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AnIntegratedIndex:Engrams, PlaceCells,andHippocampalMemory

TravisD.Goode,1,2,3,4,7 KazumasaZ.Tanaka,5,7 AmarSahay,1,2,3,4,* andThomasJ.McHugh6,* 1CenterforRegenerativeMedicine,MassachusettsGeneralHospital,Boston,MA02114,USA 2HarvardStemCellInstitute,Cambridge,MA02138,USA 3DepartmentofPsychiatry,MassachusettsGeneralHospital,HarvardMedicalSchool,Boston,MA02114USA 4BroadInstituteofHarvardandMIT,Cambridge,MA02142,USA

5MemoryResearchUnit,OkinawaInstituteofScienceandTechnologyGraduateUniversity,Onna-son,Kunigami-gun,Okinawa,Japan 6LaboratoryforCircuitandBehavioralPhysiology,RIKENCenterforBrainScience,Wakoshi,Saitama,Japan 7Theseauthorscontributedequally *Correspondence: asahay@mgh.harvard.edu (A.S.), thomas.mchugh@riken.jp (T.J.M.) https://doi.org/10.1016/j.neuron.2020.07.011

SUMMARY

Thehippocampusanditsextendednetworkcontributetoencodingandrecallofepisodicexperiences. Drawingfromrecentanatomical,physiological,andbehavioralstudies,weproposethathippocampalengramsfunctionasindicestomediatememoryrecall.Webroadenthisideatodiscusspotentialrelationships betweenengramsandhippocampalplacecells,aswellasthemolecular,cellular,physiological,andcircuit determinantsofengramsthatpermitflexibleroutingofinformationtointra-andextrahippocampalcircuitsfor reinstatement,afeaturecriticaltomemoryindexing.Incorporatingindexingintoframeworksofmemoryfunctionopensnewavenuesofstudyandeventherapiesforhippocampaldysfunction.

Introduction

AfewkeywordstypedintotheGooglesearchbarwill,moreoften thannot,immediatelyleadtotheexactpieceofinformationwe areseeking.Whilethedetailsofhowthismagichappensareproprietary,thegeneralideaistransparent;Googlehasmanagedto indexvastswathsoftheinternetandusesoursearchtermsto quicklypointtothemostappropriateinformation(https://www. google.com/search/howsearchworks ).Thesystemissurprisinglyflexible,usinghistory,context,orlocationtohoneresults; completingoranticipatingpartialbitsofinformation;andfinding andseparatingsimilaritemsbydetectingsmalldifferences. Theseproperties,whichunderliebothitsefficiencyandpopularity,echotheabilitiesofthememorysystemsoperatingin ourownbrains,particularlytheepisodicmemorycircuitsdependentonthehippocampus(Squireetal.,2004; Tulving,2002).The hippocampusiscrucialfortheencodingofmemory,itisadeptat integratingandinterpretingcontextualcuestodriverecall,andit isefficientatbothdiscriminationandassociation(Marenetal., 2013).Thus,muchlikehowGoogleworksasanindexofinformation,oneparsimoniousexplanationforhippocampalfunctionis thatitfunctionsasanindexofmemories(Guoetal.,2018; Miller andSahay,2019; Tanaka,2020; TanakaandMcHugh,2018; Tanakaetal.,2018; Tonegawaetal.,2018).Thisisnotanew idea(McClellandetal.,1995; TeylerandDiScenna,1985, 1986; TeylerandRudy,2007);however,recentworkhasbegun tolenddirectexperimentalevidencetothistheoryandedifying putative,underlyingcircuitmechanisms.Here,wewillexplore andexaminethesefindingsindepthanddiscusspossibleindexingmechanisms,aswellashowtheseideascouldshapeabetter understandingofmemoryprocessesinboththehealthyand diseasedbrain.

SpaceandMemory

Thediscoveryofplacecells,neuronsinthehippocampusthat havereceptivefieldstunedtodiscretelocationswithinacontext (O’KeefeandDostrovsky,1971),revolutionizedtheexperimental approachtostudyinghippocampalfunction(Moseretal.,2017). Oneoftheearliestandmostinfluentialtheoriestoemergelinking placecellactivityandepisodicmemorywasthecognitivemap theory(O’KeefeandNadel,1978),positingthattheprimary roleofthehippocampusistoprovideaspatialframeworkthat permitsthelocationandassociationoftheitemsandevents thatconstituteagivenexperience.Whiletheauthorssuggested thatthiscognitivemapmaynotbelimitedtophysicalspaceand couldbeappliedtomapepisodicexperiencemorebroadly, placecellsandtheirpropertieshaveprovedausefulsubstrate toexamineandtesttheseideas.Forexample,asdifferentensemblesofneuronsarerecruitedtorepresentdifferentspatial experiences,thehippocampuscouldcontinuouslyprovidea newunderlyingscaffoldingacrossspace(andtime)thatwould allowmemoriestoremainbothrelatedanddistinct.Subsequent retrievalcouldthenbetriggeredbyareinstatementoftheoriginal spatialmaptriggeredbythecuesthatdefineagivencontext(WikenheiserandRedish,2015).Overtime,othertheoreticalmodels havebuiltonandexpandedtheseideas,and,asnotedinother sectionsbelow,haveproposedanatomicalsubstratesforhippocampalfunctionsthatincludenoveltydetection(LismanandOtmakhova,2001; Vinogradova,2001),rapidencoding,and patterncompletionandseparation(KesnerandRolls,2015; KnierimandNeunuebel,2016; NadelandMoscovitch,1997), aswellastheirrelationtospatialcoding.Moreover,frameworks thatencompasstheplacecelldatabutarenottiedtoaspecific spatialfunctionofthehippocampushavealsobeendescribed.

Thesepositthatplacecellsmayreflectabroaderfunctionality relatedtogeneral-purposesequencegeneration(Buzsakiand Tingley,2018)orrelationalmemory(CohenandEichenbaum, 1993; Eichenbaumetal.,1994),allowingtheextensionofboth hippocampalmemoryandphysiology(Aronovetal.,2017; MacDonaldetal.,2011; Pastalkovaetal.,2008)beyondthedomain ofphysicalspace.

Handinhandwiththegrowingcharacterizationofplacecell physiology,theredevelopedadeeperunderstandingofthe anatomical,behavioral,andcomputationalpropertiesofthehippocampalcircuit.Thisresultedinspecificmnemonicfunctions beinglinkedtotheanatomicandphysiologicalpropertiesof discretesubregionsofthestructure(FanselowandDong,2010; Nadeletal.,2013; Strangeetal.,2014).Inthisframework,the classicmodelofsequentialprocessingalongthetrisynaptic loophasthelargenumberandsparseactivityofgranulecellsin thedentategyrus(DG)providingorthogonalizationofsimilar corticalinputsleadingtopatternseparation(HainmuellerandBartos,2020; LealandYassa,2018; McHughetal.,2007),theDG providinginputtotherecurrentCA3networktofacilitateautoassociationandpatterncompletion(Cayco-GajicandSilver,2019; KesnerandRolls,2015; KnierimandNeunuebel,2016; McHugh etal.,2007; Nakazawaetal.,2002),andfinally,CA1broadcasting theresultsbacktothecortex(SolteszandLosonczy,2018; Valero anddelaPrida,2018).Thisframeworkhasservedasthebackboneofrelatingplacecellactivitytomemoryprocessing,insofar asplacecellsmaycoordinatethepatternseparation,completion, andreinstatementpropertiesnotedabove.

Buildingonthisframework,advancesingeneticapproaches haveledtoactivity-dependentmemorytaggingsystemsin rodentmodels,whichallowfortheexaminationandartificialreactivationorinhibitionofdistinctmemorytraces(Josselynand Tonegawa,2020).Thesetracesfulfillthepropertiesofthememoryengram,amonikerforthephysicalbasisofmemoryfirstproposedbyzoologistandbiologistRichardSemon(Schacteretal., 1978; Semon,1921),inthattheycanbeviewedasthephysical instantiationofanexperienceregisteredinenduringchanges insynapticconnectivityandphysiologyofanensembleof neurons.Taggingsystemsemployedincludethetetracyclineregulatedtranscriptionalactivationsystem,inwhichtime-locked expressionofactuatorssuchasopsinsorchemogeneticreceptorsareinducedviaactivity-dependentpromoters(e.g.,c-FosorArc-expressing; Liuetal.,2012)orimmediateearlygene (IEG)-bindingelements(Sunetal.,2020),aswellastheCreERT(estrogenreceptorT2)transcriptionsystem(targeted recombinationinactivepopulations[TRAP]mice),whichutilizes atamoxifen-sensitivemodifiedestrogenreceptortodriveexperience-drivenexpressionofCre-dependentconstructsinactivatedcells(Guenthneretal.,2013).Suchmethodshaveallowed fortheartificialtriggeringofmemory-relatedbehaviorevenin contextswherenosuchbehaviorwouldbeexpected.While thesemethodsarenotwithouttheircaveats(discussedfurther insectionsbelow),engram-labelingstudieshaveshownthatoptogeneticorchemogeneticstimulationorinhibitionofexcitatory neuronsintheDG(e.g., Guoetal.,2018; Lacagninaetal.,2019; Liuetal.,2012),CA3(e.g., Dennyetal.,2014),orCA1(e.g., Ghandouretal.,2019; Ryanetal.,2015; Tanakaetal.,2014)reinstatesorimpedes(respectively)behavioralrecallofthatexpe-

Perspective

rience.Whilemanyengramstudieshavefocusedonmeasures ofconditionedfear,theseandotherstudiesreporthippocampal engram-drivenbehaviorforavarietyofcontext-specificbehaviors,includingplace(e.g., Ramirezetal.,2013)orsocialavoidance(e.g., Zhangetal.,2019),aswellasappetitiveconditioning andplacepreference(e.g., Redondoetal.,2014).

Thesefindingsraiseseveralkeyquestions.First,howcana smallnumberofexperience-taggedhippocampalengramcells, muchfewerinfractionthanthatofactiveplacecells(e.g., Tanaka etal.,2018),encodeacomplexbehavioralexperience?Indeed, activationofaverysmallpercentage(2%–3%)ofDGgranule cellslabeledduringlearningcanreproducecontext-appropriate behaviors(forexamples,see Liuetal.,2012).Further,activation intheDGcouldharnessthepatterncompletionabilitiesofthe downstreamCA3networktoamplifytheiractivityviaattractor dynamics(Colginetal.,2010; KnierimandNeunuebel,2016) andleadtoarobustbrain-widereinstatementofamemoryrelatedensemble.However,reactivationofasubsetofCA1 neurons,whichlackrecurrentconnectivity,canalsotrigger behavioralreinstatementandpresumablymemoryrecall(e.g., Ryanetal.,2015).Itisplausiblethatdownstreamactivationof theentorhinalcortex(EC)and/orre-entrantexcitationofthe DGandCA3addthesefeatures,therebyfunctioninglikearecurrentnetwork,althoughexperimentalevidencesupportingthis interpretationislacking.

Additionally,50yearsofhippocampalphysiologyinrodents hasrevealedthatplacecellactivityisexquisitelystructured acrossnotonlyspacebutalsotime(HowardandEichenbaum, 2015).Duringexploration,thedominantthetaoscillationinthe hippocampallocalfieldpotential(LFP)organizesensemblesof placecellswithspatiallyadjacentreceptivefieldsintosequences,expressedonthetimescaleofasinglethetacycleof 125ms(BurgessandO’Keefe,2011).Thesesequencescan bere-expressedduringsharp-waveripples(SWRs)thatoccur duringpausesinmovementonanevenshortertimescale,compressedintofasteventslastingonly10sofmilliseconds(Foster, 2017).Thisprecisetemporalarrangementofactivityhasmade thegapbetweenplace-cell-andengram-basedmemorystudies difficulttobridge,asthelatterhavedemonstratedthat simultaneousoptogeneticactivationofensemblesofneurons intemporalandspatialpatternsthatarenotobservedundernaturalphysiologicalconditionsaresufficienttoevokebehaviors mimickingmemoryrecall.Onecaninterpretthisgapinthetemporaldynamicsbetweenopticallyinducedbehavioralreinstatementandplacecellactivityasreflectingthedispensabilityof thesetemporalpatternforbehaviorsdrivenbycontextualrecognition,orperhapsthisdisconnectcouldsimplybeduetotechnicallimitationsintheplacecellrecording,astheretrievalofa hippocampal-dependentmemorycanoccurveryrapidlyandin theabsenceoftheexplorationneededtodriveextensiveplace cellactivity,precludingarobustsamplingofactivity.For instance,whenrodentsreceiveafootshockimmediatelyafter placementinapreviouslyexposedchamber,contextexplorationmaybeminimal,yetanimalssuccessfullyretrievethe contextualmemoryandassociateitwithshock,resultingin context-dependentbehaviorduringsubsequentmemorytests (Wiltgenetal.,2001).Onepossibilityisthatreinstatementof evenasingleplacefieldissufficientformemoryreinstatement.

Figure1.ComparingandContrastingHippocampalPlaceCellsand Engram-Tagged(ImmediateEarly-Gene-Expressing)Neurons Placecellactivityhasprecisetemporalstructureduringbothexplorationand rest,whereasengram-taggedneuronsaresimultaneouslyandexperimentally reactivated.Placecelldensityappearsmoderate,andthisdensityofactive placecellsisrelativelystableinanygivencontext.Conversely,engram-tagged cellsinthehippocampusaresparse,withfamiliarcontextsexhibitinglow levelsoftaggedexpression.Engramcellsexhibitconsiderablylessstabilityin theircontext-dependentreinstatementovertimeascomparedtoplacecells, althoughbotharehighlyunstablewithtime.Remotetimepointsforreactivationofexperience-dependentengramcellsremainunknown.While thereareoverlappingbehavioralcorrelatesofplaceandengramcellactivity, placecellresearchhasledthefieldinitscorrelationtobehavior.

However,long-termmonitoringofthestabilityofplacecell representationsacrossrepeatedvisitsonthetimescaleof weeks,nowpossibleduetoadvancesin invivo imagingapproachesinmice,suggeststhatthereexistsahithertounappreciatedhighdegreeofinstabilityinthespatialrepresentationofa familiarenvironment(Zivetal.,2013;but,alsosee(Gonzalez etal.,2019).Ifonlyafraction( 15%)ofplacecellsshowstability

acrossseveralweeks,itbecomesmoredifficulttodrawadirect connectionbetweenmemoryrecallandacompletelystable spatialrepresentation.Itispossiblethatasmallfractionofstable placecellscanserveasapartialcuetoreinstatethefullrepresentationofmemorythroughaprocessofpatterncompletion indownstreamregions;however,thisviewischallengedbya recentstudyexaminingthephysiologicalnatureofengramcells inthehippocampus,whichisdiscussedbelow(Tanakaetal., 2018).Thus,whileplacecellstudieshaveprovidedinsightinto theanatomicalorganizationandpotentialmemorymechanisms ofthehippocampus,we,likemanyothers,struggletoreconcile thesepotentialspatialcodingpropertieswiththecoreroleofthe systemasamemorystoragedevice(Tanaka,2020).Further,itis important,boththroughhypothesisandexperiments,toattempt toidentifytherulesoftransformationthatallowsimultaneous activationofhippocampalengramstogenerateappropriatepatternsofdownstreamactivationandbehavior(Lismanetal., 2017).Perhapsthen,weshouldreconsiderwhattheactivityof hippocampalneuronsduringmemoryformationandrecalltruly representsandhowplacecellsthathavedrivenmuchofthe thinkinginthefieldforthelast50yearscaninformusabout thehippocampusasamemorysystem(Figure1).

InstantiatingtheHippocampalIndex

Thehippocampalmemoryindexingtheorypositsthatthehippocampusdoesnot‘‘contain’’theepisodicmemoryitself;rather,it generatesacodeor‘‘index’’thatbindsneuronalactivitypatterns underlyinganexperientialevent,whichisstoredacrossdistributedneocortical(andpotentiallysubcortical)modules(Teyler andDiScenna,1985, 1986; TeylerandRudy,2007).Inother words,thehippocampusencodesalinkedrepresentationof brainactivityatthetimeofanexperienceorepisode,which cansubservesubsequentrecallviaactivationofthathippocampalrepresentation.Whatispresumedtomakethesepatternsof activityuniquefromotherexperience-inducedpatternsinthe brain,suchasensembleactivityinthesensorycortexactivated byastimulus,istheirconjunctiveandassociationalnatureand theabilityofthehippocampalensembletoreinstatetheoriginal spatialandtemporalpatternsofcortical/subcorticalactivityofan experience(McClellandetal.,1995; TeylerandRudy,2007). Importanttonoteisthattheindexingtheoryisnotmutually exclusivetothecognitivemaptheory.Instead,itsimplyremains agnostictowhat,ifanything,thehippocampalneuronsinvolved inmemoryindexingmustrepresentintermsofbehaviorallyrelevantinformation;spatialcodingwouldbeacceptableifthese neuronshadpropertiesconsistentwiththatofanindex,assummarizedandpresentedin Figure2.Inthefollowingandsubsequentsections,weelaborateonthesefeaturesanddiscuss howthebrain’scircuitarchitecturesupportsaviewofhippocampalfunctionthroughthelensofindexing.

EngramsasIndices

Numerousstudieshavenowshownthatphotoactivationofa sparsehippocampalengramdrivesIEGactivityinselectdownstreambrainregionsthoughttobeinvolvedintheoriginal learning(e.g., Ramirezetal.,2013, 2015; Royetal.,2017). Suchobservations,togetherwiththereinstatementofbehavior followingoptogeneticengramstimulation,supporttheidea thatthehippocampusiscapableofindexingandtriggering

memoryrecallbyreinstatinglearning-dependentactivityin memory-relatedextrahippocampalbrainsystems.However, increasedIEGinductioninextrahippocampalstructures followingartificialactivationofengram-bearinghippocampal cellsdoesnotnecessarilyindicatethatthesearethepreciseextrahippocampalneuronsinvolvedintheoriginallearning.Moreover,specificcontrols,suchasuntagged,context-exposed andnonreinforcedanimals,areoftenessentialtoaddressissues ofmemoryversusperformance;indeed,animalsmaybeableto usealternativelearningorgeneralizationstrategiestoachieve task-dependentbehavior(e.g., Wiltgenetal.,2010),evenin theabsenceofthehippocampus(fordiscussion,see Maren etal.,2013).So,whatistheevidenceforlearning-specificand hippocampus-dependentreinstatementofneuralactivity?To thisend,onestudyhasshownthatphotoinhibitionoflearningtaggedCA1pyramidalcellsresultedinthereductionoffear behaviorinashock-associatedcontextandthatthiscoincided withreducedreinstatementofc-Fosexpression,specificallyin otherc-Fos-taggedand,presumably,engram-bearingcortical andsubcorticalcellsofthebrain(Tanakaetal.,2014).Inaseparatestudy(Guoetal.,2018),itwasfoundthatcontextualfear

Perspective

Figure2.KeyFeaturesofHippocampal MemoryIndexingTheory

learningincreasedmossyfibersynaptic contactsoftaggedengram-bearingdentategranulecells(DGCs)withparvalbumin-positive(PV+)stratumluciduminhibitoryneurons(SLINs)toasignificantly greaterextentthanarandompopulation ofDGCs.Thisengram-dependent recruitmentofPV+ SLINsreturnedto pre-learninglevelswithtime-dependent memorygeneralization.Bygenetically enhancingthecouplingofengrambearingDGCswithPV+ SLINs,theauthorsincreasedfeed-forwardinhibition inDG-CA3circuitryandstabilizedthe hippocampalengram.Critically,thiswas showntoconferoptogeneticbehavioral reinstatementandcontext-specificreactivationofadistributedfearmemorytrace inhippocampal-cortical-subcorticalnetworksatremotetimepoints.Collectively, thesefindingsmirrornaturalrecall,insofarascontextualfearmemoryretrieval intheoriginallearningcontextisassociatedwiththespecificreactivationof learning-dependenttagginginthehippocampusandsomeextrahippocampaltargets.However,formaldemonstrationfor howhippocampusmayinfactcoordinate extrahippocampalreinstatementinan experience-dependentmannerisabsent.

Inlightofunderstandinghippocampal engramfunctionsthroughthelensofindexing,itisimportanttoemphasizethatalthoughbehavioral reinstatementdoesnotequatetoneuralreinstatement,the behavioraloutcomesofoptogeneticmanipulationsofhippocampalengramsappearexperiencedependent.Forexample, asnotedabove,stimulationofcontextualfear-conditioningtaggedcellsintheDGresultsinincreasedfreezinginasafe (noshock)context(e.g., Liuetal.,2012).However,ifsuchstimulationoftheDGoccursforneuronsthatweretaggedfollowing theextinctionoffearinashock-associatedcontext,thenthis manipulationresultsinreducedfreezinginashock-associated contextanddecreasedspontaneousrecoveryofcontextual fear(Lacagninaetal.,2019).Likewise,inhibitionofcontextfear-taggedDGcellsattenuatesfreezinginashock-associated context(Tanakaetal.,2014),butinhibitionofextinction-tagged DGcellscanincreasedefensiverespondinginapreviouslyextinguishedcontext(Lacagninaetal.,2019).Theseexperience-specificfindingsarecomplementedbyotherstudieswherethe behavioralresponse(beyonddefensivebehavior)ofDGengram reactivationreflectsthevalenceofthereinforcingstimuliassociatedthecontextorengram(e.g., Ramirezetal.,2015; Redondo etal.,2014).Also,considerthatsimplyreactivatingengramcells

thatweretaggedduringhomecageexplorationorexposuretoa contextinwhichshockneveroccurreddoesnotappearto induceabnormallocomotionorovertdefensivebehaviors(e.g., Ghandouretal.,2019).

Whileindexingmayexplainthecapacityforphotoactivationof taggedhippocampalengramcellstotriggermemory-specific behaviorindifferentcontexts,reinstatementofbehaviorisoften notablylessthanwhatwouldbeexpectedthroughnaturalrecall (i.e.,returningtheanimaltotheoriginaltrainingcontext).Inthe frameworkofindexing,weproposetherearenumberofreasons whythismaybethecasebeyondthefactthatnaturalrecallis presumablymosteffectiveinreactivatingtheindex.Importantly, theabovementionedtaggingsystems,whileexperimentallytime locked,arestillthoughttoopenawindowoftaggingthatmaybe ontheorderofatleastseveralhours.Thus,whenartificiallyreactivatingthesecells,itispossiblethattheexperimentermayalso betriggeringactivationofothernonspecificindicesand/or experiencessuchasactivityinthehomecageandpre-or post-traininghandling.Thesepatternsarenotspecifictotheprimarylearningepisodeinquestionandthusmaycompetefor behavioralexpression.Contextualstimulipresentinthetest contextmayalsotriggerinterferenceaswell,actingasexternal inhibitors.Thereby,anumberofcontrols(e.g.,nonreinforced, homecage)forbetterisolatingandassessingthedegreeof experienced-dependentbehavioralreinstatementshouldbe performed.Additionally,hippocampalindicesnotonlyareproposedtoencodetherelevantbrainsystemsactivatedduring anexperiencebutalsomayrepresentthesequentialpatterns ofsuchactivation(BuzsakiandTingley,2018).Currentmethods ofoptogeneticreactivationofhippocampalengramcellslack suchsequence-basedreactivation(Carrillo-Reidetal.,2019), beyondwhatisinherentlystructuredinthelinkageofhippocampalengram-bearingcircuits.Furthertechnologicaladvancements,whichmaybetterconstrainthewindowoftaggingtoa particularexperienceormaybeabletoreactivatecellsin sequence-dependentmanners,arecrucialtoimprovethereadoutsandinterpretationofthisreinstatement.

Isthehippocampusaloneinitspotentialcapacityforindexing?Associationcorticessuchasthesensoryassociationcortex andECmayalsoexhibitindexingpropertiesduetotheirconvergenceofsensoryinput,therebycontributingtoahierarchicalindexingscheme(McClellandetal.,1995).Thus,thehippocampus mayservetosomeextentasanindexofindicesintheECand otherinputstructuresasinformationisroutedinandthenback outagain.Assumingsuchhippocampalsignalscanbedecompressedtoreinstateactivityincorticalandsubcorticalnuclei (asnotedabove),anexactone-to-onerepresentationinthehippocampusofcorticalmodules(forexample)seemsunlikelyand maynotbenecessary.Infact,convergenceofneuralactivityinto thehippocampusmightbeessentialforitsabilitiestoform conjunctivecontextualrepresentations(RudyandO’Reilly, 1999).Othercriticaltargetsofthehippocampus,suchastheretrosplenialcortex(RSC)(Cowansageetal.,2014; Maoetal., 2018)orlateralseptum(LS)(Benderetal.,2015; Besnard etal.,2019; TingleyandBuzsa ´ ki,2018),mayalsomaintain suchconvergenceofprocessingandmaytherebybepartofa hierarchicalindexingscheme,assumingthesestructuresare capableofreinstatingpatternsofexperience-specificassembly

patterns.Indeed,onestudyfoundthatreactivationofcellsofthe RSCthatweretaggedatconditioningissufficienttoinduce behavioralexpressionoffear,evenintheabsenceofafullyfunctionalhippocampus(Cowansageetal.,2014).Importantly,this studyshowedthatoptogeneticactivationoftheRSCengram, likenaturalrecall,recruitedoverlappingdownstreamcircuitsin theamygdalaandEC,demonstrativeofreinstatementofexperientialactivity.Thus,indexingmaynotnecessarilybeuniqueto thehippocampus;however,thehippocampusmaybeuniquely positionedtoindexepisodicevents,giventhesignificantly greaterextenttowhichitintegratescomplexandhierarchical sensoryinformationfromacrossthebrain(seesectionsbelow), aswellasduetoitsdiscriminativecodingandcircuitarchitecture.Unpublishedfindingsindicatingthatreactivationof engram-taggedneuralstructures,outsidethehippocampus, doesnotequallyreinstatebehaviormaysupporttheparticular importanceofhippocampalindexing(Royetal.,2019).

MemoryIndexinginHumans?

Electrophysiologicalstudiesinhumanshavesuggestedthatthe humanhippocampusalsopossessespropertiesconsistentwith indexing.Forexample,inepilepticpatientswithdepthelectrodesimplantedintothemedialtemporallobe,freerecallof anaudiovisualexperiencewasshowntofollowtheselectivereactivationofhippocampalandECcellsthatwereactiveduring thepriorexperience(Gelbard-Sagivetal.,2008).Likewise, successfulretrievalinanobjectassociationtaskcoincided withreinstatementofspikingactivityinhippocampalandEC cells,hippocampalactivityprecedingECfiring,anddecoding analysesofECactivitypredictingtheidentityoftherecalledobject(Staresinaetal.,2019).Otherintracranialrecordingshave shownthatbehavioralrecallwaslinkedtocoordinatedhippocampal-lateraltemporalcorticalrepresentationalreinstatement ofitem-contextassociations(PachecoEstefanetal.,2019).In thisstudy,hippocampalreinstatementprecededthatseenin theneocortex,andhippocampal-corticalgammaphasesynchronyduringhippocampalreinstatementpredictedneocortical reinstatement.Moreover,thesefindingsaremirroredinadditionalstudiesthathavefoundmemory-relatedreinstatementin thehumanhippocampusisunderscoredbyasparseanddistributedsetofactivecells(Wixtedetal.,2014, 2018).

Humanfunctionalmagneticresonanceimaging(fMRI)studies supportasimilarinterpretation.Forexample,onefMRIstudy (Harandetal.,2012)reportedthathippocampalBOLD(bloodoxygen-level-dependent)activityduringanepisodiclearning experiencematcheditsactivityatrecall(i.e.,recognitionofpreviouslyshownvisualcues),particularlywhensubjectsreported therememberingofepisodicdetailsofthelearningevent.Interestingly,thisepisodicreinstatementofhippocampalactivity occurredforrememberedcuesat3daysandeven3months followinglearning.Moreover,forsuccessfulretrievalofexperientialmemory(intaskssuchasobjectrecallandrecognition), regionsincludingtheRSC,parahippocampalcortex(PHC),perirhinalcortex(PRC),andprefrontalcortex(PFC)haveallbeen showntoexhibitrecall-dependentreinstatementalongwithor inclosetemporalproximitytohippocampalreinstatement,suggestiveofhippocampal-dependentrouting(e.g., Arnoldetal., 2018; Jonkeretal.,2018; Schultzetal.,2019).Again,whilereinstatementandtemporalpatternsofactivationalonedonot

Figure3.Hippocampal(DG-CA3-CA2-CA1)CircuitArchitectureand AnatomicalLoopsPermitFlexibleIntegrationandRoutingof ExperientialInformation

(A)Examplesofhippocampalinputs(bluearrows). (B)Examplesofhippocampaloutputs(orangearrows).Notethattheprojectionsshownarenotexhaustive.

Brainregionsincludetheanteriorcingulatecortex(ACC),bednucleusofthe striaterminalis(BNST),basolateral/basomedialamygdala(BLA/BMA),central amygdala(CEA),dentategyrus(DG),dorsalraphenucleus(DRN),entorhinal cortex(EC),cornuammonisregions(CA1–CA3),infralimbiccortex(IL),locus coeruleus(LC),anterior/lateralhypothalamicarea(A/LHA),lateralseptum(LS), medialseptum(MS),nucleusaccumbens(NAC),orbitalfrontalcortex(OFC), prelimbiccortex(PL),nucleusreuniens(RE),retrosplenialcortex(RSC),subiculum(SUB),supramammillarynucleus(SUM),andventraltegmentalarea (VTA).BrainregionimagesweregeneratedusingBrainExplorer2.0(Lein etal.,2007).

demonstrateindexing,thesefindingsareconsistentwithdata fromrodentsandleaveopenthepossibilitythatfutureexperimentsmaydirectlytestthisideainhumans.

AnIntegratedCircuitModeloftheHippocampalIndex Inthedecadessincetheintroductionofthehippocampalmemoryindexingtheory,considerableadvanceshavebeenmadein ourunderstandingofthecomplexityanddiversityofhippocampalcircuits.Ifexperience-taggedhippocampalengramsserve asepisodicindices,howmightthecircuitarchitectureofthe brainbeemployedforencodingandrecall?Totheseends, theconjunctive,sparse,andcompressedcodegeneratedin theDGviapatternseparationwouldsupportindexingbyminimizingmemoryinterference(Figure2,featurei)(Cayco-Gajic andSilver,2019; HainmuellerandBartos,2020; Knierimand Neunuebel,2016; McHughetal.,2007),whileDGoutputs, togetherwithdirectECinputs(alongsidethediverseafferents describedbelow),ontoCA3cellswouldbiasattractordynamics intherecurrentnetworktostoreanexperienceasanewmemory,orcatalyzetheretrievalorupdatingofapreviouslyencoded memorybypatterncompletion(Figure2,featuresii–iv).Accordingly,theexperienceisregisteredinasparsehippocampalcode orengramcomposedofprincipalcells(andinhibitoryneurons [INs])acrossthedifferentsubregions(DG,CA3,CA2,and CA1),withtheircoordinatedactivitypermittingintra-andextrahippocampalreinstatementoftheoriginalexperiencethrough dynamicrouting(Figure2,featuresv–vii; Figures3 and 4).We elaborateonthisideawithrecentexamplesinthenextsections. DynamicRouting:HippocampalAfferents

Inthisframework,theDG-CA3-CA2-CA1circuitcanbeperceived asatemplateofnodes,witheachnodereceivingdiverseintra-and extrahippocampalinputsallowingfortheintegrative,dynamic, andflexibleincorporationofcognitiveandvisceralinformation intomemoryrepresentations(Figure3A).Thesepropertieswould enablethehippocampustoparticipateinmany‘‘types’’ofmemories—spatial,goal-oriented,social,future-planning—allwhich maycomprisediverseepisodicexperiences.Forexample,direct long-rangeGABAergicprojectionsfromthelateralhypothalamus (LH)toCA3havebeenrecentlyidentified(Zhouetal.,2019a); theseneuronssynapseontoCA3interneuronsandappearto havecriticalfunctionsintasksofobjectrecognitionanddiscrimination,revealingadirectpathwaybywhichCA3mayintegrate endocrinesignalsinlearningandmemoryprocesses.CA3neuronsalsoincorporatelocuscoeruleus(LC)input,andonestudy foundthatLCprojectionstodorsalCA3(dCA3;butnottoCA1 orDG)arerequiredforencoding(butnotretrieval,whichmaybe mediatedbyCA3-CA1[seebelow])ofacontextualrepresentation (asassessedbydistancetraveledinapreviouslyexploredcontext orviasingle-trialcontextualfearconditioning)(Wagatsumaetal., 2018).Additionally,parallelcircuitsprojectingfromneuronsin thesupramammillarynucleus(SUM)totheDGandCA2have beenfoundtocarrycontextualandsocialnoveltysignals,respectively,allowinghypothalamicsculptingofhippocampalmemoryin atask-specificmanner(Chenetal.,2020;seealso Lietal.,2020; Hashimotodanietal.,2018).Theserecentdiscoveriesbroadenour understandingofthediversityofmammalianhippocampalafferentsandpointtomultiplesourcesviawhichthehippocampus mayintegratesignalsformemoryformationorrecall.Activityof thesedistinctsetsofinputs(alongsideotherimportantinputs, includingfromtheamygdalaandanteriorcingulatecortex),recruitedbasedonongoingexperience,maygovernwhichhippocampalroutesaredeployedforencodingand/orrecall.

Perspective

DynamicRouting:HippocampalEfferents

Furtherarguingagainstasimplesequentialprocessingloop,itis clearthateachnodewithintheDG-CA3-CA2-CA1circuitprojectstodistinctoutputs(Figure3B).Intheframeworkofindex theory,theseoutputsmaybeflexiblydeployedbyengramcells toreinstateanexperience(whetherthatexperienceisappetitive, aversive,etc.).Anexampleofthispotentialselectiveroutingcan befoundinventralCA1(vCA1)neurons(Ciocchietal.,2015).In thisstudy,vCA1cellsweretrackedbasedontheirprojectionsto thePFC,nucleusaccumbens(NAc),andamygdaladuring behavior,revealingthatactivityintheseneuronsweretaskand pathwaydependent.ThesefindingscriticallysuggestthatsignalsoutofCA1arenotuniformlytransmittedtoitstargets; rather,itsupportstheideaofthatefferenthippocampalsignals areroutedbasedontaskandmnemonicdemands.Withparticularrelevancetoindexing,dorsalCA1(dCA1)tetroderecordings pairedwithcircuit-specificoptogeneticshaveshownthat expressionofconditionedplacepreference(CPP)dependson thereinstatementofdCA1representationsthatwereactiveduringtraining(Troucheetal.,2019).Furthermore,CPPexpression islostifdCA1terminalsinNAcarephotoinhibited,despitedCA1 pyramidalcellsmaintainingtheircontext-dependentcellassembliesduringtesting(seealso Zhouetal.,2019b).

ThisroutingabilityisnotrestrictedtoCA1;CA3outputneuronsmayrouteinformationviaprojectionstoCA1,CA2,orthe DLS(dorsolateralseptum).Indeed,brain-wideanalysesofcoactivatedcircuitsaccompanyingcontextualfeardiscrimination identifiedaCA3-DLSmodule(Besnardetal.,2019).This pathwayappearstorecruitsomatostatin(SST)+ DLScellsto gateconditionedfreezing,as invivo calciumimagingfound SST+ DLScellactivityreliablydiscriminatedshock-associated versussafecontexts.Insupportofthesefindings,optogenetic-terminal-specificsilencingofdCA3terminalsindCA1 andDLShassuggesteddistinctrolesfordCA3-CA1and dCA3-DLSprojectionstocontextualfearlearning(orconsolidation)anddiscrimination,respectively(Besnardetal.,2020).

ForCA2,itsefferentnetworkpositionsitstronglyformemories involvingsocialrecognition,discrimination,andaggression(MiddletonandMcHugh,2019).Indeed,CA2(andCA3)efferentsdo notuniformlyregulatediscrimination(Raametal.,2017).OptogeneticexperimentshavedemonstratedthatanteriorCA2/dCA2 neuronstargetingdCA1areessentialfornovelobjectrecognition, butnotfordiscriminationbetweennovelandfamiliarconspecifics. TheoppositewastruefordCA2/dCA3projectionstoposterior CA1.PhotoinhibitionofdCA2/dCA3projectionstotheDLSwere insteadshowntosomewhatenhancesocialdiscrimination,but withnoeffectonobjectdiscriminationorrecognition.Insocialbehaviors,axonsfromdCA2neuronstargetingventralhippocampus wereshowntobecriticallyinvolvedinmaintainingmemoryofa familiaranimal(Meiraetal.,2018; Raametal.,2017),whilepharmacogeneticinhibitionofCA2terminalsintheDLSattenuatessocialaggression(Leroyetal.,2018),apathwaythat,whenactive, appearedtoinvokeDLS-innervationoftheventromedialhypothalamustodriveattackbehavior.

Intotal,multiplenonoverlappingengramswithinthesediverse hippocampalroutesmaycompetethroughupdatingorongoing learningtomodifybehavioraloutput.Forexample,two-photon (2P)imagingofDGandCA3engrams invivo revealedthatthe

updatingofarewardlocationpromotedactivityremappingin CA1andCA3,butnotintheDG(HainmuellerandBartos, 2018).Recentworkhasalsodemonstratedthatcontextualfear extinctionrecruitsadistinctDGengramfromthatencodingthe originalcontext-shockassociation,whichreduceslevelsof freezinginthetrainingcontextbutcanbeovercomebytheoriginalengramtoinducerelapse(Lacagninaetal.,2019).Likewise, prolongedoptogeneticorchemogeneticreactivationofaDG fearmemoryengraminthetrainingcontextwithouttheunconditionedstimuluspromotedextinctionoftheconditionedresponse (Khalafetal.,2018).Ofcourse,thehippocampusisnotuniquein thistypeofbroadconnectedness;otherhubsinthebrain, includingtheclaustrum(Jacksonetal.,2020)andthethalamus (HalassaandSherman,2019),maysurpassitintermsoftotal connectivity.However,thesedatasuggestthatthehippocampusiscapableofintegratingandroutingcomplexinformation fromavarietyofsourcestructures,supportingitsroleinthebindingofcognitionandemotiontosubservememory(Figure4A).

DynamicRouting:InhibitoryMicrocircuits

Howmightthesediversecommunicationchannelsrunning throughthehippocampusbemanaged?HippocampalINsare wellpositionedtofunctionasarbitersofinformationflowinthe hippocampus,astheytargetdifferentcellularcompartmentsof principalneurons,arereciprocallyconnectedwithotherinterneurons,andprojectlocallywithinandacrossdifferentsubregions andlamellaeandoutofthehippocampustoassociationcortices andsubcorticalcircuits(long-rangeINs)(Caroni,2015).Moreover, hippocampalINsmodulateneuronalexcitability,summationof excitatoryinputs,andneuronalfiringinadditiontogenerationof networkoscillations(thetaandgammaoscillations)andassuch arethoughttoplaycriticalrolesinlocalcircuitcomputationsunderlyingexplorationandencoding,actionselection,memory consolidation,retrieval,andreinstatement(Cardin,2018; Makino etal.,2019; RouxandBuzsaki,2015; Sosaetal.,2018).Indeed, recentstudieshaveuncoveredadiversepopulationofINsin CA1andCA3thatexertperisomaticanddendriticinhibitionon DGCsandaremodulatedbySWRs.

LocalINsmayregulateinformationflowwithinahippocampal subregionbybiasingrecruitmentofprincipalcells,thereby creatingparallelchannelsasevidencedinastudythatidentified biasedPV+ basketcell(BC)connectivitywithdeepandsuperficialCA1neuronsoftheventralhippocampus(Leeetal.,2014). TheauthorsfoundthatPV+ BCspreferentiallyinnervateddeep CA1pyramidalneuronsbutreceivedgreaterexcitatoryinputs fromsuperficialCA1pyramidalneurons.Atthelevelofoutput, PV+ BCsexertgreaterinhibitionontobasolateralamygdala (BLA)-projectingdeepCA1neuronsthanthosethatprojected tothePFCand,inturn,receivedgreaterexcitatoryinputfrom PFCthanBLA.ThesedatasuggestthatPV+ BCsdonotuniformlyinhibitCA1;instead,itislikelythatPV+ BC-principalcell microcircuitsbiasinformationflowtodistinctvCA1outputs, includingPFC,BLA,NAc,DLS,andLH(servingdynamicand flexiblerouting).LocalINsmayalsodifferentiallyregulatetheta phase-lockingandburstfiringofCA1neuronsthroughsomatic ordendriticinhibition,respectively(Royeretal.,2012).Because burstfiringofpyramidalcellsisthoughttoincreasesynaptic communicationbyincreasingexcitationofdownstreamtargets, localINsmaymodulateCA1outputsthroughthismechanism ll

AB

(Gravesetal.,2012; Lisman,1997; TakahashiandMagee,2009). Importantly,rhythmicoptogeneticactivationofPV+ INsinCA1to mimicthatseenduringlearningenhancedtheta,delta,andripple oscillations;stabilizedfunctionalconnectivitybetweenCA1neurons;andreliablypromotedensemblereactivation(Ognjanovski etal.,2014).Theexactroletheseoscillationsplayintheabilityof anindextoreactivatedownstreamtargetsremainslargelyuntested;however,evidencesuggeststhecoherenceorcoordinationofactivitytheyprovidemayfacilitateboththeencodingand recallofmemoriesacrossvariousstructuresbyensuringtemporalcoordinationofactivity(Buzsaki,2015; Corcoranetal.,2016; Igarashietal.,2014; JooandFrank,2018; Linetal.,2017; Makinoetal.,2019; WirtandHyman,2019).

Pioneering invivo recordingsandimagingstudiesinratsidentifiedextensivelyconnectedINswithextrahippocampal(septum, subiculum,para-andpre-subiculum,andRSC)projectionsthat coordinatenetworkoscillations(Bonifazietal.,2009; Jinno, 2009).Long-rangeinhibitoryprojectionneuronsoftheLECsup-

Figure4.HippocampalEngramsMayIndex ExperiencetoReinstateExperiential Memory

(A)Distinctexperiencesarethoughttobeencoded withinDG-CA3-CA2-CA1connections,with engram-bearingcellsbeingfunctionallylinkedto otherneuronsforthesameepisode.Recallcan thenbedrivenbypartialinput(cues)thatreinstate activity(filled-incircles/squares)withinhippocampalcircuitstodriveextrahippocampalreinstatementviaitsdiverseoutputsandconnectivity tootherengram-bearingcells.PCs,principalcells; INs,inhibitoryneurons.

(B)Hippocampalcellsmayregistermorethanone experienceindistinctpatternsofconnectivity prescribedbyactivity-dependentgeneexpression(shownhereascombinationsof1sand0s).

pressCA1cholecystokinin-positive (CCK+)interneuronsthatrelayfeed-forwardinhibitionfromCA3toCA1 exvivo (Basuetal.,2016).Thisdisinhibitionof CA1interneuronsinducedenhanceddendriticspikingwithinaspecifictemporal window,amechanismbywhichsensory informationandmnemonicinformation arrivingfromexcitatoryLECinputsand CA3,respectively,maybeintegrated. Morerecently,aclassoflong-rangeinhibitoryneuronalnitricoxidesynthase (nNOS)-expressingcellsinCA1(LINCneurons)hasbeenidentifiedthatprojectboth locallyandextra-hippocampally(ChristensonWicketal.,2019).Theseneurons inhibitsuperficialanddeepprincipalcells andotherINsinCA1andproject todiverseextrahippocampaltargets, includingtheteniatecta,subiculum,hypothalamus,olfactorybulb,andEC.OptogeneticactivationofLINCneuronsentrained hippocampaloscillationsandhippocampal-frontalcortex(teniatecta)coherence. Thus,convergingevidencehasbeguntoilluminatehowcellphysiology,activity-dependentgeneexpression,andmicrocircuitconnectivitysupporthippocampalengram-cell-dependentindexing (i.e.,encodingofexperiencesandroutingofinformationtomediate reinstatement).Wediscussthesefeaturesofengramcellidentitynext.

IndexCellIdentity

Giventhelong-standingfocusonrodenthippocampalplace cells,anobviousquestioniswhataspectofcontextualmemory isencodedwithinthehippocampalengram.Behavioralstudies usingvariationsofcontextualfearconditioningsuggestthat thehippocampusgeneratesaconjunctiverepresentationof multimodalsensoryinformationformedthroughphysicalexplorationofacontext(Fanselow,2000;seealso Krasneetal.,2015). Forexample,onestudypreexposedratstoeithertheconditioningcontextorindependentfeaturesofthatcontextandfound contextfearafteranimmediateshockisfacilitatedonlywhen

Perspective

thesemultimodalcuesarepresentedtogether,suggestingthat thehippocampusrepresentstheconjunctionofthecues definingthecontext(RudyandO’Reilly,1999).Indeed,temporarypharmacologicalinactivationofthehippocampusduring thecontextpreexposurepreventsthecontextualfearconditioningofimmediateshock(Matus-Amatetal.,2004).Paststudiesof IEGexpressioninthehippocampussupportthisthisview(e.g., Huffetal.,2006; Zhuetal.,1997);thestrongestIEGresponse isachievedwhenanovelcombinationofmultimodalcuesispresentedtotheanimal.Conversely,hippocampalIEGexpression isweakornonexistentwhenahighlyhabituatedstimulusis given.Notethatimmediateshockuponcontextentryinthe absenceofpreexposure(andextensivepostexposure)does notappeartoelevatelevelsofIEGsinthehippocampusrelative toahabituatedhomecage(seealso Erwinetal.,2020).These datasuggestthatIEG-expressingengramneuronsmaynot necessarilyorexclusivelystorespatialinformation,asrodents willhaveactiveplacecellseveninthemostfamiliarofcontexts, butratherhippocampalcircuitsdetectnoveltyinthecombinationofsensorycuesandencodeitasacontextualrepresentation supportingtheepisodicexperience.Indeed,optogeneticstimulationofCA1neuronstaggedinanovelcontextthedaypriorto immediateshockdeliveryinthesamecontext,butnotadifferent context,resultedinretrievalofthecontextualfearmemory (Ghandouretal.,2019;seealso Ramirezetal.,2013).Thus,a memoryengram,definedbyactiveprincipalcellsduringcontextuallearning,maypreferentiallyencodeconjunctivecontextual information,asopposedtospecificlocationsthatcouldbe biasedbyaspecificcueorsubsetofcues.

Physiology

Keyinsightsintothepreciseidentityofengram-bearingcells camefrom invivo recordingofCA1neuronsinamouseinwhich c-Fos-positiveneuronslabeledduringanovelcontextexposure weretaggedwithchannelrhodopsin2(ChR2)andsubsequently opticallyidentified(Tanakaetal.,2018).Asexpected, 50%of allCA1pyramidalcellscouldbeclassifiedasplacecells;however, onlyone-quarteroftheseplacecellsalsoexpressedc-Fos(optogeneticallyidentified).Inshort,engramcellswereplacecells,but onlyone-quarterofplacecellswereengramcells.Duringmemory encoding,theseengram-bearing(c-Fos)cellsaredistinguishedby highermeanfiringrates(asalsoseeninacalciumimagingstudy; Ghandouretal.,2019),repetitiveburstsofactionpotentialsatthe thetafrequency,andhigherentrainmentbythelocalfastgamma oscillationcomparedtothenon-c-Fos-expressingplacecells, againhighlightingtheroleinhibitorycircuitsandoscillationsmay playintheformationoftheindex.Interestingly,whenmicewere returnedtothecontextthenextday,c-Fos-positiveengramneurons,whileremainingplacecells(meaningtheystilldemonstrated areliablein-sessionspatiallyreceptivefield),showedamuch higherdegreeofspatialinstabilitycomparedtotheencodingsession(remapping)thanthec-Fos-negativeplacecellpopulation. Thesedatacanbeseenasparadoxical;howisitthattheneurons showntobecapableofreinstatingcontext-appropriatebehavior showrelativelylowerspatialspecificitythantheremainingactive cells?Importantly,whenonlyfiringrate(andnotlocation)was considered,itwasclearthattheengramcellsfaithfullyencoded contextualidentity,butnotspecificlocation.Areturntotheencodingcontextresultedinc-Fos-positiveneuronsreinstatingtheir

averagefiringrates,whichwerehighlycorrelatedbetweenthefirst andsecondvisits,whilethefiringratesinadistinctcontextwere stronglyaltered.Itworthnotingthatthisstrongcorrelationofactivityemergesassoonasanimalisplacedintheenvironment, suggestingtheiractivitycouldsupportrapidretrievalofcontextual memory,consistentwiththeindexingtheory.Further,thesefindingsofauniquephysiologysuggesttheimportanceofthetemporalrelationshipbetweeninputfromCA3andtheECintriggering CA1pyramidalcellplasticityandactivity invivo (Bittneretal., 2017; Ketzetal.,2013).

Complementaryresultswerealsofoundinaphysiologicalstudy inwhichc-Fos-positiveCA1neuronswereinhibitedduringrecall (Troucheetal.,2016).Inthisstudy,engramneurons,definedby c-Fosexpressionassociatedwiththeacquisitionofacocaine-rewardedCPP,werelabeledwithaninhibitoryopsin.Inactivationof thisensembleduringasubsequentrecallsessionreducedCPP behaviorand,interestingly,ledtoaglobalremappingofthe c-Fos-negativeactiveplacecellpopulation.Together,these datasuggestthattheroleofthec-Fos-positiveplacecellsisto serveasacontext-specificmemoryindexandthattheiractivity iscrucialforthestablereinstatementofamoredetailedspatial map,consistingoftheremainderoftheplacecellpopulation, thatwouldpermitanimalsprecisenavigation.

Whileitmayseemoddatfirstthattheneuronsindispensable forinducingmemoryrecallinCA1showspatialinstability,recent computationalmodelinglendssupporttothisview(Bennaand Fusi,2019).Basedonanassumptionthatthehippocampusencodescorrelationsofincomingsensoryinformation,similarto theinterpretationfromcontextualconditioningandIEGstudies, themodelpredictedinstabilityinthespatialrepresentationof thehippocampus.Theirultrametrictree-likenetworkgenerated sparseandcompressedrepresentationsofinputstoefficiently storeuncorrelatedpatternsinahippocampal-likenetwork. Whenspatialnavigationina2Dopenfieldissimulated,activities ofhippocampalcellsinthemodelexhibitstrongmodulationby theanimal’slocationwithintheenvironment(i.e.,placecells). However,similartotheexperimentalobservationsabove,these placefieldssignificantlyremappedbetweenepochsinthe sameenvironment,suggestinginstabilityoffiringlocationasa reflectionofcorrelationcodingratherthanspatialcoding.Taken together,thesestudiessupportaviewthatactivityofthehippocampalengramreflectsmorethanjustspaceandsuggestatleast asubsetofneuronsarededicatedtocapturingtheconjunctive correlationsthatdefinethelargercontextoftheexperience. Activity-DependentRegulationofGeneExpressionand Connectivity

Hippocampalengramsaregeneratedandmaintainedby strengtheningormodificationofsynapsesamongactivatedcells withinandacrosssubregions.Onestudyfoundthatc-Fos-tagged CA3cellspreferentiallyrespondedtostimulationofengramtaggedDGCs(Ryanetal.,2015),resultsindicativeofexperience-drivenconnectivityofDG-CA3.Likewise,context-feardependentincreasesinthenumberandsizeofspinesin engram-bearingcellsofCA1coincidewithdirectinputfrom engram-taggedcellsfromCA3(Choietal.,2018).Additionally, DGengramcellswereshowntoexhibitgreaterconnectivity withstratumlucidumPV+ INsthannon-engramDGcells(Guo etal.,2018).Theseobservationshavemotivatedinvestigations

intohowdevelopmentalprogramsandactivity-dependentgene expressionprescribeengramformation.First,principalneurons mayhavedifferingpropensitiestowardrecruitmentintoengrams basedondevelopmentallyprogrammedintrinsicfiringproperties andconnectivity(CembrowskiandSpruston,2019; Solteszand Losonczy,2018).Second,activity-dependenttranscriptionfactorsandcombinationsthereofenableneuronstoread patternsofneuralactivityandtranscribemolecularspecifiersof connectivitytofacilitatestrengtheningormodificationofsynapses(Tyssowskietal.,2018).Forexample,cyclicadenosinemonophosphate(cAMP)-response-element-bindingprotein(CREB)overexpressionstudieshaverevealedthatenhancingbasal activityandexcitabilityofprincipalcellsinthehippocampus(or subsetsofamygdalarneurons,etc.)priortolearningcanbias theallocationandtaggingprocesstothesecellswithoutaltering theoverallsizeoftheengramperse(JosselynandFrankland, 2018; JosselynandTonegawa,2020).Arecentreportusing RNAsequencingofengram-taggedDGcells(followingcontextual fearconditioning)identifiedauniquelearning-dependentgenetic profileforengram-bearingcells,withCREB-dependenttranscriptionnetworksbeingdifferentiallyregulatedandrequiredfor consolidation(Rao-Ruizetal.,2019).Interestingly,manyofthese geneswerepreviouslyshowntoregulatesomaticinhibition(e.g., neuronalPASdomainprotein4[NPAS4],proenkephalin[Penk], andbrain-derivedneurotrophicfactor[BDNF])(Bloodgood etal.,2013).Consistentwiththesefindings,withintheDG, contextualfearlearningregulatesCREB-dependentlevelsofneuropeptideY(NPY)inSST+ hilarperforantpath-associated(HIPP) interneurons,whichmayregulateSST+ HIPP-mediatedfeedback andfeed-forwardinhibitionintheDGtogovernthesizeofthe engram(Razaetal.,2017; Stefanellietal.,2016).Notsurprisingly, differentIEGtranscriptionfactors(includingNPAS4)havebeen functionallyimplicatedinlinkingprincipalcellswithdistinctINnetworkstosupportengramformation(Sunetal.,2020).Thus,experientialinputmaydriveuniqueIEGexpressiontogovernfunctional allocationofengram-bearingcellstoworkinconcertformemory expression(Figure4B).

HippocampalIndexStabilityandMemoryFidelity Memoryconsolidationisthoughttoinvolvetransformationand reorganizationofhippocampal-linkedcognatecorticalrepresentationsandagradualdecayofthehippocampalengramover time(DeNardoetal.,2019; Guskjolenetal.,2018; Kitamura etal.,2017; Royetal.,2017; Tayleretal.,2013; Winocuretal., 2007).Consolidatedmemorieshavebeenshowntogeneralize orlackdetail,includingtheextenttowhichtheymayelicit visceralorphysiologicalreactions,leadingtothesuggestion thattherolethehippocampusplaysinmemoryistocontribute episodicdetail(Yonelinasetal.,2019).Ifthiscontributionrelies ontheinitialmemorytraceornotremainsacontestedtopic. Forexample,ithasbeenarguedthathippocampalmemory tracesremain,evenforoldermemories(MoscovitchandNadel, 2019),butothershavearguedthereisashiftintherolethehippocampusfromoneofrecalltoreconstructionintheabsenceof theoriginaltrace,withtheactivityservingtoindexconsolidated neocorticaltraces(BarryandMaguire,2019a, 2019b).These observationsraisethefollowingquestions:Isthehippocampal indexalwaysnecessaryformemoryretrieval,ordocorticalin-

dexesacquirethisfunctionovertime?Isthecorticalindexequivalenttothehippocampalindex?

Severallinesofevidencesupportthenotionthatthehippocampalindexmaybenecessaryformaintenanceandretrieval ofonlyhighlyprecisememories.First,althoughhippocampal damageatremotetimepointsmaystillpermitretrievalof detailedcontextualrepresentations,theextentofmemory retrievalisoftenmuchlessrobust(Wangetal.,2009),suggesting thatextrahippocampalindicesmaynotfullycompensateforthe lossofthehippocampalindex.Indeed,whileasimilardegreeof hippocampalactivationisseenforrecentandremotememories, thereactivationpatternsaredifferent(Tayleretal.,2013;seealso Guskjolenetal.,2018).Second,artificiallystabilizingtheengram withinDG-CA3decreasesremotememorygeneralization(and maintainsbehavioralreinstatementofremoteDGengramstimulation; Guoetal.,2018),providingadirectlinkbetweenmaintenanceofthehippocampalindexandremotememoryprecision. However,maintenanceofseparatehippocampalindicesforall episodicmemoriesisthoughttorequiresignificantlygreatercapacitythanisavailabletoavoidmemoryinterference(McClellandetal.,1995; MillerandSahay,2019; SkaggsandMcNaughton,1992).InCA1,thevariousmethodsemployedtogenetically labelengramneuronstypicallycapture 20%ofthepyramidal cellsintheregion(Tanakaetal.,2018),and invivo imagingsuggestedthattheidentityoftheseallocatedneuronsshiftsoverthe timescaleofhours(Caietal.,2016);thus,itappearsthatnatural decayofhippocampalindicesensuresthetime-dependentreorganizationofmemorytracestosupportdifferentdegreesof generalizationandgenerationofschematofacilitatenew learning.Itmaybethatsomehippocampalindexes,perhaps forsalientlifeevents,aremaintainedforlongerperiodsoftime, therebypermittingrecallofremotememorieswithhighfidelity.

Theintegrityandcompositionofthecorticalindicesdepends onhowcompetitionforrepresentationofepisodicmemoriesand abstractionofstatisticalcommonalitiesacrossensembles dictatethebalancebetweenpreservationofdetailsversusgenerationofschematofacilitatememorygeneralization.Thismay involvetime-dependentchangesintheexactnumberofcells andpatternsofefferentconnectivityofcorticalensemblesand linkageofdistinctengramsofseparateexperiencesviasome degreeofoverlappingandsynchronousactivation(duringrecall orreconsolidation)ofengram-bearingcells(Abdouetal.,2018; DeNardoetal.,2019; Kitamuraetal.,2017; Ohkawaetal., 2015; Oishietal.,2019; Pignatellietal.,2019; Ramirezetal., 2013; Redondoetal.,2014).

Whilenoonemodelcanexplainallthecurrentdata,fromthe perspectiveofengramsandindexing,wefavorthehypothesis ofatime-dependentshiftintheindexingfunctionfromthehippocampustocorticaltracesconcurrentwithasilencingorlossof theoriginalhippocampalindex(Tonegawaetal.,2018).Ultimately,time-dependentshiftsinhippocampus-dependent episodicdetailmaybeusefulinthedevelopmentofexperiential schemasandbroaderknowledge.

HippocampalDysfunction Indexopathies

Memorydeficitsandhippocampaldysfunctionaccompany traumaticbraininjury,epilepsy,age-relatedcognitivedecline,

Alzheimer’sdisease(AD),andnumerousotherpsychiatricdisorders,includingposttraumaticstressdisorder(PTSD)andschizophrenia(BesnardandSahay,2016; Habermanetal.,2017; Small etal.,2011).Canthesedisordersofexperientialmemorybeclassifiedas‘‘indexopathies,’’insofarastheyaremarkedbyan inabilitytoaccuratelyorpreciselyencodeoreffectively implementhippocampus-dependentroutingofinformation (i.e.,indexing)?Forexample,recentworkdocumenteddeclining hippocampalandcorticalreinstatementinagingindividuals (Trelleetal.,2020).Disease-oraging-inducedexcitation-inhibitoryimbalanceinhippocampalcircuits,whichmayimpedeindexing,mayunderliemuchofthesememorydysfunctions. Indeed,excitation-inhibitionimbalance(hypo-orhyperactivity) atthelevelofCA1(e.g., Ohetal.,2013)andCA3(e.g., Simkin etal.,2015; Wilsonetal.,2005)andlossoffeed-forwardinhibitioninDG-CA3(e.g., Guoetal.,2018)areassociatedwithmemoryimprecisioninpreclinicalmodelsofagingandmemorydisorders.Humanimagingstudieshavefurtherreportedsimilar activitychanges(e.g.,hyperactivity)ofhippocampalstructures (Habermanetal.,2017),suchasinpresymptomaticfamilialAD (FAD)individuals(Quirozetal.,2010)orpatientswithamnestic mildcognitiveimpairment(Bakkeretal.,2012).

Suchcellular,circuit,andnetwork-levelalterationsmay disruptthebalancebetweenpatternseparationandpattern completion,therebypromotingaberrantindex-dependentreinstatementandrecall.WhatpreviouslymayhavebeensubthresholdtotriggerCA3-dependentmemoryrecallpriortodiseasemay besufficientafterdiseaseonset,therebypromotingexcessive reinstatementandmemoryexpressionincontextsthatmay notbeoptimal.Forexample,aberrantorexcessiveretrievalof pastexperienceshasbeensuggestedtounderliepsychosisin schizophrenia(Tammingaetal.,2010).Additionally,degradation offlexibleroutingtoextrahippocampaltargets,duetoconnectivitylossesindiseaseorinjury,mayalsoimpedetheabilitiesofthe hippocampustoeffectivelyintegratecorticalandsubcorticalinformationforproperencodingandretrieval.Perhapsrelatedto

suchcircuitloss,ADisconsidered,inpart,adiseaseofmemory retrievalfailure(LealandYassa,2018; Royetal.,2016). PromotingIndexing:NewDirectionsinTherapy

Recentadvancesintechnologyandmedicinehaveledtoanumberofnewtherapeuticavenuesformemorydisorders—strategiesthatmaybeeffective,inpart,becausetheypromoteor reestablishhippocampalindexingfunctions.Forexample, growinginteresthascenteredondeep-brainandclosed-loop feedbackneuroprostheticsforsymptommanagementinindividualswhenotherlinesoftreatmenthavefailed(Grosenicketal., 2015; TakeuchiandBerenyi,2020).Perhapsbyrestoring context-dependentrouting(andtherebyindexing),theserealtimeelectrophysiological(oropto-orchemogenetic,potentially) methodscoulddynamicallynormalizeaberrantactivityorrestore cellexcitabilityinperturbedbraincircuits(e.g.,inhippocampalamygdalarorhippocampal-prefrontalloops).Additionally, recentdevelopmentsintargetedgene-editingapproaches (KnottandDoudna,2018)maypermitmolecularreallocationor respecificationofconnectivityaimedatpromotingmemoryprecisionandaccuracy.Indeed,quietingdisease-relatedhyperexcitedCA3pyramidalcellsmayinvolvetargetingfeed-forward inhibitorymechanismsofDG-CA3(Guoetal.,2018; Vianada Silvaetal.,2019)orCA2-CA3connections(Boehringeretal., 2017)orinhibitingaberrantLH-CA3activity(Zhouetal., 2019a),forexample.Otherpharmaco-orgenetherapiesthat promoteneurogenesisinagingordiseasestatesmayalsoexist asbeneficialtherapeuticavenuesformemoryimpairments (MillerandSahay,2019).

Noveltreatmentsformemoryimpairmentsmaynotbelimited toinvasivetechniquesandmayinvolvesupplementingexisting procedurestobesttapintotheindexingpropertiesofthehippocampus.Forexample,althoughtheuseofmnemonicdevicesfor memorytreatmentsisnotnew,recentdevelopmentsintechnology,suchasaugmentedrealityor3Dinteractiveenvironments, mayprovidenovelavenuesforimprovingmemoryrecallwithin andbeyondtheclinic.Indeed,thegrowingubiquityofpersonal

Figure5.OutstandingChallengesforHippocampalIndexingandMemoryResearch

handhelddevicesmaymakemobileremindersormnemonic cues(tofacilitatereinstatementofmemory)fortreatmentsor symptommanagementformemoryimpairmentsmoreaccessibleorspecialized.Whencombinedwithpsychologicaltreatmentsintheclinic,theseandtheabovementionedpossibilities mayyieldnewsuccessesintreatment-resistantmemorydisorders.Movingforward,indexingmaybeausefulframeworkfor improvingclinicaltherapiesformemorydysfunction.

Conclusions:MovingMemoryForward

Perhapsthebrain’smostpowerfulsearchengine,thehippocampus,sitsatthecenteroftheacquisitionandrecallof episodicmemory.Whilethemechanismsofhowthisis achievedhavebeenthefocusofdecadesofresearchacross manyspeciesanddisciplines,itisoftenchallengingtorelate disparatelinesofinquiry.Here,wehavehighlightedhuman andanimalworkbasedonrecentgenetic,physiological, anatomical,andcomputationalapproachesthattogethersupportanexpandedviewofthehippocampalmemoryindexing theory.Inparticular,wearguethat(1)thefunctionalrolesofputativehippocampalengramcellsincludeindexing,whichmay facilitatedetailedrecallofepisodicexperiences;(2)this episodicrecallisfacilitatedbythereinstatementofengram cellactivityandintheirexperience-sculptedconnectivity;(3)indexingmaynotbeuniquetothehippocampus,butthehippocampusmaybeuniquelypositionedtoindexexperientialmemory;and(4)diseaseofthehippocampusmayimpedetruly episodicmemorybydisruptingitscapacityforprecise context-specificreinstatement.Nonetheless,thereremaina numberofoutstandingquestionsforthefieldandforfuture work(Figure5).Futureworkthatintegratestheselevelsofanalyseswillberequiredtounderstandhowthedynamicsofinformationflowinthehippocampalcircuitcontributestotheencodingandrecallprocessesitsupports.

ACKNOWLEDGMENTS

T.D.G.acknowledgessupportfromHarvardBrainScienceInitiative.K.Z.T.acknowledgessupportfromMEXTGrant-in-AidforYoungScientists (19K16305),Grant-in-AidforJSPSfellows(19J00974),andaNakajimaFoundationresearchgrant.A.S.acknowledgessupportfromNIHBiobehavioral ResearchAwardsforInnovativeNewScientists(BRAINS)1-R01MH104175, NIH-NIA1R01AG048908-01A1,NIH1R01MH111729-01,theJamesandAudreyFosterMGHResearchScholarAward,theEllisonMedicalFoundation NewScholarinAging,theWhitehallFoundation,theInscopixDecodeAward, theNARSADIndependentInvestigatorAward,EllisonFamilyPhilanthropic support,theBlueGuitarFund,aHarvardNeurodiscoveryCenter/MADRC Centerpilotgrantaward,anAlzheimer’sAssociationresearchgrant,theHarvardStemCellInstitute(HSCI)developmentgrant,andanHSCIseedgrant. T.J.M.acknowledgessupportfromMEXTGrant-in-AidforScientificResearch (19H05646),MEXTGrant-in-AidforScientificResearchonInnovativeAreas (17H05591,17H05986,and19H05233),andtheRIKENCenterforBrain Science.

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weakness. He was trembling. The boys went marching by

“There he is!” Emily shrilled, above the din. “There he is! There he is! There he—” And waved a futile little hand. It wasn’t so much a wave as a clutching. A clutching after something beyond her reach.

“Which one? Which one, Emily?”

“The handsome one. The handsome one. There!” Her voice quavered and died.

Jo put a steady hand on her shoulder “Point him out,” he commanded. “Show me.” And the next instant. “Never mind. I see him.”

Somehow, miraculously, he had picked him from among the hundreds. Had picked him as surely as his own father might have. It was Emily’s boy. He was marching by, rather stiffy. He was nineteen, and fun-loving, and he had a girl, and he didn’t particularly want to go to France and—to go to France. But more than he had hated going, he had hated not to go. So he marched by, looking straight ahead, his jaw set so that his chin stuck out just a little. Emily’s boy.

Jo looked at him, and his face flushed purple. His eyes, the hardboiled eyes of a Loop-hound, took on the look of a sad old man. And suddenly he was no longer Jo, the sport; old J. Hertz, the gay dog. He was Jo Hertz, thirty, in love with life, in love with Emily, and with the stinging blood of young manhood coursing through his veins.

Another minute and the boy had passed on up the broad street— the fine, flag-bedecked street—just one of a hundred service-hats bobbing in rhythmic motion like sandy waves lapping a shore and flowing on.

Then he disappeared altogether.

Emily was clinging to Jo. She was mumbling something, over and over. “I can’t. I can’t. Don’t ask me to. I can’t let him go. Like that. I can’t.”

Jo said a queer thing.

“Why, Emily! We wouldn’t have him stay home, would we? We wouldn’t want him to do anything different, would we? Not our boy. I’m glad he enlisted. I’m proud of him. So are you glad.”

Little by little he quieted her. He took her to the car that was waiting, a worried chauffeur in charge. They said good-by,

awkwardly. Emily’s face was a red, swollen mass.

So it was that when Jo entered his own hallway half an hour later he blinked, dazedly, and when the light from the window fell on him you saw that his eyes were red.

Eva was not one to beat about the bush. She sat forward in her chair, clutching her bag rather nervously.

“Now, look here, Jo. Stell and I are here for a reason. We’re here to tell you that this thing’s got to stop.”

“Thing? Stop?”

“You know very well what I mean. You saw me at the milliner’s that day. And night before last, Ethel. We’re all disgusted. If you must go about with people like that, please have some sense of decency.”

Something gathering in Jo’s face should have warned her. But he was slumped down in his chair in such a huddle, and he looked so old and fat that she did not heed it. She went on: “You’ve got us to consider. Your sisters. And your nieces. Not to speak of your own—”

But he got to his feet then, shaking, and at what she saw in his face even Eva faltered and stopped. It wasn’t at all the face of a fat, middle-aged sport. It was a face Jovian, terrible.

“You!” he began, low-voiced, ominous. “You!” He raised a great fist high. “You two murderers! You didn’t consider me, twenty years ago. You come to me with talk like that. Where’s my boy! You killed him, you two, twenty years ago. And now he belongs to somebody else. Where’s my son! that should have gone marching by to-day?” He flung his arms out in a great gesture of longing. The red veins stood out on his forehead. “Where’s my son! Answer me that, you two selfish, miserable women. Where’s my son!” Then, as they huddled together, frightened, wild-eyed, “Out of my house! Out of my house! Before I hurt you!”

They fled, terrified. The door banged behind them.

Jo stood, shaking, in the center of the room. Then he reached for a chair, gropingly, and sat down. He passed one moist, flabby hand over his forehead and it came away wet. The telephone rang. He sat still. It sounded far away and unimportant, like something forgotten. I think he did not even hear it with his conscious ear. But it rang and rang insistently. Jo liked to answer his telephone, when at home.

“Hello!” He knew instantly the voice at the other end.

“That you, Jo?” it said.

“Yes.”

“How’s my boy?”

“I’m all right.”

“Listen, Jo. The crowd’s coming over to-night. I’ve fixed up a little poker game for you. Just eight of us.”

“I can’t come to-night, Gert.”

“Can’t! Why not?”

“I’m not feeling so good.”

“You just said you were all right.”

“I am all right. Just kind of tired.”

The voice took on a cooing note. “Is my Joey tired? Then he doesn’t need to play if he don’t want to. No, sir.”

Jo stood staring at the black mouthpiece of the telephone. He was seeing a procession go marching by. Boys, hundreds of boys, in khaki.

“Hello! Hello!” the voice took on an anxious note. “Are you there?”

“Yes,” wearily.

“Jo, there’s something the matter. You’re sick. I’m coming right over.”

“No!”

“Why not? You sound as if you’d been sleeping. Look here—”

“Leave me alone!” cried Jo, suddenly, and the receiver clacked onto the hook. “Leave me alone. Leave me alone.” Long after the connection had been broken.

He stood staring at the instrument with unseeing eyes. Then he turned and walked into the front room. All the light had gone out of it. Dusk had come on. All the light had gone out of everything. The zest had gone out of life. The game was over—the game he had been playing against loneliness and disappointment. And he was just a tired old man. A lonely, tired old man in a ridiculous, rose-colored room that had grown, all of a sudden, drab.

IV

OLE SKJARSEN’S FIRST TOUCHDOWN

B G F

From “At Good Old Siwash,” copyright, 1911, by Little, Brown and Company. By special permission of the author.

Am I going to the game Saturday? Am I? Me? Am I going to eat some more food this year? Am I going to draw my pay this month? Am I going to do any more breathing after I get this lungful used up? All foolish questions, pal. Very silly conversation. Pshaw!

Am I going to the game, you ask me? Is the sun going to get up to-morrow? You couldn’t keep me away from that game if you put a protective tariff of seventy-eight per cent ad valorem, whatever that means, on the front gate. I came out to this town on business, and I’ll have to take an extra fare train home to make up the time; but what of that? I’m going to the game, and when the Siwash team comes out I’m going to get up and give as near a correct imitation of a Roman mob and a Polish riot as my throat will stand; and if we put a crimp in the large-footed, humpy-shouldered behemoths we’re going up against this afternoon, I’m going out to-night and burn the City Hall. Any Siwash man who is a gentleman would do it. I’ll probably have to run like thunder to beat some of them to it.

You know how it is, old man. Or maybe you don’t, because you made all your end runs on the Glee Club. But I played football all through my college course and the microbe is still there. In the fall I think football, talk football, dream football. Even though I go out to the field and see little old Siwash lining up against a bunch of overgrown hippos from a university with a catalogue as thick as a city directory, the old mud-and-perspiration smell gets in my nostrils, and the desire to get under the bunch and feel the feet jabbing into my ribs boils up so strong that I have to hold on to myself with both hands. If you’ve never sat on a hard board and wanted to be

between two halfbacks with your hands on their shoulders, and the quarter ready to sock a ball into your solar plexus, and eleven men daring you to dodge ’em, and nine thousand friends and enemies raising Cain and keeping him well propped up in the grandstands—if you haven’t had that want you wouldn’t know a healthy, able-bodied want if you ran into it on the street.

Of course, I never got any further along than a scrub. But what’s the odds? A broken bone feels just as grand to a scrub as to a star. I sometimes think a scrub gets more real football knowledge than a varsity man, because he doesn’t have to addle his brain by worrying about holding his job and keeping his wind, and by dreaming that he has fumbled a punt and presented ninety-five yards to the hereditary enemies of his college. I played scrub football five years, four of ’em under Bost, the greatest coach who ever put wings on the heels of a two-hundred-pound hunk of meat; and while my ribs never lasted long enough to put me on the team, what I didn’t learn about the game you could put in the other fellow’s eye.

Say, but it’s great, learning football under a good coach. It’s the finest training a man can get anywhere on this old globule. Football is only the smallest thing you learn. You learn how to be patient when what you want to do is to chew somebody up and spit him into the gutter. You learn to control your temper when it is on the high speed, with the throttle jerked wide open and buzzing like a hornet convention. You learn, by having it told you, just how small and foolish and insignificant you are, and how well this earth could stagger along without you if some one were to take a fly-killer and mash you with it. And you learn all this at the time of life when your head is swelling up until you mistake it for a planet, and regard whatever you say as a volcanic disturbance.

I suppose you think, like the rest of the chaps who never came out to practice but observed the game from the dollar-and-a-half seats, that being coached in football is like being instructed in German or calculus. You are told what to do and how to do it, and then you recite. Far from it, my boy! They don’t bother telling you what to do and how to do it on a big football field. Mostly they tell you what to do and how you do it. And they do it artistically, too. They use plenty of language. A football coach is picked out for his ready tongue. He

must be a conversationalist. He must be able to talk to a greenhorn, with fine shoulders and a needle-shaped head, until that greenhorn would pick up the ball and take it through a Sioux war dance to get away from the conversation. You can’t reason with football men. They’re not logical, most of them. They are selected for their heels and shoulders and their leg muscles, and not for their ability to look at you with luminous eyes and say: “Yes, Professor, I think I understand.” The way to make ’em understand is to talk about them. Any man can understand you while you are telling him that if he were just a little bit slower he would have to be tied to the earth to keep up with it. That hurts his pride. And when you hurt his pride he takes it out on whatever is in front of him—which is the other team. Never get in front of a football player when you are coaching him.

But this brings me to the subject of Bost again. Bost is still coaching Siwash. This makes his ’steenth year. I guess he can stay there forever. He’s coached all these years and has never used the same adjective to the same man twice. There’s a record for you! He’s a little man, Bost is. He played end on some Western team when he only weighed one hundred and forty. Got his football knowledge there. But where he got his vocabulary is still a mystery. He has a way of convincing a man that a dill pickle would make a better guard than he is, and of making that man so jealous of the pickle that he will perform perfectly unreasonable feats for a week to beat it out for the place. He has a way of saying “Hurry up,” with a few descriptive adjectives tacked on, that makes a man rub himself in the stung place for an hour; and oh, how mad he can make you while he is telling you pleasantly that while the little fellow playing against you is only a prep and has sloping shoulders and weighs one hundred and eleven stripped, he is making you look like a bale of hay that has been dumped by mistake on an athletic field. And when he gets a team in the gymnasium between halves, with the game going wrong, and stands up before them and sizes up their nerve and rubber backbone and hereditary awkwardness and incredible talent in doing the wrong thing, to say nothing of describing each individual blunder in that queer nasal clack of his—well, I’d rather be tied up in a great big frying-pan over a good hot stove for the same length of time, any day in the week. The reason Bost is a great

coach is because his men don’t dare play poorly When they do he talks to them. If he would only hit them, or skin them by inches, or shoot at them, they wouldn’t mind it so much; but when you get on the field with him and realize that if you miss a tackle he is going to get you out before the whole gang and tell you what a great mistake the Creator made when He put joints in your arms instead of letting them stick out stiff as they do any other signpost, you’re not going to miss that tackle, that’s all.

When Bost came to Siwash he succeeded a line of coaches who had been telling the fellows to get down low and hit the line hard, and had been showing them how to do it very patiently. Nice fellows, those coaches. Perfect gentlemen. Make you proud to associate with them. They could take a herd of green farmer boys, with wrists like mules’ ankles, and by Thanksgiving they would have them familiar with all the rudiments of the game. By that time the season would be over and all the schools in the vicinity would have beaten us by big scores. The next year the last year’s crop of big farmer boys would stay at home to husk corn, and the coach would begin all over on a new crop. The result was, we were a dub school at football. Any school that could scare up a good rangy halfback and a line that could hold sheep could get up an adding festival at our expense any time. We lived in a perpetual state of fear. Some day we felt that the normal school would come down and beat us. That would be the limit of disgrace. After that there would be nothing left to do but disband the college and take to drink to forget the past.

But Bost changed all that in one year. He didn’t care to show any one how to play football. He was just interested in making the player afraid not to play it. When you went down the field on a punt you knew that if you missed your man he would tell you when you came back that two stone hitching-posts out of three could get past you in a six-foot alley. If you missed a punt you could expect to be told that you might catch a haystack by running with your arms wide open, but that was no way to catch a football. Maybe things like that don’t sound jabby when two dozen men hear them! They kept us catching punts between classes, and tackling each other all the way to our rooms and back. We simply had to play football to keep from being bawled out. It’s an awful thing to have a coach with a tongue like a

cheese knife swinging away at you, and to know that if you get mad and quit, no one but the dear old coll. will suffer—but it gets the results. They use the same system in the East, but there they only swear at a man, I believe. Siwash is a mighty proper college and you can’t swear on its campus, whatever else you do. Swearing is only a lazy man’s substitute for thinking, anyway; and Bost wasn’t lazy. He preferred the descriptive; he sat up nights thinking it out.

We began to see the results before Bost had been tracing our pedigrees for two weeks. First game of the season was with that little old dinky Normal School which had been scaring us so for the past five years. We had been satisfied to push some awkward halfback over the line once, and then hold on to the enemy so tight he couldn’t run; and we started out that year in the same old way. First half ended 0 to 0, with our boys pretty satisfied because they had kept the ball in Normal’s territory. Bost led the team and the substitutes into the overgrown barn we used for a gymnasium, and while we were still patting ourselves approvingly in our minds he cut loose:

“You pasty-faced, overfed, white-livered beanbag experts, what do you mean by running a beauty show instead of a football game?” he yelled. “Do you suppose I came out here to be art director of a statuary exhibit? Does any one of you imagine for a holy minute that he knows the difference between a football game and ushering in a church? Don’t fool yourselves. You don’t; you don’t know anything. All you ever knew about football I could carve on granite and put in my eye and never feel it. Nothing to nothing against a crowd of farmer boys who haven’t known a football from a duck’s egg for more than a week! Bah! If I ever turned the Old Folks’ Home loose on you doll babies they’d run up a century while you were hunting for your handkerchiefs. Jackson, what do you suppose a halfback is for? I don’t want cloak models. I want a man who can stick his head down and run. Don’t be afraid of that bean of yours; it hasn’t got anything worth saving in it. When you get the ball you’re supposed to run with it and not sit around trying to hatch it. You, Saunders! You held that other guard just like a sweet-pea vine. Where did you ever learn that sweet, lovely way of falling down on your nose when a real man sneezes at you? Did you ever hear of sand? Eat it! Eat it! Fill your

self up with it I want you to get in that line this half and stop something or I’ll make you play left end in a fancy-work club. Johnson, the only way to get you around the field is to put you on wheels and haul you. Next time you grow fast to the ground I’m going to violate some forestry regulations and take an axe to you. Same to you, Briggs. You’d make the All-American boundary posts, but that’s all. Vance, I picked you for a quarterback, but I made a mistake; you ought to be sorting eggs. That ball isn’t red hot. You don’t have to let go of it as soon as you get it. Don’t be afraid, nobody will step on you. This isn’t a rude game. It’s only a game of post-office. You needn’t act so nervous about it. Maybe some of the big girls will kiss you, but it won’t hurt.”

Bost stopped for breath and eyed us. We were a sick-looking crowd. You could almost see the remarks sticking into us and quivering. We had come in feeling pretty virtuous, and what we were getting was a hideous surprise.

“Now I want to tell this tea-party something,” continued Bost. “Either you’re going out on that field and score thirty points this last half or I’m going to let the girls of Siwash play your football for you. I’m tired of coaching men that aren’t good at anything but falling down scientifically when they’re tackled. There isn’t a broken nose among you. Every one of you will run back five yards to pick out a soft spot to fall on. It’s got to stop. You’re going to hold on to that ball this half and take it places. If some little fellow from Normal crosses his fingers and says ‘naughty, naughty,’ don’t fall on the ball and yell ‘down’ until they can hear it uptown. Thirty points is what I want out of you this half, and if you don’t get ’em—well, you just dare to come back here without them, that’s all. Now get out on that field and jostle somebody. Git!”

Did we git? Well, rather. We were so mad our clothes smoked. We would have quit the game right there and resigned from the team, but we didn’t dare to. Bost would have talked to us some more. And we didn’t dare not to make those thirty points, either. It was an awful tough job, but we did it with a couple over. We raged like wild beasts. We scared those gentle Normalites out of their boots. I can’t imagine how we ever got it into our heads that they could play football, anyway. When it was all over we went back to the gymnasium