Mapping Tumor-Induced Neuronal Hyperactivity in Glioblastoma Mouse Models
Mason Chan
Senior Thesis | 2026
Mapping Tumor-Induced Neuronal Hyperactivity in Glioblastoma
Mouse Models
MasonChan
Song/MingLab,PerelmanSchoolofMedicineatTheUniversityofPennsylvania
Glioblastoma(GBM)isthemostcommonandaggressiveprimarybrain tumorinhumanadults,withamediansurvivalofapproximately15 months.Recentstudieshavedemonstratedthatgliomacellsformfunctional synapseswithneuronsandthatneuronalactivitypromotestumor progression.Sunetal.(2025)usedviraltracingtomaptheanatomical connectivitybetweenneuronsandpatient-derivedglioblastomacells, identifyingbrainregionsthatsendsynapticinputtothetumor.Thisstudy extendsthisworkbymappingneuronalhyperactivityacrossthebrainin tumor-bearingmiceusingc-Fosimmunohistochemistry. Immunocompromisedmicereceivedstereotacticinjectionsofpatientderivedglioblastomaorganoids(UPO-10072)intothesomatosensory cortex(n=10)orshamsurgerywithHibernateAvehicleatthesame coordinates(n=10).Openfieldtestingwasconductedatweeks2and4 post-injection.Micewereperfusedimmediatelyfollowingtheweek4 behavioralsession,andbrainsectionswereprocessedforc-Fosandhuman nestinimmunostaining.Automatedcellquantificationwasperformedusing QuPathfollowingalignmenttotheAllenMouseBrainAtlas.Tumorbearingmiceshowednobehavioraldifferencesatweek2butexhibited significantlyreducedcentertimeatweek4,indicatingananxiety-like phenotype.Brain-widec-Fosmappingrevealedregion-specificchanges, withtheamygdalashowingsignificantlyincreasedc-Fosexpressionin tumor-bearingmicecomparedtocontrols.Mostotherquantifiedregions showednosignificantdifferencesbetweengroups.Ourfindingsindicate thatGBMinducesincreasedneuronalactivityintheamygdala,which providesapotentialneuralcorrelatefortumor-associatedanxiety-like behavior.
Glioblastoma Background
Glioblastoma(GBM)isthemostaggressiveprimarybraintumorinadults.GBM alsorepresentsthemostcommonmalignantbraintumor,havinganoccurrence rateof3.2casesper100,000peopleannually.Despitedecadesofresearchand therapeuticdevelopment,theprognosisforGBMpatientshashadlimited improvement.Thecurrentmediansurvivaltimeis15monthspost-diagnosis,even withthecurrenttherapeuticintervention.Thiscurrentstandardofcareconsistsof surgicalresectionwithconcurrentradiotherapyandtemozolomidechemotherapy. Thisapproach,however,onlyprovidesmodestsurvivalbenefitsofanadditional2.5 months.GBMcellsmigratealongwhitemattertractsandinvadenormalbrain parenchymabeyondthevisibletumormargins,whichmakescompletesurgical resectiondifficult.Becauseofitsinfiltrationmethod,combinedwiththetumor's therapeuticresistance,GBMisknownasoneofthemostintractabletumorsin oncology.
Evidencebeginningtoaccumulatein2019indicatesthattherelationship betweenglioblastomaandsurroundingbraintissueismorethanthehistoricalmass effectmodel,whichattributedthepatientdamagetothetumorbeingaforeign masscompressingthesurroundingtissue.Gliomacellsintegrateintotheneural microenvironmentandcommunicatebidirectionallywiththenervoussystemin additiontothephysicaldamageandpressureofthetumor.Venkateshetal. demonstratedin2019thatgliomacellsformglutamatergicsynapseswithneurons. Usingelectrophysiologicalrecordingsfromgliomacellsinacutebrainslices, combinedwithimmunoelectronmicroscopyandoptogeneticstimulationin tumor-bearingmice,theyestablishedthatneuronsformfunctionalAMPA receptor-mediatedsynapsesontogliomacells.Theseneuron-to-gliomasynapses exhibitconventionalsynapticarchitecture,includingpresynapticvesicleclustering insynapticcleftsandpostsynapticreceptoraggregatesongliomacellmembranes. Neuronalstimulation,eitherelectricaloroptogenetic,evokespostsynapticcurrents ingliomacells.Thesynapticinputdepolarizesgliomacellmembranesandinduces calciuminfluxthroughAMPAreceptors.Theirinvivooptogeneticexperiments demonstratethatneuronalactivitypromotesgliomacellproliferation,while pharmacologicalblockadeofAMPAreceptorswithperampanelsuppressestumor growthandprolongssurvivalinxenograftmodels.Venkateshetal.identifiednonsynapticcommunicationmechanismsaswell.Gliomacellsextendcellularprocesses termedtumormicrotubesthatformgapjunction-couplednetworks,propagating depolarizingcurrentsbetweencells.Neuronalactivityevokedpotassiumcurrentsin gliomacellsareamplifiedthroughthisnetwork,extendingmembrane depolarizationbeyondcellsreceivingdirectsynapticinput.Theirworkestablished neuronalactivityasamicroenvironmentaldriverofgliomaprogressionandshowed thatintrinsicelectricalpropertiesofneuraltissuecontributetotumorbiology.
TamimiAF,JuweidM.EpidemiologyandOutcomeofGlioblastoma.In:DeVleeschouwerS,editor.Glioblastoma[Internet]. Brisbane(AU):CodonPublications;2017Sep27.Chapter8.Availablefrom:https://www.ncbi.nlm.nih.gov/books/NBK470003/ doi:10.15586/codon.glioblastoma.2017.ch8
1 StuppR,MasonWP,vandenBentMJ,WellerM,FisherB,TaphoornMJ,BelangerK,BrandesAA,MarosiC,BogdahnU, CurschmannJ,JanzerRC,LudwinSK,GorliaT,AllgeierA,LacombeD,CairncrossJG,EisenhauerE,MirimanoffRO; EuropeanOrganisationforResearchandTreatmentofCancerBrainTumorandRadiotherapyGroups;NationalCancerInstitute ofCanadaClinicalTrialsGroup.Radiotherapyplusconcomitantandadjuvanttemozolomideforglioblastoma.NEnglJMed.
2 2005Mar10;352(10):987-96.doi:10.1056/NEJMoa043330.PMID:15758009. Stuppetal.(2005)
3 TamimiAF,JuweidM.(2017)
4 Venkatesh,H.S.,Morishita,W.,Geraghty,A.C.,Silverbush,D.,Gillespie,S.M.,Arzt,M.,Tam,L.T.,Espenel,C., Ponnuswami,A.,Ni,L.,Woo,P.J.,Taylor,K.R.,Agarwal,A.,Regev,A.,Brang,D.,Vogel,H.,Hervey-Jumper,S.,Bergles,D. E.,Suvà,M.L.,…Monje,M.(2019).Electricalandsynapticintegrationofgliomaintoneuralcircuits.Nature,573(7775),539–545.https://doi.org/10.1038/s41586-019-1563-y
6
5 Venkateshetal.(2019)
ThedemonstrationthatgliomacellsformfunctionalsynapsespromptedHuangHobbsetal.’sinvestigationintowhetherneuronalactivitycouldinfluencetumor behavioracrosslongdistanceswithinthebrain.Huang-Hobbsetal.testedthis possibilityusingamousemodelofglioblastomawheretumorswereimplantedin onehemisphere.Theythenusedchemogenetictoolstoselectivelyactivatecallosal neuronslocatedintheopposite,tumor-freehemisphere.Contralateralneuronssend axonalprojectionsthroughthecorpuscallosumtoreachthehemispherecontaining thetumor.Activationofthedistantneuronsquickenedgliomaproliferationand invasion,whichdemonstratedthatthetumorrespondstoremoteneuralactivity. Toidentifythemediatorsoftheobservedremoteinfluence,theyperformedhighthroughputscreeningofgenesenrichedinthepopulationofgliomacellsfoundat theleadingedgeoftumorinvasion.Thisscreenidentifiedseveralaxonguidance molecules.SEMA4Fwasspecifiedasakeyregulator.Contrarytotheconventional viewofaxonguidancemoleculesasneuronalproducts,theyfoundthatgliomacells themselvesexpressSEMA4F,andthatthisexpressionincreasesinresponseto neuronalactivity.SEMA4Fsecretedbytumorcellsactslocallytoremodelsynapses inthesurroundingbraintissuewhichshiftthebalancetowardincreasedexcitatory transmissionandelevatedactivity.Thiscreatesaself-reinforcingcyclewhere remoteneuronalactivitystimulatesSEMA4Fproductioningliomacells,which thenamplifieslocalneuronalactivitythroughsynapticrestructuring.Supporting thefunctionalimportanceofthismechanism,aknockoutofSEMA4Finglioma cellsreducedbothtumorgrowthandinfiltration.Surgicaltransectionofthecorpus callosumeliminatedthegrowth-promotingeffectsofcontralateralneuronal activation,alsoconfirmingthatphysicalconnectivitybetweenhemispheresis requiredforremoteneuronstoinfluencetumorprogression.Theirexperiments establishedthatgliomasintegrateintobrain-wideneuralnetworks,respondingto neuronsintheirimmediatevicinityandalsotoactivitypatternsdistributedacross moredistantbrainregions.
Huang-Hobbs et al.
Huang-Hobbs,E.,Cheng,Y.T.,Ko,Y.,Luna-Figueroa,E.,Lozzi,B.,Taylor,K.R.,McDonald,M.,He,P.,Chen,H.C., Yang,Y.,Maleki,E.,Lee,Z.F.,Murali,S.,Williamson,M.R.,Choi,D.,Curry,R.,Bayley,J.,Woo,J.,Jalali,A.,…Deneen, B.(2023).RemoteneuronalactivitydrivesgliomaprogressionthroughSEMA4F.Nature,619(7971),844–850. https://doi.org/10.1038/s41586-023-06267-2
Sun et al.
7 Whilethefunctionalconsequencesofneuronalactivityongliomacellshadbeen establishedthroughsynapticandparacrinemechanisms,thecomprehensive anatomicalarchitectureunderlyingtheseinteractionsremainedincompletely characterized.Sunetal.mappedtheconnectivitybetweenpatient-derived glioblastomacellsandneuronsthroughoutthemousebrainusingviraltracing techniques.Theytransplantedpatient-derivedglioblastomaorganoidsthathad beengeneticallyengineeredtoexpresstheTVAreceptorandrabiesglycoprotein intoimmunocompromisedmice.Followingtumorengraftment,theyinjected EnvA-pseudotyped,replication-incompetentrabiesvirusengineeredtoexpress fluorescentreportersintothetumormass.Thevirusselectivelyinfectedcells expressingtheTVAreceptor,ensuringinitialinfectionremainedrestrictedto tumorcells.Thebrainthenunderwenttrans-monosynapticretrogradetracing, crossingbackacrosssynapsestolabelneuronsthatformdirectpresynaptic connectionsontothegliomacells.Thisapproachenabledcomprehensive identificationofallbrainregionssendingsynapticinputtotheglioma.Their analysisconcernedpatternsspanningbothlocalandlong-rangeconnections, includingcorticalareasandneuromodulatorycenters,suchasthebasalforebrain cholinergicsystemanddorsalrapheserotonergicneurons.Theconnectivityprofile variedwithtumorlocation,indicatingthatgliomacellsintegrateintopre-existing neuronalnetworksinamannerdependentonlocation,withdifferenttumor positionsexposingcellstodifferentactivity.Theirresearchprovidedtheanatomical foundationforunderstandinghowbrain-wideneuralactivityconvergesontothe tumormicroenvironmentandestablishedthatgliomacellsbecomeembedded withinstructuredcircuitarchitecture.
Krisnha et al.
Krishnaetal.directlytestedwhetherneuron-gliomaconnectivityinfluences patientoutcomesbymeasuringfunctionalconnectivityandcorrelatingitwith survivalinbothmousemodelsandhumanglioblastomapatients.Theyemployed magnetoencephalographywithacoherenceanalysis,aneuroimagingtechnique thatquantifiesconnectivitybetweenbrainregionswhilereducingthecommon artifactsfromvolumeconduction.Theirapproachallowedforanon-invasive measurementofintegrationbetweenthetumorandtherestofthebrain.Usingthis method,theywereabletoidentifyregionswithintumorsthatexhibitedhigh functionalconnectivity(HFC)versuslowfunctionalconnectivity(LFC)withthe broaderneuronalnetwork.Theythenperformedsite-directedbiopsiesduring
surgicalresection,samplingtissuespecificallyfromHFCandLFCtumorregionsto LFCxenografts.Extendingthesefindingstohumanpatients,theyanalyzed preoperativemagnetoencephalographydatafromglioblastomapatientsand quantifiedthefunctionalconnectivitybetweeneachpatient'stumoranddistributed brainnetworks.Patientswhosetumorsexhibitedhigherfunctionalconnectivity showedsignificantlyshortersurvivaltimes,evenaftercontrollingforother prognosticfactors,includingage,tumorsize,extentofresection,andmolecular markers.Theconcordancebetweenmousemodelsandhumanpatientsestablished thatneuron-gliomafunctionalconnectivityhasdirectclinicalrelevanceforpatient outcomes.Theirworkstrengthenedthebiologicalandclinicalsignificanceof neuron-gliomainteractionsandsuggestedthatdisruptingneuron-glioma communicationmayrepresentapromisingtargetfornoveltherapeuticstrategies.
Research Goal
Thestudiesdescribedpriorestablishedthatneuronsformfunctionalsynapseswith gliomacellsandthatneuronalhyperactivitypromotestumorprogressionthrough synapticandparacrinemechanisms.Whilethesestudieshaveestablishedneurongliomasynapticintegrationanddemonstratedelevatedneuronalexcitabilitywithin aGBM-infiltratedcortexspecifically,acomprehensivespatialmappingofneuronal hyperactivityhasnotbeendone.Toaddressthisgap,ourstudyemployed immunohistochemicaldetectionofc-Fos,animmediateearlygenewhose expressionactsasaproxyforrecentneuronalactivity.Thec-Fosproteinisa transcriptionfactorthatdimerizeswithJunfamilyproteinstoformtheAP-1 transcriptionfactorcomplex,anditsexpressionisrapidlyinducedfollowing neuronaldepolarizationandcalciuminfluxthroughNMDAreceptorsandvoltagesensitivecalciumchannels.Thec-FosmRNAisdetectablewithinminutesof neuronalactivationandpeaksapproximately30to60minutesafterstimulation, whilec-Fosproteinlevelsreachtheirmaximumbetweenoneandthreehourspoststimulationbeforegraduallydeclining. Thismakesc-Fosimmunostaininga snapshotofneuralactivitypriortocollectionofthetissue.Thisallowsfordetection atsingle-cellresolutionforquantificationofactiveneuronsaccuratelywithin specificareas.Theprimarylimitationofc-Fosmappingisthesamerestrictiontoa singletemporalwindow,whichprecludesanalysisofactivityovertime. Additionally,c-Fosexpressiondoesnotdistinguishbetweenexcitatoryand inhibitoryneuronpopulationsandonlyallowsforcompositemeasuring. 8 9 10 11
8 Chung(2015)
ChungL.ABriefIntroductiontotheTransductionofNeuralActivityintoFosSignal.DevReprod.2015Jun;19(2):61-7.doi: 10.12717/DR.2015.19.2.061.PMID:27004262;PMCID:PMC4801051.
9 Kovács,K.J.(2008),MeasurementofImmediate-EarlyGeneActivation-c-fosandBeyond.JournalofNeuroendocrinology, 20:665-672.https://doi.org/10.1111/j.1365-2826.2008.01734.x
10 Kovács(2008) 11
Sunetal.,inmappingtheanatomicalconnectivitybetweenneuronsand glioblastomacells,promptedtheobjectiveofthisstudy.Thehypothesiswasthat brainregionsidentifiedbySunetal.assendingsynapticinputtogliomacells wouldexhibitincreasedneuronalactivity.Thiswastestedbycomparingc-Fos expressionbetweentumor-bearingandsham-injectedcontrolmiceacrossmultiple brainregions.Openfieldtestingrevealedananxiety-likephenotypeintumorbearingmiceatfourweekspost-injection,characterizedbyreducedcentertime. Centertimeisdefinedastheextentthemousespendswithinadefinedregionin thecenterofthefield.Thisbehavioralfindingdirectedadditionalattentiontobrain structuresinvolvedinanxietyprocessing,includingtheamygdala,duringc-Fos quantification.
Animals
Immunocompromisedalbinojuvenilemicewereusedforallexperiments.Mice wererandomlyassignedtoeitherthetumorgroup(n=10)orshamcontrolgroup (n=10).Followingsurgery,themicerecoveredfortwoweeksbeforebehavioral testingbegan.(Fig1)
[Fig.1:Timelineschematicshowingday0asthetumorimplantationtime,followedby openfieldtestinginweeks2&4,andthesubsequentperfusion,staining,andimagingin week4.]
Tumor Introduction
Themicewereanesthetizedwithketamine(50μL).Onceunresponsive,themice weresecuredinastereotacticframeandmaintainedonisoflurane.Theireyeswere lubricatedtopreventdryingduringtheprocedure.Thesurgicalsitewassterilized withalternatingbetadineandethanol.Averticalincisionwasthenmadealongthe midlineofthescalp,and10%hydrogenperoxidewasappliedtodissolve connectivetissueandvisualizeskulllandmarks.Thebasiccoordinateswereset usingbregmaandlambdaaslandmarks.ANanojectsystemwasprefilledwith mineraloil,thenloadedwithtumorcellsuspension.Asmallholewasdrilledat coordinatestargetingthesomatosensorycortex.TheNanojectwasloweredtothe injectionsiteandtumorcellsofline10072weredeliveredinfoursequential injectionstotaling100μL.Controlmiceunderwentidenticalprocedureswith HibernateAvehiclesubstitutedfortumorcells.Theincisionwasclosedwith suturesandmiceweremonitoredduringrecovery.
Open-Field Behavioral Testing
Anxiety-likebehaviorwasassessedusingtheopenfieldtestattwoandfourweeks post-surgery.Theopenfieldparadigmmeasuresananimal'swillingnesstoexplore anexposedcentralareaversusremainingnearthewallsofanarena,abehavior knownasthigmotaxis. Micewithheightenedanxietytendtoavoidthecenterof thearenaandspendmoretimealongtheperiphery.Foreachsession,amousewas placedinthearenaandallowedtoexplorefreelyfor10minuteswhileanoverhead camerarecordeditsmovement.Testingwaslimitedtotwosessionspermouseto preventhabituationtothearena.Mousepositioncoordinateswereextractedfrom thevideorecordingsusingDeepLabCut,amachinelearning-basedposeestimation toolthatidentifiesandtracksanatomicallandmarksacrossframes.Centertime, definedasthecumulativedurationspentinthecentralzoneofthearena,servedas theprimarymeasureofanxiety-likebehavior.
Brain Processing
Themicewereperfusedtranscardiallyat4weekswithphosphatebufferedsaline (PBS)followedby4%paraformaldehyde.Brainswerepost-fixedin4% paraformaldehydeonarotator,thentransferredtosucroseuntilsaturated.Brains weresectionedcoronallyat40μmthicknessmanually.Everysixthsectionwas collectedintoantifreezemediumandstoredinsix-wellplates.
Free-floatingsectionswereincubatedin0.3%TritonX-100inPBSfor1-2 hoursatroomtemperature,thenblockedfor1houratroomtemperaturein blockingbuffercontaining1%mouse-on-mouseblockingreagent.Thesections werethenincubatedovernightat4°Cwithprimaryantibodiesagainstc-Fos (rabbit,1:500)andhumannestin(mouse,1:500).FollowingthreewashesinTBST, thesectionswereincubatedfor1-2hoursatroomtemperaturewithAlexaFluor 488-conjugateddonkeyanti-rabbitandAlexaFluor555-conjugateddonkeyantimousesecondaryantibodies(1:500),withDAPIincludedfornuclear counterstaining.AfterthreeadditionalwashcyclesinTBST,thesectionswere mountedonslideswithFlouromount.
FluorescentimagingwasdoneonaZeissLSM800confocalmicroscopewitha DefiniteFocus2.Whole-sectiontilescanswereimagedat10×magnificationusing the488nmlaserlinetovisualizec-Fosimmunoreactivity.Thelow-magnification imagesprovidedbrain-widecoverageforregionalquantificationandatlas alignment.Forcloserexaminationoftheamygdala,highermagnificationimages weredoneat20×usingboththe405nmchannelforDAPInuclearstainingandthe 488nmchannelforc-Fos,allowingquantificationofc-Fosexpressionatcellular resolutionwithintheregion.
Image Processing Pipeline
Rawimagefiles(.czi)acquiredfromtheZeissLSM800confocalmicroscopewere processedusingFiji(ImageJ).Theywerethencompressedviaaz-stackand
maximumintensityprojection.Brightnessandcontrastwereadjustedforeasier alignment.Acustomscriptthenstandardizedimagedimensionsandconvertedfiles to.tifformat.ProcessedimageswereimportedintoQuPath(v0.5.1)andloaded intotheABBAplugin(v0.10.4)viaFijiforregistrationtotheAllenMouseBrain CommonCoordinateFramework(CCFv3).Eachtissuesectionunderwentaffine transformationandwasmanuallyassignedtoitscorrespondinganterior-posterior positionbasedonanatomicallandmarks.Thesectionswerethenwarpedtoits matchedatlasreferenceusingmulti-pointdeformationtoalignitsbiological structurestoatlas-definedregionalboundaries.Registeredsectionswerethen
Tumor Engraftment
Humannestinimmunostainingwasperformedtoverifysuccessfulengraftmentof patient-derivedglioblastomaorganoidsintumor-injectedmice.Allmiceinthe tumorgroupdisplayednestin-positivestainingattheinjectionsite,confirmingthe presenceofhumantumorcellsatfourweekspost-implantation.Tumorswere centeredinthesomatosensorycortexatthetargetedcoordinates,withvariable degreesoflocalinfiltrationintothesurroundingbrainparenchymaobservedacross animals.Theextentoftumorspreadvariedbetweensubjects,althoughitwas functionallyconsistent.ControlmicethatreceivedshaminjectionswithHibernate Avehicleshowedcompleteabsenceofnestinimmunoreactivity,confirmingthat nohumancellswerepresentandthatanydifferencesobservedbetweengroups couldbeattributedtotumorpresenceratherthansurgicalprocedurealone(Fig.2).
Anxiety-Like Behavior Emerges by Week 4
Toassesswhethertumorpresenceaffectedanxiety-relatedbehavior,micewere evaluatedusingtheopenfieldtestattwoandfourweekspost-surgery.This paradigmquantifiesananimal'stendencytoavoidtheexposedcenterofanarenain favoroftheperiphery,abehaviorindicativeofanxiety-likestatesinrodents. Centertimeservedastheprimarydependentmeasure,withreducedcentertime reflectingincreasedanxiety.
[Fig.2:20xofacoronalsectionshowinghumannestinimmunostaining(red)intumorbearingmousebrain.DAPInuclearcounterstain(blue).]
Atweek2post-injection,tumor-bearingandcontrolmiceshowedno significantdifferenceincentertime(p>0.05).Bothgroupsspentcomparable amountsoftimeinthecenterzoneofthearena,suggestingthatearly-stagetumor developmentdidnotproducedetectablechangesinanxiety-likebehavior.Byweek 4,however,tumor-bearingmiceexhibitedamarkedreductionincentertime comparedtocontrols,andthisdifferencereachedstatisticalsignificance(p<0.05). Tumormicedisplayedpronouncedthigmotaxis,spendingmuchofthesessionin theperipheralzoneadjacenttothearenawallswhileavoidingthecenter.
Todeterminewhetherthisbehavioralchangereflectedaspecificanxiety phenotyperatherthangeneralmotorimpairmentorsicknessbehavior,total distancetraveledwasanalyzedatbothtimepoints.Therewasnosignificant differenceinactivitybetweentumorandcontrolgroupsateitherweek2orweek4 (p>0.05),indicatingthattumor-bearingmiceretainednormalambulatorycapacity. Theselectivereductionincentertimewithoutcorrespondingchangesinoverall movementsupportstheinterpretationthattumor-bearingmicedevelopedan i lik h h f k id i idi ih h
[Fig.3:Openfieldbehavioralanalysis.(a)Representativetrackingmapsshowingmouse movementtrajectoriesintheopenfieldarenaforcontrolmiceatweek2.(b)Total distancetraveledatweek2,control(right)andtumor(left).(c)Timeincenterzoneat2 weeks,control(right)andtumor(left).(d)Representativetrackingmapsshowingmouse movementtrajectoriesintheopenfieldarenafortumormiceatweek2.(e)Numberof crossestocenteratweek2,control(right)andtumor(left).(f)Totaldistancecoveredin centerzone,control(right)andtumor(left).(g)Representativetrackingmapsshowing mousemovementtrajectoriesintheopenfieldarenaforcontrolmiceatweek4.(h)Total distancetraveledatweek4,control(right)andtumor(left).(j)Representativetracking mapsshowingmousemovementtrajectoriesintheopenfieldarenafortumormiceat week4.(k)Numberofcrossestocenteratweek4,control(right)andtumor(left).(l) Totaldistancecoveredincenterzone,control(right)andtumor(left).(m)Representative trackingmapsshowingmousemovementtrajectoriesintheopenfieldarenaforcontrol miceatweek4.ns=notsignificant;p<0.05.]
Distinct Activity Between Tumor and Control
Mappingofc-Fosexpressionshoweddistinctdifferencesinactivitybetweentumor andcontrolgroups.Controlmicedisplayedc-Fosexpressionconsistentwith patternsexpectedfollowingopenfieldexposureandcageactivity.Thedistribution ofc-Fos+cellsincontrolanimalswasconsistentacrosssubjectsandcorrespondedto regionsknowntobeengagedduringexploratorybehaviorandspatialnavigation.
Tumor-bearingmiceexhibitedanaltereddistributionofc-Fosexpression comparedtocontrols.Importantly,thesechangeswerenotuniformacrossthe brain.Someregionsshowedelevatedc-Fosexpressionintumormice,whileother areasappearedcomparablebetweengroups.Theregion-specificpatterning suggestedthattumorpresenceselectivelymodulatedneuronalactivityinparticular braincircuitsratherthanproducingaglobalincreaseordecreaseinneural activation(Fig.4).
Seibenhener,M.L.,Wooten,M.C.UseoftheOpenFieldMazetoMeasureLocomotorandAnxiety-likeBehaviorinMice. J.Vis.Exp.(96),e52434,doi:10.3791/52434(2015).
[Fig.4:Brain-widec-Fosdistribution.(a)Singletumorsamplewithatlasregion boundariesoverlaid.(b)Representativecoronalsectionsatmatchedanterior-posterior levelsfromcontrol(left)andtumor-bearing(right)miceshowingc-Fosimmunoreactivity density.]
Regional Quantification Identifies Amygdala
Hyperactivity
Toquantifyregionaldifferencesinneuronalactivitybetweengroups,automated celldetectionwasperformedinQuPathusingauniformintensitythresholdapplied acrossallsamples.Thenormalizedpositivepercentageofc-Fossignalwas calculatedforeachatlas-definedbrainregion,andvalueswerecomparedbetween tumorandcontrolgroups.
Quantitativeanalysisacrossmultiplebrainregionsrevealedthattheamygdala showedthelargestandmostconsistentelevationinc-Fosexpressionintumorbearingmice.Thenormalizedc-Fos+percentageintheamygdalawassignificantly greaterinthetumorgroupcomparedtocontrols(p<0.05).Thisfindingwas consistentacrossmiceandwasobservedbilaterally,thoughthedatawasanalyzed withthebrainhemispheresparsedseparatelytoassesspotentiallateralizationeffects.
Incontrasttotheamygdala,mostotherbrainregionsexamineddidnotshow statisticallysignificantdifferencesbetweentumorandcontrolgroups.Theregions wequantifiedincludedhippocampalsubfields,somatosensoryandmotorcortices, thalamicnuclei,andvariousposteriorbrainstructures.Whilesomeregionsshowed trendstowardincreasedordecreasedc-Fosexpressionintumormice,specifically thesomatosensorycortexinthecontrol,thesedidnotreachstatisticalsignificance
atthep<0.05threshold.Thespecificityoftheamygdalafindingsuggeststhat tumor-inducedchangesinneuronalactivityareconcentratedincircuits,potentially thosemostrelevanttothebehavioralphenotypeobserved(Fig.5).
[Fig.5:Regionalc-Fosquantification.Graphshowingnormalizedc-Fos+percentagein theamygdalaforcontrol(left)versustumor(right)groups.GraphshowingnormalizedcFos+percentageintherightlateralizedamygdalaforcontrol(left)versustumor(right). p<0.05,unpairedt-test.Datapresentedasmean±SEM.]
Brain-widevisualizationofc-Fosexpressionpatternswasgeneratedusing BrainrendertomapthenormalizedpositivepercentagesontotheAllenCCFv3 atlas.Heatmaprepresentationrevealedtheregionalspecificityoftumor-induced changes,withtheamygdalaprominentlyhighlightedasasiteofelevatedactivityin tumor-bearinganimals.Thisvisualizationprovidesacomprehensiveoverviewof thespatialdistributionofneuronalactivitychangesassociatedwithglioblastoma presence(Fig.6).
[Fig.6:Heatmapvisualizationofbrain-widec-Fosexpression.Three-dimensional renderingoftheAllenCCFv3atlaswithregionscoloredbynormalizedc-Fos+percentage forcontrol(left)andtumor(right)groups.Colorscaleindicatesexpressionlevel. GeneratedusingBrainrender.]
Highermagnificationconfocalimagingoftheamygdalawasperformedtoexamine c-Fosexpressionatcellularresolution.Imagesacquiredat20×magnificationwith bothDAPInuclearcounterstainandc-Fosimmunofluorescenceconfirmedthe presenceofelevatedc-Fos+celldensityintumor-bearingmicecomparedto controls.Individualc-Fos+nucleiwereclearlyidentifiable,andtheincreasedsignal intumoranimalscanbeseeninrepresentativeexamplesections(Fig.7).
[Fig.7:20x(b,d)and10x(a,c)fluorescenceimagesoftheamygdalafromcoronalbrain sectionsshowingc-Fosactivity(green)withDAPInuclearcounterstaining.Control(c,d) andtumor(a,b).]
Discussion
Theamygdalaisthecentralnodeinmediatingfearandanxiety-relatedbehaviors, andhyperactivityspecifictotheareahasbeenlinkedtostatesofanxietyacross species.Theco-occurrenceofamygdalahyperactivityandincreasedthigmotaxisin tumor-bearingmicesuggeststhatglioblastomamaydriveanxiety,asitmaybeable todowithotherregions,throughaberrantactivationoflimbiccircuitry.Whether thisrepresentsadirecteffectofthetumor-causedsignalsoranindirect
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consequenceofalteredsignalingfromotherbrainregions,orevenaresponseto othersystemicfactorsassociatedwiththetumorremainstobedetermined. Thesefindingsextendthepriorworkcharacterizingneuron-glioma interactions.Venkateshetal.demonstratedthatneuronsformfunctional glutamatergicsynapsesontogliomacellsandthatneuronalactivitypromotestumor proliferation. WhereasSunetal.identifiedwhichneuronsprojecttothetumor, ourcurrentworkaddresseswhichregionsexhibitalteredactivity. The combinationofanatomicalconnectivityandfunctionalhyperactivitywould supportthehypothesisthatneuron-gliomasynapticintegrationproduces measurablechangesinbrainactivitypatternsandpotentiallyanimalbehavior.
AnxietyandaffectivedisturbancesareprevalentinGBMpatients,though theiretiologyinthispopulationisconsideredmultifactorialandsoincompletely understood.Boeleetal.reportedaprevalenceofanxietyrangingfrom24to48% ingliomapatientsassessedbyself-reporting,withthesesymptomscarrying significantimpactonqualityoflifeandoverallsurvival. Thecontributingfactors aregenerallyunderstoodtoincludeacombinationofpsychologicalresponsesto diagnosisandtreatmentaswellasneurophysiologicalchangesassociatedwith tumorpresenceandprogression.Theopenfieldtestweusedmeasuresthigmotaxis, whichisawell-establishedindexofanxiety-likebehaviorinrodents,andthe behavioraldifferencebetweentumor-bearingandcontrolmiceemerged specificallyatweekfourwithoutcorrespondingchangesintotallocomotion, supportinginterpretationasananxiety-likephenotyperatherthangeneralsickness behaviorormotorimpairment.Whethertheamygdalahyperactivityobservedhere contributestothatbehavioralphenotype,andwhetheranalogouscircuit-level changesarepresentinhumanGBMpatientswithanxietysymptoms,arequestions ourdataraisebutcannotresolve.Thetranslationalrelevanceofopenfield thigmotaxistoclinicalanxietyinhumanpatientsislimited,andanyconnection betweenthemousefindingsandthehumanliteraturewouldrequiredirect investigationinpatientpopulations.
Severallimitationsconstraintheinterpretationofourfindings.Ourstudy usedc-Fos,which,first,onlycapturesasinglewindowofneuronalhyperactivity andcannotresolvechangesacrosstime,andsecond,makesdistinguishing inhibitoryandexcitatoryneuronsimpossible.Observingthecorrelationbetween amygdalaandhyperactivityandanxiety-likebehaviordoesnotestablishcausality, asthehyperactivitymaydrivethebehavioralphenotype,resultfromit,orarise fromaseparatefactor.Thestudyutilizedimmunocompromisedmiceforless omplicationswithtumorintroduction,howeverthechoicemayinfluencetumor
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Venkateshetal.(2019) 13 Sunetal.(2025) 14 Boele,F.W.,Klein,M.,Reijneveld,J.C.,&Heimans,J.J.(2023).Depressionandanxietyingliomapatients.Neuro-Oncology Practice,10(4),335–344.https://doi.org/10.1093/nop/npad013
biologyandneuralfunctioncomparedtoimmunocompetentmice.Thesample size,whileadequatefordetectingtheamygdalaeffect,mayhavebeentoosmallto detectsmallerdifferenceinotherregions.Additionally,GBMisinfiltrative,and tumorsspreadvariablyfromtheinjectionsiteacrossanimals.Differencesinthe eventualtumorlocationandextentofinfiltrationcouldinfluencewhichbrain regionsareaffectedandmaycontributetovariabilityinc-Fosexpressionacross mice.Storingthebrainsectionsfree-floatingincryoprotectantmakes distinguishingtheorientationofhemispheresimpossiblebeforemounting, meaningthelateralaccuracyoftheheatmapcouldsufferifaspecificsectionwas hardtodefineasflippedoraccuratelyalignedwhenimaging. Futureresearchshouldtestwhetheramygdalahyperactivitydrivestumor progressionorreflectsthedisruptioncausedbythetumor.Specifically, chemogeneticsilencingofamygdalaneuronswouldaddressthisdirectly.If silencingtheamygdalareducesbothanxietybehaviorandtumorgrowth,this wouldsuggesttheamygdalatobeofevenmoreinterest.GiventhatSunetal. identifiedwidespreadconnectivityfromdiversebrainregionstoglioblastomacells, theamygdalamaycontributetoalargercircuitarchitecturethatcollectively regulatestumorbehavior.
Thetimingofamygdalahyperactivityrelativetoanxietysymptomsandtumor growthisalsounresolved.Ourstudycapturedactivityatfourweekswhen behavioralphenotypeshadappeared,butwedonotknowwhetheramygdala activationprecedesanxietyordevelopsalongsideit.Longitudinalc-Fosmappingat earliertimepointswouldshowthetemporalsequenceofevents.Weekoneortwo mappingmaypotentiallyshowwhichregionsactivatefirstduringtumor developmentandwhetheramygdalahyperactivitypredictssubsequentbehavioral changes.Beyondtheamygdala,usinganothermethodofneuronalactivity monitoringwithalargersamplesizemaybeabletodeducethefunctioneffectsof glioma-brainsynapsesmoreconfidentlyandconsistently.
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