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BehavioralCoreUserManual

BroadwayResearchBuildingSuiteB15

MikhailV.Pletnikov,MD,PhDBehavioralCoreDirector

JenniferL.Yocum,MA

BehavioralCoreCoordinator

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TableofContents

GeneralInstructions: SOPforBRBBehavioralSuite………………………………………………… 3 CleaningtheBehavioralSuite……………………………………………….. 4 GeneralExperimentalDesign………………………………………………….6ProtocolsforBehavioralExperiments ActivityMonitor/OpenField………………………………………………… 7 ElevatedPlusMaze(withouttrackingsystem)……………………….8 ElevatedPlusMaze(withtrackingsystem)……………………………. 9 Pre‐pulseInhibition………………………………………………………………10

Y‐Maze………………………………………………………………………………… 11SocialInteraction………………………………………………………………….15Intruder/Resident………………………………………………………………..17Sociability&PreferenceforSocialNovelty…………………………..18Habituation/Dishabituation………………………………………………….20Olfaction…………………………………………………………………………….. 21NovelObjectRecognition……………………………………………………..23MorrisWaterMaze…………………………………………………………….. 24UltrasonicVocalizationDetector…………………………………………..28ForcedSwimTest…………………………………………………………………30FearConditioning………………………………………………………………..31TailSuspensionTest……………………………………………………………. 33Rotarod……………………………………………………………………………….34T‐Maze………………………………………………………………………………..35Passive‐andActive‐Avoidance…………………………………………….38

ContactInformation Animalresources……………………………………………………………….. 40 BRBBehavioralsuite………………………………………………………….. 40

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StandardOperatingProcedures(SOP)fortheBRBBehavioralSuiteBeforebeginningyourexperiment

1. BesuretocontactanimalresourcestoreceivepropertrainingfortheBRBfacility.2. WhenactivatingyourcardyouwillneedpermissiontoaccesstheBehavioralsuite,whichissuiteB15,

andthehighriskroom,B16.YouwillhavetofillouttheBRBAnimalFacilityformthatcanbefoundunderthe“Forms”sectionontheirweb‐page(www.jhu.edu/animalcare)andgetitsignedbyDr.M.Pletnikov,BehavioralCoreDirector.

3. Besurethatthereisspaceforyouranimalsinthehighriskroombecauseallmicemustbehousedinthehigh‐riskroomafterbeinginthebehavioralsuite.

4. ContactDr.M.Pletnikov(2‐3760)forpermissiontousethesuite,andJenYocum(2‐4259)fortraining,tobooktherooms,andifanytechnicalproblemsarise.

DuringtheExperiment

1. ThebehavioralsuiteisusedbymanyinvestigatorsthroughoutJohnsHopkins;therefore,treattheequipmentandthefacilitywithrespect.

2. Beneatandconscientiouswhileusingthefacility.3. Cleanupeverydayevenifyourexperimentwilltakemorethanoneday(seethesectiononcleaning).4. Maintainaworkenvironmentthatisassterileaspossible.

a. WhencollectingyourmicespraythecagesandthetransportationcartwithVimoba.b. AfterbringingyourmiceintoSuiteB15,leavethecartinthehallwayandcarrythecagesinside.c. Placeabluepad/clothunderyourcages.d. SprayyourhandswithVimobaeachtimeyouhandleanewmouse.e. DonotputANYTHINGonthefloor.f. WhenyoureturnyourmicespraythecagesandcarrythemtothecartintheB15hallway.g. Inthehigh‐riskroom,spraythewaterspoutontheracks(whereyouwillreturnyoumice)with

Vimoba.5. Foranyexperimentlastingmorethananhouryouwillneedtoprovideventilatedcagetopsandwater

bottlesforyourmice.CagelidsCANNOTbeproppedopen,andanimalsshouldnotbeexposedtoopenairinthehallways.Youcannotrunanimalexperimentsinthehallwayandyoushouldnotbeinjectinganimalsinthehallwayseither.

6. Cleanwaterbottlescanbefoundintheprocedureroomandshouldnotbeusedmorethanonce.7. Usedwaterbottlesshouldbeplacedoutsideofthesuitenexttothedirtycagecarts,onawaterbottle

rack.8. Itisgoodpracticetosprayandcleantheequipmentwith70%ethanolandVimobabetweenmiceas

thiseliminatesanysmellsorstresshormonesthatthepreviousmousemayhaveleftanddecontaminatesproperly,accordingtoBRBregulations.

9. Keeptheroomfreeofdistractions.Foropenfieldexperiments,leavetheroomwhileyourmicearerunning,ifpossible.

10. Thereshouldbenonoise,andthe"ExperimentinProgress"signshouldbeonthedoortoeliminateanyinterruptions.

11. Itisalsoadvisabletonotwearanystrongsmellingperfumesorhandlotions.

Mostly,usecommonsense.Whenworkingintheroomsyouwantanenvironmentascleanaspossiblesothatyourmicedon'tgetsick,andsothattheexperimentcanrunsmoothly.Eliminateanyconfoundingvariablesbykeepingthetestingsituationasstableaspossibleandrandomizingyourexperimentalandcontrolgroups.

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Foranyquestions,commentsorsuggestionspleasecontactDr.M.Pletnikov(2‐3760)orJenYocum(2‐4259).

CleaningtheBehavioralSuiteEventhougheachexperimentlistshowthesuiteshouldbecleaned,aseparatesectioniswarrantedtofurtherexplainwhatisexpectedofeveryoneusingtheroom.TheBRBanimalresourcesstaffdonotcleanthisroomatallsoitistheresponsibilityofeachindividualresearchertomaintainacleanresearchenvironment.Mostdaysthereismorethanoneresearcherusingtheroom.Withthisinmind:

• Berespectfulofothers.• Evenifyourexperimentwillcontinueoverthenextfewdays,leavetheroomintheconditionyou

wouldliketofinditifyouwereabouttostartanewexperiment.• Turnoffallequipment,exceptcomputerssothattheycanrunanti‐virussoftware,andlights.

Shutthedoor.GeneralCleaning:

1. Replaceanysupplyitemyousignificantlydepleted,e.g.papertowels.2. Wipeallsurfacesclean.3. Discardusedgloves,foodpellets,bedding,waterbottlesandblueworkpads/clothsinthetrash.4. Disposeofallsharpsinthesharpscontainer.Ifthesharpscontainerisnearthefillline,notifythe

personinchargeofbooking.5. Sweepthefloorintoadustpananddiscardthecontentsinthetrash.6. Thebroomandpancanbefoundbytheentranceofthesuite,aswellasamopforbiggermesses.7. Youshouldmopthefloorifyouhaveleftdirtthatcannotberemovedwithabroom.Youshouldalso

mopupanywaterspills‐NEVERleavewaterspillsinthesuite.8. Thehallwayoutsidethesuiteisalsoourresponsibility.Ifyoufinditmessy,makeanoteandmentionit

tothepersoninchargeofbookingtherooms.Whenyoutransportanimals,cleanupanymessyoutrackintothehallway.

9. Emptythetrash.Thebagmustbeclosedandleftinfrontoftheeuthanasiastationoutsidethesuite.Obtainanewbiohazardbagandputitinthetrashcan.

Equipment:

1. Itisgoodpracticetocleantheequipmentbetweeneachmousetoeliminateanysmellsorstresshormones.

2. Besuretothoroughlycleanitafteruse.3. Alwaysturnoffallequipment,exceptcomputers.

TurnoffthelightandreturnyouranimalstotheHighRiskRoom.Mostcleaningcanbekepttoaminimumifyoucarryoutyourexperimentsinacarefulandconscientiousmanner.Thankyouforyourhelpinthisregard.

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ProtocolsforBehavioralExperiments

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GeneralExperimentalDesignWhendesigningexperiments,youmustdoyourbesttocontrolforallpossiblycovariatesandconfoundingvariables.Thisisaccomplishedbykeepingallconditionsthesameforallmice,asbesttoyourability,andintroducingrandomizationintheorderofwhenmicearerun.

• Ifyouaretestingbothmaleandfemalemice,oronegroupversusanother(drugvs.vehicle,knockoutvs.wt,etc),youwanttomakesurethatyouhaverepresentativesfromallgroupsbeingrunatthesametime,iftherearemultipletestchambers.Forexample,ifyouarerunninganimalsintheopenfieldin4chambers,andyouhavemalesandfemales,thenrun2malesand2femalesduringonesession,andcontinueinthatway,sothatthereislessofaneffectoftimeorparticulareventsonthedata.

• Asbestyoucan,youshouldrunallexperimentsatthesametimeoneachgivenday,orminimallywithinthesametimeframe.Circadianrhythmcanbealargefactorinyourresults,especiallyifyoutestsomeanimalsduringthelightcycle,andothersduringthedarkcycle.Eventhedifferencebetween7amand4or5pmcanbestriking.

• Youshouldrunallofaparticulartypeofexperimentinthesameroom,especiallyiftheyhaveaspatiallearningcomponent.Forexample,ifyouarerunningtheY‐maze,itisbesttorunallsessionsinthesameroom,evenifthemicearedifferent,becauseoneroommayhavecuesthatareeasierformicetorememberthananother.IfyouareinasmallroomsuchasB15B,youmaybeclosertothemiceanditmayhaveadifferentfeel,smells,andmanyotherthingsthatmayaffecttheirbehaviorascomparedtoiftheywereruninB15G.Keepingthisthesamebetweenallyourmicewillhelpensurethatthiswillnotfactorintoyourresults.

• Aspossible,ifthesamepersoncanrunallexperiments,itisideal.Micegetusedtothesmellsandtouchofparticularpeople,andthatpersonislikelytohandlemostmiceinthesameway.Themorepeoplethatarerunningtheanimals,themorechancethereisforinconsistencies,andthusthingsthatmayaffectyourdata.

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ActivityMonitor/OpenFieldThisexperimentassessesgeneralmotoractivity.Itwilltellyouhowmuchyourmicemove,stereotypicalmovementandexploratoryactivity(forexample,rears)andyouwillbeabletoassesshowmuchtimespentinthecenterversustheperipherytoevaluateanxiety.Makesuretorandomizeyourmice.Wehavefouropenfieldchamberssobesuretomixupmalesandfemales,andcontrolversusexperimentalgroupsasmuchaspossible.

1. CollectyourmiceandbringthemtotheOpenFieldroom.Cleanoutactivitychamberswith70%ethanolandVimoba.

2. Turnonthecomputer(thepowerstrip,thenthecomputerbutton,computerscreen,andchambers).3. Logintowindowsasadministrator(nopassword)byhitting“Enter”4. DoubleclickthePASshortcuttostarttheprogram(PhotobeamActivitySystem)

User=pasadminPassword=pletnikov

5. Goto“File”“CreateNewSessionDB”“ChangeNameorDirectory”Createfolder&nameforeasyaccesstoyourdataHit“Create”

6. Goto“Session”Tab”CreateNewSession”,selectnamefordata”Create”7. Goto“Session”Tab“Editthissession””Chambers”

ChangethenamesofthesubjectstocorrespondwithyourmiceHit“Start/StopControl”Changetheintervallengthtohowlongyouwouldliketheintervaltobebetweentotalsofactivityevents.ChangetheIntervalperPhase(howmanyintervalsyouwouldlikepersession).Crosscheckwithtotaltime,then“Save&Close”

8. Gotothe“Equipment”TabTestthechambers(NB‐testbetweeneachpairofmice)CheckthatblocksarewhiteBlockswillbeyellowifthereisaproblemoriftherearemovingobjectsdetectedThenhit“x”toclose

9. Hit“Session”tab”Runsession”PlaceyourmiceinchambersHit“Startall”

10. Yoursessionwillendautomaticallyatthetimeyoudesignated.Removeyourmiceandwipedowntheinsideofthechamberswith70%ethanolandVimoba.

11. Repeatsteps6‐10foreachsetofmiceyouwouldliketorun.12. ToexitPAS,hit“File”CloseQuit13. WhenyouarefinishedyourexperimentopenyourfileinMicrosoftAccess.

DoubleclicktheAccessicononthedesktop. Select“OpenExistingFile”FindyourdataClick'Open'SelectIntervaldataSelect“File”ExportChangethedocumenttypetoExcelSaveinExcel.Filescanbesavedonfloppy,CDorjumpdrive.

14. Cleanupthebehavioralsuite,includingchambers,surfaces,floor,andtrash.15. Turnoffthepowerstripandinterfaceboardthatcontrolsthechambersbeforeleaving.

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ElevatedPlusMaze(withouttrackingsystem)

Thistestmeasuresanxietylevels.Itworksbyevaluatingnaturalconflictinmicebetweenexplorationofnewenvironmentandfearofopenandwelllitspace(i.e.,openarm).Exploratorybehaviorbetweenexperimentalandcontrolanimalswillbecompared;lessexplorationoftenmeansmoreanxiety.

1. Collectyourmicefromtheirrespectiveroomsandbringthemtothebehavioralsuite(toallowforacclimation).

2. SetuptheMazewiththevideocamerapositionedinawaythatallowsyoutoseeasmuchofthemazeaspossible.

a. Aimthecamerafromahighangledirectlyattheclosedarmsb. Adjusttoallowaclearviewofbothendsoftheclosedarms

3. Providewaterbottlesandventilation4. Useanotecardtokeeptrackofyouranimals.Writeeachanimal'snumberdownonacardbefore

theirturninthemaze.Holdituptothevideocameraandrecordforafewseconds‐enoughtimetoallowyoutoreadthenumberwhenreviewingthedata.

5. Placeyourfirstmouseonthestartingsquare.Thisisthesquaredirectlyinthemiddleoftheplusmazethatisneitherintheopenarmsnortheclosedarms.

6. Hitrecordassoonaspossible.7. Leavemouseinmazefor5min.8. Hittherecordbuttonagain,whichwillputthecameraonstandby.9. Removethemouseandreturntoittoitscage.10. Cleanthemazetoremoveanyodororstresshormonesthatmayhavebeenreleased.11. Repeatsteps4‐10untilyouhaverunallofyoursubjects.12. ReturnthemicetothehighriskreturnroominBRBandreturntothebehavioralsuitetocleanup.13. Cleanupthebehavioralsuite,includingthemaze,surfaces,floor,andtrash.14. Turnoffthecomputer,monitor,powerstripandlightsbeforeleaving.

DataAnalysisYouwillanalyzethedatabywatchingthevideorecording.Youwilltracktheamountoftimespentintheclosedarmsversustheopenarms.Youwillalsocountheaddips,rearsandstretchmovementstoevaluateriskassessmentbehaviors.Youmayalsochoosetocountnumberofbolileftbyeachmouse,whichisconsideredanautonomicmeasureofstress/anxiety.Thiscountmaybebesttodoinperson,aftereachmousehascompleteditstrial.Analysisofmovementsmayrequiremultipleviewingssobesuretouseacomputerprogramthatallowsforeasyreviewandfreezeframing.

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ElevatedPlusMaze(withtrackingsystem)

1. Collectyourmicefromtheirrespectiveroomsandbringthemtothebehavioralsuite(toallowforacclimation).

Howtooperatethetrackingsystem

2. Makesurethecameraispluggedin,theyellowvideowireisconnectedtothetrackingsystemcable,andthecomputeristurnedon.

3. Createafilefolderforyourdataonyourflashdrive,oronthecomputertobedirectlyburnedtoCDandremovedaftertheexperiment.

4. OpentheprogramSMART.5. Configuration:Paths:chooseyourfolder.ThenselectConfiguration:Timings:Changeto5

min.6. Under"DataAcquisition"selectsinglesubjecttrackingFileopenzoneElevatedplus

maze.zne7. Aligntheplusmazeaccordingtotheimageonthescreen.8. Selectdarkorlightsubject,dependingonyourmousecolor.HitStart.Thetrackingsystemwillwait

tostartrecordinguntilthefirstsignofmovement.Ifthesystemhasahardtimetrackingyouranimal,youmayneedtoadjustthethreshold(default=80)andcontrast(default=3).

Youarenowreadytorunyourexperiment

9. Open"Eventtracker".Aboxofeventswillappear.Numbers1and3havebeenassignedtorearsanddips,respectively.Whileyourmouseisinthemazeyouwillneedtowatchthescreenandhit1eachtimethemouserearsand3eachtimeitdipsovertheedgeoftheopenarm.

10. Placeyourfirstmouseonthestartingsquare.Thisisthesquaredirectlyinthemiddleoftheplusmazethatisneitherintheopenarmsnortheclosedarms.HitStart.Recordrearsanddips.

11. Theprogramstopsautomaticallyrecordingafter5min.12. Fillinthedetailsofyoursubjectandexperiment.13. HitFileSave(foreachmouse).14. Closethewindow.15. SelectDataacquisitionSinglesubjecttracking.Alsobesurethattheeventtrackerisopen.16. Putthenextmouseonthestartsquare.17. Repeatsteps11through18foreachsubject.18. Closethewindow.Youarenowreadytoanalyzeyourdata.

ElevatedplusmazeAnalysis

19. SelectFileOpenZoneelevatedplusmaze20. FileOpensinglesubjecttrack.21. SelectthefilesyouneedfromyourfolderHit√HitGo.22. Checkthat"Subjectcode","zonename","permanencetime",and"events"areselectedunderthe

"Standard"tabtobeincludedinthereport.23. Alsolookunder"ZoneTransitions".Thesamefouritemsshouldbeselected.24. Theboxthatsays"Include"shouldbeselectedunderbothscreens.HitOK.Saveresultsandanalyze.

Forstatisticalanalysis,youwillcomparethetimespentintheclosedarmscomparedtothetimespentintheopenarms.Youwillalsocomparethenumberofrearsanddips.

25. Cleanupthebehavioralsuite,includingthemaze,surfaces,floor,andtrash.26. Turnoffthecomputer,monitor,powerstrip,cameraandlightsbeforeleaving.

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Pre‐pulseInhibitionofacousticstartleresponseThistestallowsyoutoevaluateseveraldomainsofmousebehavior.Evaluationofthemagnitudeofstartleresponsecanbeusedasameasureofoverallreactivitytoaversivestimuliandanxiety.Repeatedpresentationofstrongauditorystimulususuallyproducesadeclineinstartleresponses,suggestinghabituationshownbyreductionofthestartleresponse.Habituationassessedwithinasinglesessioncanindicateshort‐termmemory,whilehabituationassessedbetweensessions(overhoursordays)canindicatelong‐termmemory.Pre‐pulseinhibition(PPI)ofthestartleresponsetestssensorimotorintegration.Thisisastressfultestandshouldberunaftertestsforanxiety,learningandmemoryorsocialinteraction.Themicewillbeinchambersonaplatformthatautomaticallyrecordstheirstartleresponses.Theprograminthecorehasbeendesignedtoevaluatestartleamplitude,short‐termhabituationandPPIusingaseriesoftonesofvaryingvolumes,frequenciesandintervals.Miceshouldbekeptinthehallwayonacartduringthistestifpossible,inordertominimizestressandnotexposeyourotheranimalstostrongauditorystimuli.

1. Turnonthecomputer.2. SelectSRlabiconondesktop.3. PoweronPPIboxesusingthelabeledswitchesonthepowerstrip.4. Select“Configuration”andselectthepaththatwillgotoyourpersonaldatafolder.Thisfoldershould

containthesessionandtrialfilesthatyouneedtorunyourmice.Click“OK”afteryouarefinished.5. Select"RunSession",thenselectthefile"hab_ppi_brb"(oryourownSASfileifyouchoosetousea

differentone).ThisfilehasbeendevelopedwithappropriatesettingstorunmicewithourcalibratedPPIequipment.

6. Enteryourmiceinformationintoboxes1and2(orjust1ifyouareonlyrunningonemouse),andhit“OK.”

7. Youmaywanttoweighyourmiceifyouareawareofsignificantdifferencesinbodyweightbetweenyouranimals.Bodyweightdifferenceswillinfluenceyourdatabutcanbefactoredintoanalysis.

8. Collectthefirsttwosubjects,putoneineachbox.Holdthemicebytheirtailandgetthemtocrawlintothetube. Makesure"partitions"areplacedasclosetogetheraspossible.

9. Thecomputerwillaskyouifthesubjectsareready.Hitthe'OK'button.ThePPIsystemwillrunandrecorddata.

10. Oncecomplete,removemiceandreturnthemtotheircage.Cleantheboxes.11. HitRuntostartanewpair.12. Repeatsteps9‐11foreachpairandremembertoenternewsubjectnames.13. AfterthecompletionofyourexperimentselectReportsConcatenateDatafromthedropdown

menu.14. HitOpen.15. SelectoneofyourfilesclickOpen(thedatawillappearintheboxonthetopright).16. HitAdd(thedatawillnowmovetothebigboxonthebottomofthescreen).17. Repeatsteps15‐17foreachofyourfilescontainingdata.18. Onceyouhaveconsolidatedallofyourdatafromthisexperiment:

HitExcelandsavethedataasanExcelfile.19. Cleanupthebehavioralsuite,includingchambers,surfaces,floor,andtrash.20. TurnoffthePPIchambersbeforeleaving.Returnyourmicetothehighriskroom(B16).

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Y‐Maze

TheYmazemeasuresspatialworkingandrecognitionmemorybymakinguseofarodent'snaturalexploratoryinstincts.TheY‐mazeconsistsofthreearmsofequallengthinterconnectedat1200.Thetestincludestwosessions.ThefirstsessionmeasuresworkingmemoryinmicebyscoringthenumberofalternationsthemousedoesinY‐mazewhentheanimalvisitsallthreearmswithoutgoingintothesamearmtwiceinarow.Thesecondsessionmeasuresspatialrecognitionmemory.Duringthetestingphase,onearmwillbeblockedwhilethemouseisallowedtoexploretheothertwo.Aftera10‐15minbreakthepartitionisremovedandthemouseisallowedtoexplore.Ifworkingmemoryisintact,themouseshouldrememberthefirsttwoarmsandbemoreinquisitiveaboutthethird(originallyblockedarm).Inthiswayyoucancompareexploratorybehaviorofcontrolversusexperimentalanimalsinordertodetermineofworkingmemoryisimpaired.

Ourpreliminarystudiesinmiceindicatedthatthefirst2minoftestingarethemostsensitivetomeasurespatialpreferenceforanewarm,sothetimeandnumberofvisitstothenewarmwillbecalculatedforthe1st2minoftesting.Motoractivitywillberecordedasnumberofarmsvisitedforawholedurationofthetest.

1. Beforethetest‐behavioraltestingshouldbestartedaftereverymousehasbeenextensivelyhandled.Ifyourmicehavenotbeenhandled,handleeachmouse5minsforatleast3daystodecreasestresslevels.

RunningtheY‐mazeusingSMARTtrackingprogram(automated)

Howtooperatethetrackingsystem:

2. Makesurethecameraispluggedin,theyellowvideowireisconnectedtothetrackingsystemcable,andthecomputeristurnedon.



min.6. Under"DataAcquisition"selectsinglesubjecttrackingFileopenzoneYmazezones2.zne7. AligntheYmazeaccordingtotheimageonthescreen.8. Selectdarkorlightsubject,dependingonyourmousecolor.HitStart.Thetrackingsystemwillwait

tostartrecordinguntilthefirstsignofmovement.Ifthesystemhasahardtimetrackingyouranimal,youmayneedtoadjustthethreshold(default=80)andcontrast(default=3).

Runningtheexperiment:Theexperimentconsistsof3trials.Thesecondandthirdtrialsareconducted5‐7daysafterthefirsttrial.Trial1(spatialworkingmemory):allthreearmsofthemazeareopen.Specifically,micewillbeplacedontotheendofonearmandallowedtoexplorefreelyfor5min.Thestartpositionforeachtrialwillbechangedtothedifferentarm.Thesequenceofthearmentriesisrecorded.Duringthefirstsession,allthreearmsoftheYmazewillbeopen.Thespontaneousalternationbehavioriscalculatedasthenumberoftriads(threedifferentarms,ABCnotABB)thatcontainentriesintoallthreearms

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dividedbythetotalvisits.Thefirstsessionrepresentsaclassicspontaneousalternationtestofspatialworkingmemory.

9. Collectyourmiceandbringthemtothebehavioralsuite.Setupthetrackingprogram.10. Putthemouseattheendofanarm.Thestartarmshouldbealternatedinasemi‐randomfashion.

HitStart.11. Theprogramstopsautomaticallyrecordingafter5min.12. Fillinthedetailsofyoursubjectandexperiment.13. HitFileSave(foreachmouse).14. Closethewindow.15. SelectDataacquisitionSinglesubjecttracking16. Putthenextmouseattheendofanarm.Thestartarmshouldbealternatedinasemi‐randomfashion17. Repeatsteps12through18foreachsubject.18. Closethewindow.Youarenowreadytoanalyzethepreliminarydata.

Analysis

19. SelectDataAnalysisSelectFileopenzoneYMAZEZONES2.zne20. Fileopensinglesubjecttrack.21. SelectthefilesyouneedfromyourfolderHit√HitGo.22. Checkthat"Subjectcode","zonename"and"permanencetime"areselectedtobeincludedinthe

report.23. Alsolookunder"ZoneTransitions".Thesame3shouldbeselected.24. Theboxthatsays"Include"shouldbeselectedunderbothscreens.HitOK.Saveresultsonyour

flashdriveandanalyze.25. Thespontaneousalternationisanalyzed/calculatedasthenumberoftriadscontainingentriesintoall3

armsdividedbythemaximumpossiblealternations(entriesminus2)andexpressedinpercentages.PartIIoftheexperiment‐Trials2and3:Thesecondsessioniscarriedout5–7daysafterthefirstsession.Thesecondsessionincludestwotrials.Duringtrial1,oneofthearmsofthemazewillbeblocked,allowingfora5‐minexplorationofonlytwoarmsofthemaze.Aftera30mindelay,trial2isstarted.Duringtrial2,allthreearmsareavailableforanother5‐minexploration.Trial2takesadvantageoftheinnatetendencyofmicetoexplorenovelunexploredareas(e.g.,thepreviouslyblockedarm).Micewithintactrecognitionmemorywillprefertoexploreanovelarmoverthefamiliararms,whereasmicewithimpairedspatialmemorywillenterallarmsrandomly.Thus,trial2representsaclassictestforspatialrecognitionmemory.Thespatialcuesaroundthemazeshouldbechanged(objectsonthewallsaroundthemaze,tapecolorinthemaze,woodchipsorbeddingonthefloorofthemaze).It’smostconvenienttoorganizethemiceingroupsofthreeforthisphaseoftheexperiment.Trial2Onearmmustbeblockedallowingexplorationofonly2arms,alsoalternatedinasemirandomfashion(whichyoushouldkeeptrackofonapieceofpaper).Run3miceatatime,thenmoveontotrial3.

26. Collectyourmiceandbringthemtothebehavioralsuite.Changethespatialcues.Setupthetrackingprogram.

27. Putthefirstmouse(fromthegroupofthree)attheendofoneofthetwounblockedarms.Thestartarmshouldbealternatedinasemi‐randomfashion.

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HitStart.28. Theprogramstopsautomaticallyrecordingafter5min.29. Fillinthedetailsofyoursubjectandexperiment.30. HitFileSave(foreachmouse).31. Closethewindow.32. SelectDataacquisitionSinglesubjecttracking33. Putthesecondmouseattheendofadifferentarm.Thestartarmshouldbealternatedinasemi‐

randomly.34. Repeatsteps12through18formousetwoandthreeofyourtriad.

Trial3(spatialrecognitionmemory)Opentheblockedarmandrunthetriadthatjustfinishedtrial2.Thisshouldgiveeachmouse10‐15minbetweentrial2and3).Amousewithanormalmemoryshouldspendmoretimeexploringthe“new”unblockedarm.

35. Putthefirstmouse(fromthegroupofthree)attheendofoneofthetwoarmsthatwasoriginallyunblocked.Thestartarmshouldbealternatedinasemi‐randomfashion.HitStart.

36. Theprogramstopsautomaticallyrecordingafter5min.37. Fillinthedetailsofyoursubjectandexperiment.38. HitFileSave(foreachmouse).39. Closethewindow.40. SelectDataacquisitionSinglesubjecttracking41. Putthesecondmouseattheendofadifferentarm.Thestartarmshouldbealternatedsemi‐

randomly.42. Repeatsteps12through18forthesecondandthirdmiceofyourtriad.43. Repeatsteps26‐42foreachofyourgroupsof3.44. Closethewindow.Youarenowreadytoanalyzeyourtrial2and3data.

Analysis

45. SelectDataAnalysisFileopenzoneYmazezones2.46. Fileopensinglesubjecttrack.47. SelectthefilesyouneedfromyourfolderHit√HitGo.48. Checkthat"Subjectcode","zonename"and"permanencetime"areselectedunderthe"Standard"tab

tobeincludedinthereport.Alsolookunder"ZoneTransitions".Thesame3shouldbeselected.Theboxthatsays"Include"shouldbeselectedunderbothscreens.HitOK.

49. Saveyourresultsonyourflashdriveandanalyze.50. Calculatethespontaneousalternations(asyoudidintrial1).Calculatethealternationsforthefirst2

min.Calculatetheoverallpercentage,andthepercentageforthefirsttwominutes.Findavalueforthetimespentineacharmoverall,andthetimespentineacharmforthefirsttwominutes.Alsocalculatethepercentageforthetimespentineacharmoverall,andthepercentageforthetimespentineacharmforthefirsttwominutes.

RunningY‐mazeusingStopwatchProgram(Manualmode)

51. Youwillfollowthesameprotocolforrunningtheactualanimals,andyoumayormaynotchooseto

videotapetheanimalsforlateranalysis.Double‐clicktheStopwatchprogram.

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52. YoumayeitherselectapreviouslyexistingprogramforY‐maze,orestablishyourown.Ifenteringyourown,enter3forthenumberofevents.

53. Definetheeventsas“armA,”“armB,”and“armC.”Youmayputtheseinwhateverorderismostcomfortableforyoutorememberwhenpushingthebuttons.

54. Makesurethefollowingboxesareclicked:trackduration,holdkey(thisallowsyoutoholddownthecorrespondingkeyforaslongastheanimalisinthearm),andgetlatency.

55. Setthefixedsessionlengthfor5min.Youcanchoosewhetherornotyouwanttoselectforittobeepeverysooften,butmostcomputersaresetsothattheydonotmakenoise,sothisisusuallynotnecessary.

56. Youmaythenchoosetosavethisdefinitionfileforfutureuse.Onceyouaredonetestingeachanimal,hit“save”andnameyourfile.Itisbestifyousavedirectlytoyourflashdrive,butyoumaytemporarilysavetothedesktop/harddriveandthentakethefilesoffthecomputerbeforeyouleave.

57. Analysisisdoneasearlierdescribed,andyoumayalsochoosetoanalyzeifthereisapreferenceforaparticulararmornot.

Hereisadiagram/keyforthelabelingoftheYmaze.Youwillneedthiswhenlookingatthedata.

corner

1

2

3

1

2

3

1 2

3

C

A

B

10

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SocialInteractionThistestexaminesexploratorybehaviordirectedatanew/unknown“stranger”mouse.Youwillbeabletoanalyzevarioussocialinteractions,includingaggression,sniffing,grooming,etc.Beforethetestyoushouldorganizeyourmice,makingalistofthepairsyouwillrun.Themicewillneedtobeisolatedbetween5daysto7daysbeforethetest.ItisbesttouseWTornon‐littermatemiceasthe“stranger”mouse,andnotusethestrangermousemorethanacoupletimes,orelseitsbehaviormaybeaffectedandinfluencetheexperiment.Besuretohavemarkersandnotecardswithyoutorunthisexperiment.Youshouldalsorandomizeyourmiceasthisexperimentwillberuninoneopenfieldchambersobesuretomixupyourcontrolversusexperimentalgroupsasmuchaspossibletoeliminateconfoundingvariables.

1. Collectyourmiceandbringthemtotheroom.Cleanoutthechamberwith70%ethanolandVimoba.2. Setupthevideocamerabymountingitonthetripodsothatyoucanseetheentirefieldfromabove.3. Insertthecardboarddividerdiagonallyintothechamber.

4. Besureyourmicehavedistinguishingcharacteristics,ormarktheirtails.5. Placeeachmouseintheirownhalfofthechamberandallowthemtohabituatefor5min.6. Writetheirnumbers,groupsandotherdataonanotecard.7. Hitrecordonthevideocameraandshowthecardforabout2‐5seconds.8. Removethepartitionandallowthemicetointeractfor10minutes.9. Removethemiceandreturnthemtotheirrespectivecages.10. Cleanthechamberswith70%ethanolandVimoba.11. Selectthemiceforthenextgroupandrepeatsteps5‐10foreachpairofmiceyouwouldliketorun.12. Cleanupthebehavioralsuite,includingchambers,surfaces,floor,andtrash.13. Turnoffthelights.14. ReturnyourmicetotheHighRiskRoom.

AnalyzingtheData:

15. Youwillanalyzesocialactivitiesversusnon‐social/aggressiveactivities.BasicDatatoanalyze:

SocialActivities:(Doneforeachmouse)Timespentsocial(sec) Timespentinvestigating,grooming,following,sniffingetc.

Sniffing(sec) Timespentsniffingpartnerwithinaninchandahalf Following(sec) Timespentfollowingpartnerwithin5inches

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AggressiveActivities:(Doneforeachmouse) Attacks(#) Numberoftimesthemouseattacksitspartner Bites(#) Numberoftimesthemousebitesitspartner

TailRattles(sec/#) Time/numberoftailrattles

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Intruder/Resident

Beforethetestyoushouldorganizeyourmice,makingalistofthepairsyouwillrun,whowillbetheintruderandwhowillbetheresident.Themicewillneedtobeisolatedfor5daysto7daysbeforethetest.Alsomakesuretohavemarkersandnotecardswithyoutorunthisexperiment.Youshouldrandomizeyourmicetobesuretomixupyourcontrolversusexperimentalgroupsasmuchaspossibletoeliminateconfoundingvariables.

1. Collectyourmiceandbringthemtotheroom.2. Setupthevideocamera,mountingitonatripodsothatyoucanseetheentirefieldfromaboveor

fromtheside.3. Positionthe"resident's"cageonacartorsurfaceinfrontofthecamera.4. Besureyourmicehavedistinguishingcharacteristics,oryoucouldmarktheirtails.5. Writetheirnumbers,groupsandotherdataonanotecard.6. Hitrecordonthevideocameraandshowthecardfor2‐5seconds.7. Placethenewmouseinthe"resident's"cage.8. Allowthemicetointeractfor10minutes.9. Hitrecordagaintostopfilming.10. Removetheintruderandreturnittoitscage.11. Cleanthechambers.12. Setupthenewintruderandselectthenewresidentmouseforthenextgroup.13. Repeatsteps4‐13foreachpairofmiceyouwouldliketorun.14. Cleanupthebehavioralsuite,includingchambers,surfaces,floor,andtrash.15. Turnoffthelightsandpackawaythecamerabeforeleaving.16. ReturnyourmicetotheHigh‐Riskroom.

AnalyzingtheData:

17. DownloadthefootagetothecomputersothatyoucanputitonyourflashdriveoraCD/DVD.18. Toscorethedata,youwillanalyzesocialactivitiesversusnon‐social/aggressiveactivitiesbyviewing

thefootage.Youwillalsolookatsubmissiveactivities.Thiswillprobablyrequiremultipleviewings,andacomputersystemwithslowmotion/freezeframecapabilities.

BasicDatatoanalyze: SocialActivities:(Doneforeachmouse)

Timespentsocial(sec)Timespentinvestigating,grooming,following,sniffingetc. Sniffing(sec) Timespentsniffingpartnerwithinaninchandahalf Following(sec) Timespentfollowingpartnerwithin1cm

AggressiveActivities:(Doneforeachmouse) Attacks(#) Numberoftimesthemouseattacksitspartner Bites(#) Numberoftimesthemousebitesitspartner TailRattles(sec/#) Time/numberoftailrattles SubmissiveActivities:(Donejustforintruder)

Freezing(sec) Timespentfreezing

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Sociability&PreferenceforSocialNovelty

Beforethetestyoushouldorganizeyourmice,makingalistofthepairsyouwillrun.Youwillneedafew(4‐6)non‐experimentalmiceofthesamegender.Youshouldrandomizeyourmice(controlversusexperimentalgroups)asmuchaspossibletoeliminateconfoundingvariables.Thistestexaminestheamountoftimespentexploring/interactingwithanewmouse.Themouseispresentedwithanunknownor“stranger”mouse,andtimespentinteractingwithitisrecorded.Asecondtrialisadministeredwiththesamestrangermouse,whichisnowaknownmouse,andasecondmouseisplacedinadifferentpartofthechamberandthatmouseisnowthe“stranger”mouse.Inanormalanimal,themousewillspendmoretimeinteractingwiththenewstrangermouse.AcclimationPhase:For3consecutivedays,placeeachmouseyouwanttotest(notincludingstrangermice)intothe3chamberedapparatus,openallthegates,andallowthemousetoexplorefor10mineachday.Alternatively,micecanberunintheopenfieldsetup,asdescribedabove.TestingPhase:Settingupthesoftware:

1. Double‐clickontheSocialInteractionicon.Adjustthetrialtimeto10min.EnterintheanimalIDandanyotherinformationthatisimportanttoyou.Push“start”andthiswilltakeyoutoapop‐upwindowwhereyoucanenterinthefilenameyouwanttosaveandanynotesorotherinfo.Donotpressthe“start”inthispop‐upwindowuntilyouarereadytostart(step10).Thissoftwarewillonlytrackhowmanytimestheanimalcrossesthrougheachgateandthetotaltimespentineachchamber.

2. Double‐clickontheStopwatchicon.Youwillusethisprogramtomanuallytrackhowmuchtimethemousespendsinteractingwitheachoftheothermice.

3. Fornumberofevents,enter2.Defineeachoftheeventsas“rightchamber”and“leftchamber”or“mouse”vs.“empty,”or“newmouse”vs.“oldmouse”orhoweveriseasiestforyoutoremember.

4. Inthe“Code”box,enter1forthefirstboxand2forthesecondbox,orwhateverkeysyouwanttopushinordertocounteachoftheevents.

5. Makesurethefollowingboxesareclicked:trackduration,holdkey(thiswillallowyoutoholdthekeyaslongastheinteractionistakingplace,andreleasewhenitisdone),andgetlatency.

6. Enterafixedsessionlengthof10min.Thesoundisturnedoffsoyoudonothavetoclicktheboxthatdealswithbeeping.

7. Youcansavethisfileforuseinlatertrials,orcreateanewoneeachtime.Trial1:

8. Closebothofthegates.Placethe“stranger”mouseunderoneofthewirebasketsandmakesurethebasketisinthemiddleback(furthestawayfromyou)oftheleftorrightchamber.Intheotherchamber,youwillplacetheotherbasketinthemiddlebackbutwithnoanimalinit.

9. Placethetestmouseinthecenterchamberandallowittoexplorefor5min.10. StartbothSocialInteraction11andStopwatchandimmediatelyliftbothgates.11. Everytimethemousespendstimeinvestigatingthenewmouseortheemptybasket,holddownthe

correspondingbuttonstorecordtheeventintheStopwatchprogram.Releasethebuttonwhentheeventisover.Anexploratoryeventonlycountswhenthemouse’sheadisdirectedtowardstheotheranimal(oremptybasket),orthemouseisdirectlysniffingorstretchedout/touchingtheotheranimal/animal’sbasket/emptybasket.

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12. Whenthetimehasfinished,ascreenwillpopupwiththeresults.Push“Save”andthenchoosethedirectorytosavethedataontoyourflashdrive(orontotheharddriveandthenimmediatelypulloffalldataafteryouarefinishedfortheday),andnameyourfilewhateveryouwant.Push“NewSession”andyouarereadyforthenextmouse/trial2.

Trial2:

13. After5min,repeatsteps7‐10exceptthistimeplaceanewmouseunderthesecondbasket.Thepreviousstrangermousewillremainunderthefirstbasket.Closebothgates,andrepeatsteps9‐12.

Analysis

14. Stopwatch+outputissavedasa.csvfile,whichcanbeopenedinExcel.Cutandpasteyourdataasnecessaryandanalyzelatency.

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Habituation/Dishabituation(SocialRecognition)ThistestissimilartotheSocialNoveltyandResident/Intrudertasks,inthatitexaminestheamountoftimethatamousespendsinvestigatinganovelmouse.Thistestalsohasamemorycomponent,liketheSocialNoveltytest,inthatananimalisrepeatedlyexposedtoonemouse,andtheneventuallyexposedtoanewmouse,andbehaviorismonitored.Normalmiceshouldhabituatetothefirstmouse,andthendishabituatetothenewmousepresentedinthelasttrial.SettinguptheExperiment

1. BringyourmicefromSuite16totheBehavioralCore.2. Thisexperimentcanberunineitherafreshmousecage,intheopenfieldcage(freestanding),orin

the3chamberedapparatus.Putyourtestmouseinthenovelcageandallowittoexplorefor2min.3. Makesurethecomputerison,anddoubleclickontheStopwatch+icon.Youwillusethisprogramto

manuallytrackhowmuchtimethemousespendsinteractingwitheachoftheothermice.4. Fornumberofevents,enter1(unlessyouwouldliketobreakdownspecificbehaviors,inwhichcase

youmayenterasmanybehaviorsasyoumaylike).Definetheeventas“interaction”orwhateveriseasiestforyoutoremember.


6. Enterafixedsessionlengthof2min.Thesoundshouldbeturnedoff(doublecheckonthecomputer)soyoudonothavetoclicktheboxthatdealswithbeeping.

7. Youcansavethisfileforuseinlatertrials,orcreateanewoneeachtime.8. Whenthemouseisdoneexploring,placethenovelmouseintothecagewiththetestmouseandstart

thetimerforStopwatch+.9. Recordallinstanceswherethetestmouseisinteractingwiththenovelmousebypressingthe

appropriatebuttonsonStopwatch+.Behaviorsthatcountasinteractionsinclude:sniffingoftheheadandanogenitalregion(shouldbewithin1cmofthemouse,andpreferablybeingabletoseewhiskersmoving),orcloselyfollowingandpursuingthemouse.

10. Whenthetimeisup,removethemiceandcleanthecagewith70%ethanol.11. AscreenwillalsopopupinStopwatch+withtheresults.Push“Save”andthenchoosethedirectoryto

savethedataontoyourflashdrive(orontotheharddriveandthenimmediatelypulloffalldataafteryouarefinishedfortheday),andnameyourfilewhateveryouwant.Push“NewSession”andyouarereadyforthenextmouse/trial.

12. Wait20minandthenrepeatsteps2‐11,withoutthetestmouseacclimationtimeperiod.13. Repeatstep12.14. Twentyminfollowingstep13(trial3),setupStopwatch+(steps3‐7)andplacethetestmouseinthe

cage.PlaceanewnovelmouseinthecagewiththetestmouseandstartthetimerforStopwatch+.15. Repeatsteps9‐11.Thetestanimalisnowfinishedwithtesting.

Analysis

16. Outputissavedasa.csvfile,whichcanbeopenedinExcel.Cutandpasteyourdataasnecessaryandanalyzelatency.

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OlfactionOlfactioncanbetestedintwogeneralways;latencytoanovelodorortimespentsniffingthenovelodor.Forthenovelodor,Swisscheesecanbeusedandshouldbeobtainedpriortotheexperiment.Thecheeseshouldbecutintocubesroughly1cmx1cmandbroughttothebehavioralroomontestday.Fortimespentsniffing,vanillaextractcanbeused.LatencytoaNovelOdor

1. Collectyourmiceandbringthemtothebehavioralsuite.2. Obtaincleancagesandextrabeddingfromtheanimalresourcesstaff.3. Placetheanimalsthatarewaitingfortheirturnontheoppositesideoftheroomtowhereyouwillbe

doingtheolfactiontests.4. Eachmousewillneedtheirownnewcleancagefortheexperiment.Addtheextracleanbeddingto

eachofthecleancages.Thenewlevelofbeddingshouldbe2‐2.5inches(besurethenestingmaterialisremoved).

5. Youcanmoveyourexperimentalongmoreefficientlyifyoustaggerthedifferentstages.Eachmouseneedstohabituateinacleancagefor5minutessoyoucanstartafewmicehabituatingatthesametimebecausetheolfactionexperimentusuallytakeslessthantwominutespermouse.Youshouldrunapilotexperimenttoassesshowyourmicewillbehave.

6. Placeyourmouseinanewcleancagewithbeddingat2‐2.5inchesdeep,andallow5minforhabituation.

7. Haveastopwatchready,andusingtweezersafterthehabituationperiod,insertacubeofcheesedeepintothebeddingontheoppositesideofthecagetowherethemouseissitting.

8. Timehowlongittakesforthemousetofindthecheese(createanoperationaldefinitionfor"findingthecheese",forexample,noseandwhiskersburiedinthebeddingabovethecheese).

9. Recordthetime.10. Repeatsteps6‐9forallofyourmice.11. Youmayleavethemintheircleancagesbeforereturningthembacktotheircolonyifyouwouldliketo

givethemicetimetoeatthecheese.12. Onceallofthemicearebackintheircages,cleanthebehavioralsuite,includingthesurfaces,floor,

andtrash.13. TurnoffthelightsandreturnyourmicetotheHighRiskRoom.14. Inordertoanalyzethedata,simplycomparethetimesbetweenyourtwogroupstodetermineifthere

areanydifferences.TimeSpentSniffingaNovelOdor

1. Collectyourmiceandbringthemtothebehavioralsuite2. Obtaincleancagesandextracleanbeddingfromtheanimalresourcesstaff.

Test area

Waiting

Habituation

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3. Placetheanimalsthatarewaitingfortheirturnontheoppositesideoftheroomtowhereyouwillbedoingtheolfactiontests.

4. Eachmousewillneedtheirownnewcleancagefortheexperiment.5. Youcanmoveyourexperimentalongmoreefficientlyifyoustaggerthedifferentstages.Eachmouse

needstohabituateinacleancagefor5minutessoyoucanstartafewmicehabituatingatthesametimeastheolfactionexperimentusuallytakesjustovertwominutespermouse.Youshouldrunapilotexperimenttoassesshowyourmicewillbehave.

6. Placeyourmouseinanewcleancageandallow5minforhabituation.7. Haveastopwatchready.Using2disposablepipettesplaceoneortwodropsofvanillaextractonone

sideofthecageandoneortwodropsofwaterontheothersideofthecage.Thiseliminatestheconfoundofinvestigatingthenovel"object"versusnovelsmellasyouwillcomparehowmuchtimethemousespendssniffingthevanilla.

8. Allowthemouse2minutesandtimehowmuchtimethemousespendssniffingthevanillaoutofthetwominutes(within1.5inchesoftheliquid).

9. Recordthetime.10. Returnthemousetoitsoriginalcage.11. Repeatsteps6‐10forallofyourmice.12. Onceallofthemicearebackintheircages,cleanthebehavioralsuite,includingthesurfaces,floor,

andtrash.13. TurnoffthelightsandreturnyourmicetotheHighRiskRoom.14. Inordertoanalyzethedata,simplycomparethetimesbetweenyourtwogroupstodetermineifthere

areanydifferences.

Test area

Waiting

Habituation

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NovelObjectRecognition

Thistestisusedtoexaminememoryinanimals.Themouseispresentedwithanobjectduringacclimationandthenintestingpresentedwithanovel(new)object,andtheamountoftimetheanimalspendsexploringthenovelobjectisrecorded.Themouseisthenchallengedfurtherwithasubsequenttestwherethenovelobjectismoved.Note:Asyougetmoreexperiencedwiththisprotocol,youmaybeabletoruntwomicesimultaneously.Youwouldrunonemouseinoneendofthe3chamberapparatusandoneintheoppositeend.Forthestopwatchprogram,youwouldhave4eventsandlabelthemsothatitiseasyforyoutotrackthem.Wedonotrecommendattemptingtobalancetwoanimalsuntilyouareverycomfortablewithrunningoneatatime.Acclimation

1. BringallanimalstotheBehavioralsuite.2. Placetheanimalinthe3chamberedapparatus,withbothgatesup.Noobjectswillbeinthechamber.

Allowtheanimaltoexplorefor10mins.3. Repeatsteps1&224and48hrslater,foratotalof3daysacclimation.

ExperimentSetup‐Day4

4. OnDay4,bringyouranimalstotheBehavioralSuite.Inthe3chamberedapparatus,placetwoidenticalobjectsinthetestchamber,liningthemupwiththemarkersontheoutsideofthechamber.

5. Double‐clickontheStopwatchicon.Youwillusethisprogramtomanuallytrackhowmuchtimethemousespendsinteractingwitheachoftheobjects.

6. Fornumberofevents,enter2.Defineeachoftheeventsas“rightobject”and“leftobject”orhoweveriseasiestforyoutoremember.


8. Enterafixedsessionlengthof10min.Thesoundisturnedoffsoyoudonothavetoclicktheboxthatdealswithbeeping.

9. Youcansavethisfileforuseinlatertrials,orcreateanewoneeachtime.10. Placetheanimalinthemiddlechamberwiththegatetothetestchamberopen,andpushthestart

buttonontheStopwatchprogram.11. Eachtimethatthemouseinteractswiththerightorleftobject,holddownthecorrespondingkeysfor

thetimethattheanimalisincontactwiththeobject.Theanimal’snosemustbewithin1cmoftheobjectand/ordirectedattheobject,withwhiskersmovingifyoucanseethat,oractuallytouchingtheobjectforittobeconsideredtimespentexploringtheobject.Justbeingclosetoitisnotgoodenough.

12. After10minsisup,removetheanimalandputitbackinitshomecage.Cleanoutthechamberandobjectswith70%ethanoltoremoveanysmells.

13. Repeatsteps4‐12witheachmouseuntilyouaredonefortheday.

ExperimentSetup‐Day514. Repeatsteps4‐12,exceptthatyouwillputoneoldobjectandonenovelornewobjectinthechamber

now,andthetimeshouldbe5mininsteadof10min.YoumaychoosetolabelyoureventsinStopwatchas“old”and“novel”insteador“rightobject”and“leftobject,”butit’swhateveryourpreferenceis.

15. Afteranintertrialinterval(ITI)ofapproximately2hrs,youwillmovethelocationoftheoldobjectsothatitisnowlocatedonadifferentwall.Repeatsteps4‐12,againusinga5mintriallength.

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MorrisWaterMazePre‐testRoomPreparationBeforebeginningtheexperimentyoushouldhighlyorganizeanimalgroupsandscheduling,asthisisacomplexmulti‐daytest.Youshouldplantohavebetween12‐15micepergroup,andyoushouldschedulethetestssothateachgroupgetstreatedequally.Forexampleifgroup1has10daysbetweenthevisibleplatformandthehiddenplatformtest,group2shouldalsohave10daysbetweenthem.Atleastadaybeforethetestyouwillneedtocomeintothebehavioralsuiteandsetupthewatermazeatthecorrectlocationunderthevideotracking(instructionsforoperationbelow).Fillupthemazetotheindicatedlevelandletitsitovernighttoallowthetemperatureofthewatertoreachroomtemperature.

Howtooperatethetrackingsystem

1. Makesurethecameraispluggedin,theyellowvideowireisconnectedtothetrackingsystemcable,andthecomputerison.



min.Forhiddenplatform,select“After1visittozone(choosethezonewhereyourplatformis)”.Thiswillstopthetimeroncetheanimalisontheplatform.Lookatthezonemaptodeterminewhichzoneyourplatformisin.

5. Under"DataAcquisition"selectsinglesubjecttrackingFileopenzoneZoneFile_VP.zne6. Alignthewatermazeaccordingtotheimageonthescreen,orreadjustthetracingandsaveitasyour

ownzonemap.7. Selectdarkorlightsubject,dependingonyourmousecolor.Whenyouarereadytousethesystem,Hit

Start.Thetrackingsystemwillwaittostartrecordinguntilthefirstsignofmovement.Ifthesystemhasahardtimetrackingyouranimal,youmayneedtoadjustthethreshold(default=80)andcontrast(default=3).

YouarenowreadytorunyourexperimentVisiblePlatformTestSessions:

8. Collectyourmicefromtheirrespectiveroomsandbringthemtothebehavioralsuitewhileyoupreparetheroom(toallowforacclimation).

9. Addnontoxicwhitepainttothewatertomaketheliquidopaquesothattheplatformcannotbereadilyseen.Placetheplatform1cmabovethesurfaceofthewater.

10. Attachaflagtotheplatform.Eachmousewillget4attemptstofindtheplatformwiththeflag.Theplatformpositionshouldbealternatedinasemirandomfashionbutultimatelyitshouldbeplacedonceineachquadrant(permousetrial).Thetrainingconsistsofthreetrialsperday‐separatedbyaboutanhour.

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TrainingSession1(Trials1‐4)Youwillneedatimer;thetrackingsystemnotneededforthisphase.

11. Placemouseinthemazeatoneofthefourdirections(N,S,E,W)withthevisible(becauseofflag)platforminoneofthefourquadrants.Thispositionwillbethesameforthatmouseeachtrial,andyoucanchoosetostartfollowingmiceatadifferentpositionorkeepitthesameforallmice.Keeptrackofhowfastthemousefindstheplatformeverytime.

12. Allowthemousethemousetoswimfor1min.Iftheyfindtheplatformleavethemontherefor30sec.Iftheydonotfindtheplatform,pickthemupandplacethemontheplatform.Allowthemtosittherefor30sec.

13. Placethemouseinaholdingcageandmovetheplatformtonextrandomizedquadrant.14. Repeatsteps11‐13untilyouhavetestedthemousewiththeplatformonceineachofthe4quadrants,

foratotalof4trials.15. Returnthemousetoitshomecage.16. Collectanydebriswiththefishingnet.17. Selectyournextexperimentalmouseandrepeatsteps11‐14foreverymouseinyourfirstgroup

(groupA).Agroupofabout12‐15micecanberuneachday.

TrainingSession2(Trials5‐8):18. Aftera1hourintersessioninterval,repeatthestepsforthefirsttrainingsession(11‐14),usingthe

samegroupofmice.TrainingSession3(Trials9‐12):

19. Aftera1hourintersessioninterval,repeatthestepsforthefirsttrainingsession(11‐14),usingthesamegroupofmice.

20. Returnallmicetothehighriskreturnroom.21. Cleanupthebehavioralsuite,includingthemaze,surfaces,floor,anyspilledwaterandtrash.22. Turnoffthelightsbeforeleaving.23. Thisistheendofonedayofthevisibleplatformtest.

HiddenPlatformThehiddenplatformtestshouldbeadministeredatleast24hrsaftercompletionofthevisibleplatformtest.Italsoconsistsofthree4trialssessions(separatedbyaboutanhour)perday,foratotalof12trialsaday.Itisrunfor3consecutivedayspergroup,foratotalof9sessions(36trials).Thiswillbefollowedbyatestdaywhereyouperformprobetrialtesting(describedlater).Duringhiddenplatformtraining,theflagmarkingtheplatformisremovedandtheplatformismaintainedinafixedposition.Themouseshouldbeplacedaroundtheperimetersemi‐randomlyinoneof4startpositions(quadrants)andallowedtosearchfortheplatformfor60sec,thentoremainonitfor30sec.

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Preparation24. Repeat/reviewsteps1‐7,andgetthetrackingsystemready.

TrainingSession1(Trials1‐4)

25. Placemouseinoneofthequadrantswiththehidden(noflag)platformalsoinoneofthefourquadrants.Keeptrackofhowfastthemousefindstheplatformeverytime.

26. Allowthemousetoswimfor1min.Ifitfindstheplatformleaveitontherefor30sec.Ifitdoesnot,pickitupandplaceitontheplatform.Allowittosittherefor30sec.

27. Keeptheplatforminthesamequadrant.Placethemouseinadifferentrandomlyselectedquadrant.28. Repeatsteps24‐26threemoretimes.29. Returnthemousetoitscage.30. Collectanydebriswiththenet.31. Selectyournextexperimentalmouseandrepeatsteps24‐27foreverymouseinyourfirstgroup

(groupA).Agroupofabout12‐15micecanberuneachday.TrainingSession2(Trials5‐8)

32. Usingthesameanimals,repeatthestepsfromTrainingSession1(steps24‐30),randomizingthesequenceofstartquadrantsforallmice(allmicewillstartfromeachofthe4quadrantsonceasession).

TrainingSession3(Trials9‐12)

33. Usingthesameanimals,repeatsteps24‐30,randomizingthesequenceofstartquadrantsforallmice(allmicewillstartfromeachofthe4quadrantsduringeachsession).

34. Returnallmicetothehighriskreturnroom.35. Cleanupthebehavioralsuite,includingthemaze,surfaces,floor,anyspilledwaterandtrash.36. Turnoffthelightsbeforeleaving.37. Thisistheendofonedayofthehiddenplatformtest.Repeatallinstructionsunderthe"Hidden

platform"headingeachdayforthreedays.TestDay‐“ProbeTrial”Onthefollowingday,afterthethreeconsecutivedaysoftraining,themicewillbetested.

REMOVEtheplatform.Chooseoneofthedirectionstostartallmicein,andplaceeachmousethere,startthetrackingsystemandallowthemousetoswimfor90sec.ThetrackingandtimespentineachquadrantwillbedonewiththeSMARTvideotrackingsystem.Thetimespentineachquadrantwillbeautomaticallymeasuredandthetimeinthequadrant,whichpreviouslycontainedtheplatform(thetargetquadrant)willbecomparedtothetimespentintheotherquadrants.

38. Collectyourmiceandbringthemtothebehavioralsuite.39. Makesurethecameraispluggedin,theyellowvideowireisconnectedtothetrackingsystemcable,

andthecomputerison.40. Createafilefolderforyourdataonyourflashdrive,oronthecomputertobedirectlyburnedtoCD

andremovedaftertheexperiment.41. OpentheprogramSMART.42. Configuration:Paths:chooseyourfolder.ThenselectConfiguration:Timings:Changeto

90sec.Choose“whenprogrammedtimeisover”toallowthesystemtotracktheanimalforthefulltime.

43. Under"DataAcquisition"selectsinglesubjecttrackingFileopenzoneZoneFile_VP.zne

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44. Usethezonemapyousetup,andmakesureitisstillalignedwiththewatermazeaccordingtotheimageonthescreen.

45. Selectdarkorlightsubject,dependingonyourmousecolor.Youarenowreadyforthetestphase:

46. Putyourfirstmouseinthecenterofthemaze.HitStart.Thetrackingsystemwillwaittostartrecordinguntilthefirstsignofmovement.Ifthesystemhasahardtimetrackingyouranimal,youmayneedtoadjustthethreshold(default=80)andcontrast(default=3).

47. Theprogramwillautomaticallystopafter90sec.48. Fillinthedetailsofyoursubjectandexperiment.49. HitFileSave(foreachmouse).50. Closethewindow.51. SelectDataacquisitionSinglesubjecttracking52. Putthenextmouseinthecenterofthemaze.53. Repeatsteps45through51foreachsubject.54. Closethewindow.Youarenowreadytoanalyzethedata.

MorrisWaterMazeAnalysis

55. SelectDataAnalysisSelectFileopenzoneMorrisWaterMaze.zne56. Fileopensinglesubjecttrack.57. SelectthefilesyouneedfromyourfolderHit√HitGo.58. Checkthat"Subjectcode","zonename"and"permanencetime"areselectedfromthe"Standard"tab

tobeincludedinthereport.59. Alsolookunder"ZoneTransitions".Thesame3shouldbeselected.60. Theboxthatsays"Include"shouldbeselectedunderbothscreens.HitOK.Saveresultsandanalyze

bycomparingthetimespentinthetargetquadranttothetimespentintheothersbetweengroups.61. Returnallmicetothehighriskreturnroom.62. Cleanupthebehavioralsuite,includingthemaze,surfaces,floor,anyspilledwaterandtrash.63. Turnoffthelightsbeforeleaving.

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UltrasonicVocalizationDetector

Thisisatestthatisusedtodetectvocalizationinpups.Thereareseveralprotocolsthatcanberuninordertoassessdifferentstressfulsituationsforthepups.Pre‐experimentPreparationItisrecommendedthatyouhaveawaytodistinguishthepupsfromoneanother.Thiscanbedonewithvariousmarkingtechniques,includingtoe/pawtattooing.Ifyouwouldlikeinstructionintattooing,pleasetalktoJenYocumfortraining.Itisalsoagoodideatohaveatimersoyoucantestallanimalsforthesameamountoftime(seebelowfordetails).GeneralComputerInstructions:

1. Turnonthecomputer,andmakesurethepowerswitchisturnedontotheblackpowersupplybox.Themicrophoneshouldbeontherighthandsideofthesoundproofchamber.

2. DoubleclickontheUSVicon(notUSVViewer)toopentheprogram.Ifyouarepromptedtoenteranoffset,thenpushokandallowittofindtheoffset,andthenclick“lastbox.”Youshouldthenarriveattheopeningscreen.

3. Enterthebandwidthsthatyouareinterestedincapturing.Thefullrangetheprogramcancaptureis20‐100kHz.Themajorityofmousesoundswillbewithin50‐80kHz.Youcansettwodifferentbandwidthranges(forexample20‐50,and50‐100).

4. Setthedecibellevelyouwanttohaveasaminimum.Thedefaultis55db,butyoumaychoosetochangethis.Intheexampleof55db,thecomputerwillnotrecordanythingthatisquieterthan55db.

5. Click“SaveFFTdata”andmakesure“EnableMedInput”isNOTclicked.6. Push“Start.”7. Youwillthengetapopupscreenwhereyouwillenterinyourfilename.Thenclick“Save,”andtheprogramwill

immediatelybeginrecording.8. Thereisnotimerorwaytosettimeonthisprogram,soyoumaywanttouseatimersothateachofyourtrials

areapproximatelythesamelength.Pushthe“Stop”buttonwhenyouwanttostoprecording.

DataAnalysis9. InordertoreadthefilesyoumustdownloadtheMED‐USV.xlsmacro.Downloadthistoyourflashdriveand

installitonyourlabcomputer,aswellasalldatafiles.10. OpenallBINfilesontheUSVViewerontheBehavioralCoreUSVcomputerandthenconvertallfiles(oneata

time)tocommadelimitedfiles(“.CSV”files).Downloadthesefilestoyourflashdrive/CD.11. OpenExcel,andthengotoFileOpenandthenselectMED‐USV.xls.12. Select“EnableMacros.”Onceitisopen,gotoToolsMacroMacros…(orSecurityifyoudonothavethe

Macros…option).SetthesecurityleveltoMedium.13. Openthemacrolabeled“GetUSV”andhitRun.ThenselectyourCSVfile.14. AspectrogramshouldappearasanExcelworkbook.Thisspectrogramislimitedtothefirst7.5seconds.Ifyou

wanttolookatfurtherdata,thenyouwillneedtousethe“GraphUSV”macro(describednext).15. Only255cellscanbeputintoaspectrogramatonetime.Selecttheonesofinterestbyhighlightingthem,and

thengotoToolsMacroMacrosandselect“GraphUSV”andhitRun.16. Ifyouwanttouseactualnumbersforstatisticalanalysis,youcancreatea.txtfilebyopeningyour.BINfileinthe

USVVieweronthecomputerinthefacility.DoubleclickontheicontoopentheViewer,openyourBINfile,andgotoMenuSaveAsandthenchoosethedirectorytogoontoyourflashdriveandthenpush“Save.”

17. Toviewthe.txtfile,youcanthenopenthefilewithNotepad,Wordpad,oranywordprocessingprogram(Word,CorelWordPerfect,etc).

18. Toimportthe.txtfileintoExcel,openExcel,andgotoDataGetExternalDataImportTextFile…andthenchoose“space‐delimitedtransfer”byclickingthe“space”box.

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19. Alternativelytosteps15‐17,youcanopentheUSVViewerprogram,andthenselectMenuExporttoallowyoutosaveyourBINfileasa.csvfile.Chooseadirectoryandfilenameinthe“SaveUSVCSVfile”windowandclick“Save.”

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ForcedSwimTest

Theforcedswimtest(alsocalledthePorsolttestorbehavioraldespairtest)isregardedasaphysiologicalmeasureofdespairexhibitedbythemousefrombeinginanenvironmentfromwhichitcannotescape.Thisisexhibitedthroughimmobilitybehavior.Thistestisoftenusedwhentestingvariousantidepressantsandmodelsofmooddisorders.Asanote,wearerecommendingastandard6minprotocol.Therehavebeencasesorcertainmousestrainswheremorefloating/immobilityhasbeenshownduringthelast5minofa15mintrial;thereforethereisanalternativeprotocolof15minonbothdaysthatcouldbeused.PleasediscusswithMisha/Jentodeterminewhichprotocolmaysuityourneedsbest.Testing:Youwillneed:avideocamera,tripod,andabeakerorcontainer20x14cmfilledwith16cmofwater1. BringyouranimalstotheBehavioralSuiteforacclimation.2. Setupthebeakerwithwateronastabletabletopsurface.Positionthecameraonatripodsothatthe

viewisdirectlyintothebeakerfromtheside.3. Makesurethecameraispluggedin,andconnectthevideowires,withtheyellowvideowire

connectedtothetrackingsystemcable.Turnthecomputeron.4. Createafilefolderforyourdataonyourflashdrive,oronthecomputertobedirectlyburnedtoCD

andremovedaftertheexperiment.5. OpentheprogramSMART.6. Configuration:Paths:chooseyourfolder.ThenselectConfiguration:Timings:Changeto6

min.7. Under"DataAcquisition"selectsinglesubjecttrackingFileopenzoneforced swim test.zne8. Alignthebeakeraccordingtotheimageonthescreen,orreadjustthetracingandsaveitasyourown

zonemap.9. Selectdarkorlightsubject,dependingonyourmousecolor.Whenyouarereadytousethesystem,Hit

Start.Thetrackingsystemwillwaittostartrecordinguntilthefirstsignofmovement.Ifthesystemhasahardtimetrackingyouranimal,youmayneedtoadjustthethreshold(default=80)andcontrast(default=3).

10. Theprogramwillautomaticallystopafter6min.Removethemousefromthebeaker,dryitoff,andreturnittoitshomecage.

11. Fillinthedetailsofyoursubjectandexperiment.12. HitFileSave(foreachmouse).13. Closethewindow.14. SelectDataacquisitionSinglesubjecttracking

Putthenextmouseinthebeaker.15. Repeatsteps7‐14untilyouaredonewithallmice.Youshouldchangethewatereitherafterevery

mouseoratleastafterevery2‐3miceifpossible.16. 24hourslater,repeatsteps1‐15forday2oftesting.

DataAnalysis:

17. Youwillanalyzethelast4minsofday2’strialtoseethepercentageofimmobility,aswellasthelatencytothefirstepisodeofimmobilityonbothdays.Theseareusedasameasureofdespairintheanimal.

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FearConditioningThistestusesaclassicalconditioninglearningandmemoryparadigmtoexaminefreezingbehaviorinanimalswhenpresentedwithtoneandshock.Thesechamberscanalsobeusedtoexaminevariousotherparadigmsthatuseshockand/ortonepresentation,suchasstep‐downavoidance,orlatentinhibition.ExperimentalSetup

1. BringyouranimalstotheBehavioralsuite.2. Makesurethecomputerison,anddoubleclickontheFreezeScansoftware.3. Clickthebuttonthatlookslikeacamera.Click“livevideo”andthenclick“OK.”4. Clickoffthe“saveas”button,thenclickitbackon(thisisaglitchwiththeexistingsoftware).Makea

newfolderorfindyourfoldertosavetheinformation.Itispreferredthatyouputtheinformationonyourflashdrive(eitherthroughthisdirectory,orimmediatelyfollowingtheexperimentconclusion).YoumaychoosetoburnaDVD,howeveryoucannotreanalyzethedataifyouchoosethismethod.

5. Afterselectingthedirectory,click“save.”6. Tocheckthecamerasineachofthechambers,pushtherecordbutton(reddot)ontheprogram,and

thenpushthestopbutton(blacksquare).Seeiftheboxesarerecording.7. Turnonthepowerbox(controlsthelights)andturnontheshockers.8. Resaveoverwhatyoudidbefore,repeatstep6,andthenyoushouldseeonscreenthatyoucanseein

theboxesnow.Youcantestitwithyourhandtomakesurethateverythingisrecordingsmoothly.9. Nowyouwanttomakesuretogetridoftheblacklinesaroundthepicturesoftheboxes.AdjusttheT,

B,L,andR(top,bottom,leftandright)untilthelinesaregone.Itmaybevaluessomethinglike:T:2,B:250,L:10,R:20.Keeppushing“adjust”anddosountilthelinesaregone.

10. Ontheshockbox,fliptheswitchto“manual”andtheotherswitchto“set/test”totestthelevelofshock.Noshockisactuallybeingdelivered,butadjustthedialsothatyoucandelivertheamountofshockyouwant.

11. Readjusttheswitchesto“remote”and“subject”,andmakesuretheotherdialisonMan’l/Hi.Repeatsteps10and11fortheotherchamber.

12. Onthecomputerprogram,gotoArenaSetArenaNum2.13. ArenaEnterArenaDesignMode2.InArenaDesignboxthatpopsup,selectCurrentArena1.14. Select“polygon”inArenaDesignTools.Clickonarena1tocreatetheboxforwheretolookforthe

mousebehavior.RightclickwhendoneFinishZoneOperation.15. ClickCurrentArena2inArenaDesignbox,andrepeatstep14forarena2.ClickFinish.16. ClickSetting.ForFrameundertheAnalyzingheading,3‐5isrecommended.Inter‐framemotionshould

belessthan20pixels(thedefaultof400istoohigh).ClickOK.17. Clickon“StartRules”tab“attime”buttonOK.CurrentArena2,click“attime”andthenclick

OK.Click“StopRules”tabduration,enter300OK.18. CurrentArena2,andrepeatstep17forarena2.19. ClickontheProtocolstab.Click“attime”andput30,clickOK.Thisdeterminesafterwhatamountof

timethatthesoundwillstart.Anewwindowwillpopup.ClickSoundselectTonefrequency2000Hz20secdurationOK.

20. Click“attime”andwrite48,clickOK.Thisiswhentheshockwillbedelivered(duringthelast2secoftone).

21. Clicktheport0/line2boxwhichshouldalsosay“cage1shock”totherightofthat,andthenchangethedurationto2.ClickOK.

22. Selectcurrentarena2andrepeatsteps19‐21forarena2,makingsurethistimetoselectport0/line6(cage2shock)instep21.ClickOK.

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23. Clickoffthe“saveas”boxandthenclickbackon(again,aglitchwiththesystem),andthensavethefiles.ClickOK.

24. GotoArenaSaveArenaProfilethenputitintoyourfolderandsave.Ifyouneedtoaccessyourarenainthefuture,youwouldgotoArenaLoadArenaProfile.

25. Placeyouranimalsinthecorrespondingchambers,makingsurethatthelatchesaresecureandthenclosethesoundattenuatingchambers.Whenyouarereadytobegin,CLICKTHE“ANALYZE”BOX,andpushtherecord(reddot)button,anditwillbegin.Ifyoudonotclick“analyze,”theprogramwillnotrecord/start.

26. Whenyouaredonewithyouranimals,removethemandplacethembackintotheirhomecages.Cleanoutthechamberswith70%ethanolandrepeatsteps25‐26foryourconsecutiveanimals.

DataAnalysis

27. ClicktheSaveboxSaveenterthemouseIDforarena1.Selectarena2SaveentermouseIDsavetosavetrack.

28. ClickBin,selectarena1,clicksecond(canselect10‐60sec).Push“cancel”whenExceltriestoopen(againaglitch)andthenExcelwillopen.SelectBinArena2andrepeatforarena2.

29. Youcanthencopyandpastethesecondmouse’sdataintothefirstdocumentandsavethatway,orsavethemasseparatefiles.

30. Alternatively,ifyouonlywantthetotalfreezingtime(notbrokenintobins),youcansimplypushtheExportbutton,whichsendstheinfotoExcel,andthensavethosefiles.

31. Putalldata,includingvideosandotherdataontoyourflashdriveandthendeletethemfromthecomputer.Turnoffallboxesandthelightswhenyouleave.

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TailSuspensionTestThistestistypicallyusedtostudytheeffectsofantidepressants.Thisisassessedbylookingatdifferencesinmovement,brokenintomeasuresof“agitation”andimmobility.Thetailsuspensionmodelisthoughttobealessstressfulcorrelatetotheforcedswimtest.Inthistest,amouseissuspendedbyitstailtoasmallhook,andthecomputerthenrecordswhentheanimalmovesvs.remainsstill.

Test:

1. BringyouranimalstotheBehavioralSuiteforacclimation.2. Makesurethecomputerisonandthatthepowersupply/connectorboxison.3. DoubleclickontheTailSuspensionicononthecomputer.GotoExperimentStartExperiment.4. Clickthe“AddExperiment”buttonandenterinyourexperimentID.Push“OK.”5. Selectthe“ModifyExperiment”buttontomakeanychangestoyourprotocol.Therecommendedtime

is6min.Youmayselectthenumberofblocksyouwant(likebins),whichdeterminesyouroutputandhowyouwillbeabletoanalyzeyourdata.Themoreblocksyouhave,themorebreakdownofyouranalysesyoucando,butyou’llalsohavemoredatatodealwith.Iwouldrecommendstartingwith30seconds,andifyoufindyouwanttoanalyzeyourdatain15or10secondincrements,youcanthenshortenit,orextenditto1minormoreifyouarenotinterestedinsmallerblocks.Click“OK”whenyouarefinishedenteringinyourparameters.

6. EnterinyourgroupIDandSubjectID,aswellasanycommentsyoumaywanttoadd.7. Click“Copydatatoallchambers.”ThiswillcarrythegroupIDandotherinfotothesecondchamber.

Clickonthe#2boxandenterinthesubjectIDforthatanimal.8. Clickthe“StartExperiment”button.The“SelectChambertoStart”boxwillappear.Makesurethat

Chambers1and2areselected,unlessyouareonlyrunningonechamber.9. Usingapieceoflabtape,secureeachmouse’stailtothehookinthechamber.10. Click“Start”whenyouarereadytobeginrecording.Youwillthenseethe“ExperimentRunning”

screen.11. Onceyourtimeisdone,thedatawillautomaticallysave.ClickExperimentStartExperimentand

repeatsteps4‐11foranyremainingmiceyoumayhave.DataAnalysis:

12. ToviewthefileintheViewerprogram:FromtheTailSuspensionprogram,clickonViewViewRawFile.Afileopendialogueboxwillopen.Browseandfindyourfile.

13. TocreateafiletoexportintoExcel,gotoCreateCreateCSV(RawFile,SummaryFile,orBatchSummaryFile).RawFile=asinglemouse,SummaryFile=anexperiment’sfile,BatchSummaryFile=severalexperimentsworthoffiles.Choosewhichoneisappropriate.Thisfilecanthenbeexportedintoexcel.Youwillanalyzethepercentageimmobilityduringthelast4minoftesting,aswellasthelatencytothefirstperiodofimmobility.

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Rotarod

Thistestisusedtoexaminesensorimotorfunctioninginanimals,andcanalsobeusedtotestphysicalstamina.Youareabletorunupto4animalsatatimeonarotatingrodthathasthecapacitytoaccelerateupto99.9RPM.Photobeamsrecordwhentheanimaldropsoffoftherod.1. GetyouranimalsfromSuite16andbringthemtotheroomforacclimation.Computer/ExperimentalSetup:2. TurntheRotamex‐Von,andmakesurethecomputerison.TherotarodMUSTbeonbeforeyouclick

ontheicontostarttheprogram.3. Double‐clickontheRotamexicononthecomputerdesktop.Itwillfindtheportautomatically,and

thenscanforalldevices(whichshouldreadOKforthefirstport)andtakeyoutothemenuscreen.4. GotoExperiment.Thisshouldpopupascreenwhereyoucanenterinparameters.5. FirstmakesurethatspeedisinRPM,andyouhaveselectedthecorrectwheel(mouseorrat).This

shouldcorrespondwiththetypeofrodthatisonthemachine.6. Enteryourexperimentdurationinseconds.Formostprotocols,thiswillbe300sec(5min).Then

enteryourstartingspeed.Thisisrecommendedtobe5.0RPM.YourMaxspeedwillbeupto99.9RPM.

7. AccIntmeanstheaccelerationinterval;inotherwords,howoftentherotarodisgettingfaster.Werecommendincreasingitevery5seconds.Accstepmeanshowmuchfasteritwillgeteveryinterval,whichwerecommend1.0RPM.

8. Push“SubjectID”foreachanimalyouwanttoload(youwilldothisoneatatime).EnterinthesubjectIDinformationandpushokforeachanimal.EachChannelcorrespondstoChannels1‐4ontherotarod,somakesurethatyouloadeachanimalwhereyouenteredtheirinformation.

9. Whenyouareready,loadtheanimalsontherotarod,andpush“startexperiment.”10. YoumustpusheachchannelstartbuttonandthenPUSHENTER.Thetime/recordingwillnotstartuntil

youpushenter.Asyoupusheachbutton,itwillstatethatitisloaded.11. Thetrialwillendwhenallanimalshavefallen.Push“StopExperiment”whenallanimalshavefallen.

Saveyourdatatoyourflashdrivebyselectingthecorrectdirectoryandifyouaredoinganothertrial,thenyouarenowreadytorepeatsteps8‐11.Ifyouarerunningthesameanimals,youdonotneedtore‐enterthesubjectdata,onlytodosteps9‐11.

DataAnalysis:

12. DatafilesaresavedasCSVfilesandareeasilyimportedintoExcel.YoumaywanttolookataverageRPMspeed,orlatencyontherotarodwhendoinganalyses.

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T‐mazeTheclassicversionoftheT‐mazeassesseshippocampal‐dependentspatiallearningability.Thisversionrequiresanimalstobeonfooddeprivation,sinceitusesfoodasarewardandmotivationforlearning.MemorycanbeexaminedintheT‐mazebyusingaspontaneousalternationprotocol,similartothatexplainedpreviouslyintheY‐mazeprotocol.Spontaneousalternationdoesnotrequirefooddeprivation.Themaindifferencebetweenthesetwoprotocolsisthatthespatiallearningversioncandistinguishbetweenthelearningthattakesplaceinthehippocampusbasedonwhatcuesareintheenvironment,orlearningthattakesplacebasedonasimplebodyturn.Spontaneousalternationreliesontheanimal’snaturaltendencytosearchforfoodorexploreinaplacedifferentthanwhereithadpreviouslytraveled,andcannotdistinguishwhichsearchstrategytheanimalisusinginordertomakethedecision.Itisabletodeterminedeficitsinshort‐termmemory.

SpatialLearningProtocolPre‐experimentPreparationPriortotraining,animalsmustbeplacedonfooddeprivationforseveraldaystogettoabout85‐90%oftheirnormalbodyweight.Youmaychooseagreaterorlesserweightloss.AllfooddeprivationprotocolsmustbereviewedandapprovedbytheACUC.Inordertoachievetheweightloss,animalsmustbeindividuallyhousedandfoodmustbeweighedoutdailyandrationedoutaccordingly.Animalsmustbeweigheddailyandmonitoredforoverallhealth.Iwouldrecommendatleast1weekoffooddeprivation.Atleast2‐3daysbeforestartingtraining,begintoincorporatetherewarditemintothediet(forexample,ifyouareusingrewardpellets,thengivethemouse2‐3pellets),andincludethisinthetotalweightofthefoodthatyouaregivingtheanimal.Thiswayyougettheanimalusedtoeatingtherewardfood.Fooddeprivationwillcontinueuntilyouaredoneallphasesoftesting.Animalsshouldalsobehandledatleast5minsdailyforatleast5dayspriortothestartoftheexperiment,andthroughouttheexperiment.Thisexperimentreliesonspatialcues,andsoyouwillneedanassortmentofcuestoplacearoundthemaze.Thisshouldbesetuppriortothestartoftheexperiment(beforeexposuresessions,incasethisinfluencestheanimal’spreferenceforanarm).ExposureSessions:Extraequipment/itemsyouwillneed:timer,rewardpellets,spatialcues1. BringallfooddeprivedanimalstotheBehavioralSuite.2. Setupyourcuesaroundtheroom,inanareaclosetothemazesotheanimalcanseethemreadily

duringtheexperiment.Thesecueswillremainfixedthroughouttheexperiment.Youalsoactasacueandyoumustremaininthesameplaceforalltrialsandalldaysoftheexperiment.

3. Setupthevideocamera,mountingitonatripodsothatyoucanseetheentiremazefromaboveorfromtheside.Setyourtimerfor10mins.

4. PlacerewardpelletsthroughouttheentireT‐maze,includinginthefoodwellsattheendoftheleftandrightarms.Thisencouragestheanimaltoexploreandhabituatetothemazeenvironment.

5. Press“record”onthecamera,andafterpresentinganyidentifyinginformationfortheanimal,placetheanimalonthemazeatthebottomofthe“T”inthestartarm,andpressthestartbuttononyourtimer.

6. Once10minsisup,theexposuresessionisover.Recordthenumberofrewardpelletseaten.7. Repeatsteps3‐5again30mins(ormore)laterforasecondtrainingsession.Thisisbestaccomplished

byrunninganimalsingroupsof4,suchthatwhenyouaredonewiththe4thanimal’s1sttrainingsession,itwillbetimeforyoutorunthe1stanimal’s2ndtrainingsession.

8. 24hourslater,repeatsteps1‐6fortrainingsessions3and4.

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ExposureSessionanalysis:9. Youhave2optionsforcalculatingthearmpreferenceduringtraining.Oneistoactuallyrecordthis

withatimerthathas3programmabletimersonit.Duringtraining,youwouldpushstopandstarteverytimethatanimalwentintotherightarmforonetimer,andthesamethingfortheleftarm,whiletheremainingtimeriskeepingtrackoftheoveralltime(10min).Thiswouldgiveyoumoreofaninstantread,andyoucanrecordthetimeinadatasheet.Thedisadvantagetothismethodisthatitcanbesomewhatinaccurateandifyoumissanentryorexit,thenthatcanthrowyourtimingsoff.Theotheralternativeistoanalyzeyourvideorecordingtothenseethetotalamountoftimespentineacharm.

10. Onceyouhavecalculatedthetimespentineacharmandthentotaleditoverthe4trainingsessions,youwillseewhicharmtheanimalhadapreferencefor.Thiswillbethearmyouwilltrainagainstduringthetestingphase.Iftheanimalhasnopreference(bothsidesarealmostequal,whichwouldbedefinedaslessthan60secdifferencebetweenthetwosides),thenyoucanflipacoinorperformsomeotherrandomwaytofigureoutwhichsideyouwilltrainthemagainst.

11. Youshouldrunananalysisforarmpreferenceamonggroupsofanimals,becausethismaybesignificant(andsometimesalsocorrespondswithwhereyouaresitting,sinceyoumaybeapositiveornegativeinfluence).Youalsowanttoanalyzeiftherearedifferencesinhowmanyrewardpelletswereeatenineachgroup.

Training:Youwillneed:atimer,rewardpellets,cues12. Trainingbeginsthedayafterthelastexposuresession.BringtheanimalstotheBehavioralSuiteand

allowthemtoacclimate.13. Animalswillreceiveupto20trialsaday.Place4rewardpelletsinthefoodwellthatyouaretraining

theanimaltogotowards(thiswillbetheoppositeoftheirpreferredarm,asstatedearlier).Makesurethecameraispositionedsoyoucanseetheentiremaze,andthatyourtimerissetto60sec.

14. Press“record”onthecamera,andaftershowingidentifyinginformationtothecamera,placetheanimalontheendofthestartarm(thebottomofthe“T”),facingawayfromthecenterplatform.Pushstartonthetimer.

15. Theanimalhasupto60secondstochooseanarm.Whenall4pawscrossoverthestartofthearm(right,left,orbackintothestartarm),thiscountsasachoice.Iftheanimalchoosesthecorrectarm,itisthenallowedtoeatthefoodattheendofthearmandisthenplacedbackintoitscageforanintertrialinterval(ITI)of60sec.Iftheanimaldoesnotchoosethecorrectarm(ordoesnotchooseanarm)duringanyofthefirst6trials,youshouldthengentlyguideittothecorrectarmandallowittoeat.Afterthefirst6trials,ifitmakesanincorrectchoiceordoesnotchooseanarm,youmustremoveitfromthemazeimmediately,placeitbackinitscage,anditdoesnotgetanyfoodreward.

16. Runtheanimalforupto20trials,ortoacriterionof7correctchoicesoutof8consecutivetrials.Oncetheanimalmakescriterion,skiptoTesting.

17. Repeatsteps11‐15onday2iftheanimaldoesnotreachcriterionontrainingday1,fortrials21throughupto40.Donotcorrectanyincorrectchoices,sincethisonlyappliestothefirst6trialsthattheanimaldoes.Again,oncetheanimalmakes7outof8consecutivecorrectchoices,skiptoTesting.

18. Again,iftheanimaldidnotreachcriteriononday1or2,itisgivenafinaldayoftrainingfortrials41throughupto60.Iftheanimaldoesnotreachcriterionafter60trials,itisthrownoutofthestudy(thesearereportedinthemanuscript).Onceitmakescriterion,skiptoTesting.

19. Ofnoteforsteps15‐17:Iftheanimalisinthemiddleofapotentialcriterionrunandyouonlyneedtorunanother3‐4trialsbeyondthe20scheduledfortheday,thengoaheadandkeeprunningthemto

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completethecriterion.Iftheanimalthenmessesupinsteadofmakingcriterionin23trialsforexample,goaheadandstoprunningitforthatday.

TrainingAnalysis:

20. Youwillwanttoanalyzeaveragearmchoicelatencybetweengroups,aswellastheaveragenumberoftrialstocriterionforeachgroup.

Testing:Youwillneed:atimer,rewardpellets

21. Assoonastheanimalmakescriterion,placeitinitsholding/homecageforanITIof60seconds.22. Turnthemazearound180°sothatthestartarmisnowdirectlyacrossfromwhereitwas(ifyouthink

ofa+sign,thenthebottomoftheTwouldnowbeatthetopoftheplus,insteadofthebottom).23. Baitbotharms’foodwellswithfood.Setthetimerfor180seconds.24. Placetheanimalinthestartarm,facingawayfromthecenterplatform.Startthetimer.25. Recordthearmentered,andaftertheanimalisdoneeating(orchoosesnottoeat),thenremovethe

animalandyourexperimentisdoneforthatanimal.Iftheanimalchoosesthearmthatistheoppositebodyturn,butinthesameplaceintheroombasedoncuelocation,itisrecordedasa“place”strategy.Iftheanimalmakesthesamebodyturninsteadofgoingtothesameplaceintheroom,itisrecordedasa“response”strategy.Placestrategiesrepresentahigherformoflearningthatrequirehippocampalinvolvement,andthemajorityofcontrolanimalsshouldexhibitthistypeoflearning.

TestingAnalysis:

26. Youwillwanttoanalyzethenumberoftrialstocriterionforeachgroup,aswellasnon‐parametricanalysisforplacevs.responsestrategiesforeachgroup.

SpontaneousAlternationProtocolInthisversionoftheT‐maze,youarelookingattheanimal’snaturaltendencytoalternateinitssearchforfoodorexploration.Ifyoudonotseeanalternatingpattern,thenitisindicativeofshort‐termmemorydeficits.Thereisnofoodinthemaze,andanimalsdonotneedtobeonfooddeprivationforthistask.Testing:

1. Youmayormaynotchoosetorecordthis.Youwillbepurelycheckingoff/writingwhatchoicetheanimalmakeseachtrial.

2. Placetheanimalinthestartarm,facingawayfromthecenterplatform.Waituntiltheanimalchoosesanarmbyplacingallfourpawsacrossthestartofthatarm.Recordthechoice.

3. Immediatelyremovetheanimalandplaceitdirectlybackintothestartarm,andrepeatsteps2‐3ninemoretimesforatotalof10trials.

4. Removetheanimalandplaceitbackinitshomecage.Analysis:

5. Calculatetheoverallalternationrate (percentageofresponsesinwhichtheratshiftedsidecomparedtothelastresponse)andthepercentagepositionbias(percentageofrespondingtothesideofthemazepreferredbythatindividualrat).

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Passive‐orActive‐Avoidance

Thepassive‐avoidancetestisasingletrainingtrialthatusesaclassicalconditioningparadigmtopairdarkwithanelectricshock,andthenmemoryretentionistestedwithatrial24hourslater.Inactive‐avoidance,theanimallearnstopairatonewiththeinevitabledeliveryofshock,anditmustthencrosstotheothersideinordertoavoidtheshock.Theanimalshuttlesbackandforthforseveraltrialspersession.

Passive‐AvoidanceNote:YouCANNOTrunanythingelseonthecomputerwhilethisprogramisrunning,becauseitmayloseyourdataifithascompetition.Nointernet,nootherprograms,noMicrosoftOffice…NOTHING.Testing:1. BringyouranimalstotheBehavioralSuiteandallowthemtoacclimate.2. TurnonthecomputerandthepowerswitchontheHabitestLinkPowerStation.Youwillseeabluelightcome

ononthegreencableboard.3. Onthecomputer,doubleclickonthe“GraphicState”softwareicon.4. Whenyouarepromptedforausernameandpassword,enterusername:ADMIN,password:PASSWORD.Click

“OK.”5. Wehavesetupseveralprotocolsforyouruse.Clickon“PASingle”soitishighlighted,andthenpush

“Select.”6. GotoFileRunaSession.ClickOKwhenyougetthewarningmessage.Youmaynotwantthecomputer

connectedtotheinternetatall,ifitis,soatthistimeyoumaychoosetounplugtheEthernetcordifitispluggedin.

7. Pushthe“check”buttontogettheprogramtochecktheLincstation.Itshouldfindtheonestationandstatethatallstationshavebeenchecked.ClickOK.

8. EnterintheSessionDescription,definingthesessionassomethingthatyoucaneasilyidentify.Youmaywanttoputyournameandwhateveranimalsorgroupsyouarerunning.

9. Intheboxthatsays“Firstprotocolnumbertoberun”,enter9(shouldbeapulldownboxthatonlygivesyouthatoptionanyway).

10. Clickthe“Run”button.Youshouldnowgetascreenthatlookslikeaspreadsheet.11. ThereisapinkcolumnthathasanAafterthenumber,andthepinkboxsays“?SUBJID?”Doubleclickonthis

andthenenteryouranimal’sIDnumber.ClickOK.Theboxshouldchangefrompinktogreenandnowsay“READY”onit.

12. Placethemouseinthelightside(clearplexiglass)ofthechamberandsnapthedoorshut.Currentlythereisnoacclimationtimebuiltintotheprotocol.Ifyouwouldliketoallowthemousetoacclimatebeforethedooropens,timethemouseforthe1‐5minofacclimationandthenstarttheprogram.

13. Doubleclickonthegreen“READY”box,anditwillchangetoredandsay“RUNNING.”Afteranacclimationperiod,thegatewillopenandthemouseshouldcrossover.Atthispointthegatewillcloseandthemousewillreceiveamildshock.Thisendsday1oftesting.Theboxwillonthespreadsheetwillnowchangetoblueandsay“FINISHED.”

14. Ifyouhavemoremicetorun,clickonthe“FINISHED”boxanditwillresettothepink“?SUBJID?”box.Repeatsteps11‐14untilyouhavecompletedallmice.MakesuretocleanthechamberanddroppanwithethanolorVimobabetweeneachmousetoremoveanyscentleftbehind.

15. Whenyouarefinishedwithallmicefortheday,clickon“End/Terminate”andyouwillbereturnedtothemainmenu.SeeDataAnalysissectiontofollowsteps18‐23attheendoftheday.

16. Returnallmicetosuite16.

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Day2testing:17. Twenty‐fourhourslater,repeatsteps1‐16,exceptthistimeselect“PAsingleday2”forstep5,andthennumber

1forstep9.Thedifferencebetweenday1andday2trialsisthatiftheanimalcrossesovertothedarkchamberonday2,itwillnotreceiveashock;thetrialwillsimplybeover.Iftheanimalhasintactmemoryretention,itshouldnotcrossovertothedarkchamber.

DataAnalysis:

18. Dataanalysiselementsarealreadybuiltintotheprograms.Attheendofyoursession,itwillautomaticallypromptyouifyouwanttolookatyourdata.SelectAnalyzeandReview.

19. Select“AnalyzeALLprotocolsandstructures”OK.20. Afterafewsecondsitwilltellyouthatanalysisiscomplete.PushOK.ClickonIndividualRuns,andthiswill

produceaspreadsheetwithallofthedata.Selecttherun(s)youwanttolookat,andclickSelectElement.21. ThenyoucanclickonViewGraphorViewNumbers,dependingonwhetheryouwanttoseeagraphorviewthe

actualnumbers.22. Whenlookingatthenumbers,youwillcometoaViewListpopupandthenhavetheoptiontopushExportto

exportyourdataintoExcel.Writeinthenameyouwanttogiveyourdatawhenprompted,makesureitissavingtoyourfolder/flashdrive,andthenpushSave.

23. YoumaynowexittheGraphicStatesoftwareaftersavingallofyourdata.Thefilehasbeenexportedana.txtfile,soyouwhenyougointoExcel,youwillopenyourfileandbepromptedtoaTextImportWizard.Allofthedefaultsettingsshouldbecorrect,sothenpushNextNextFinish.

Active‐Avoidance

Testing:24. Followsteps1‐15above,includingallwarnings,exceptforstep5select“AAmultiple,”andforstep9enterthe

number12.25. Dataanalysiswillbedoneinthesamewayasmentionedaboveinsteps18‐23.

40

UsefulInformationAnimalResources AnimalCareandUseTraining http://www.jhu.edu/animalcare/training1.html ResearchAnimalResources Ross459(ph:5‐3273) DirectorofLaboratoryAnimalManagement

LindsayBarnes [email protected],(ph:2‐1933)BRBBehavioralSuite Dr.MikhailPletnikov‐BehavioralCoreDirector [email protected],(ph:2‐3760)

JenYocum‐BehavioralCoreCoordinator [email protected]@gmail.com,(ph:2‐4259)

behavioral core user manual - brain science institute · 4 for any questions, comments or...

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