeingwashedwithPBSfor3×10min,thesectionswereincubatedwiththesubstrate(0.05%3,3’-diaminobenzidineand0.01%H2O2inPBS)for5min.Thenthesectionsweredehydratedbygradientalcoholandxylene,andcoveredwithgelatin.ThesectionscontainingunilateralPVN(bregmabetween-1.4mmto-2.12mm)orCeA(bregmabetween-2.12mmto-2.8mm)wereselectedaccordingtotheRatAtlasofPaxinosandWatson(fourthedition).ThebeginningandtheendofPVNandCeAwereidentifiedbythioninstainingofevery10thsectionfromrostraltocaudal.The10consecutivecoronalsectionsofeachanimalatthesamelevelwereselectedforanalysis.ThesizeoftheareacontainingtheCRHcellswasassessedbyoutliningandmeasuringPVNorCeAregionofeachsection.DataofCRHneurondensitywereanalyzedbynonparametricMann-WhitneyU-test.Medianswereusedtorepresentthecelldensity.Dataoflocomotoractivityandsucrosepreferencewereanalyzedbytwo-wayanalysisofvariance(ANOVA).Thebodyweightgainwasanalyzedbyt-test.Testsweretwotailed.Resultswereexpressedasmean±SEM.P<0.05wasconsideredtobesignificantlydifferent.TheCUMSmodelratsdisplayedareductioninbodyweightgain.CUMSsignificantlyaffectedthebodyweightgain.Thebodyweightgainofthestressedratswas(23.8±3.83)g(n=6),whilethatofthecont