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Silent learning

Citation for published version:Rossato, JI, Moreno, A, Genzel, L, Yamasaki, M, Takeuchi, T, Canals, S & Morris, RGM 2018, 'Silentlearning', Current Biology, vol. 28, no. 21, pp. 3508-3515.e5. https://doi.org/10.1016/j.cub.2018.09.012

Digital Object Identifier (DOI):10.1016/j.cub.2018.09.012

Link:Link to publication record in Edinburgh Research Explorer

Document Version:Peer reviewed version

Published In:Current Biology

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Download date: 21. Mar. 2021

Rossato et al - 1

Draft66:04September20181

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Silent learning 3

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JanineIRossato*1,AndreaMoreno*1,2,LisaGenzel1,MiwakoYamasaki3,Tomonori5

Takeuchi1,SantiagoCanals2andRichardGMMorris1,26

71CentreforCognitiveandNeuralSystems,8

EdinburghNeuroscience,9UniversityofEdinburgh,1GeorgeSquare,EdinburghEH89JZ,UK10

112InstitutodeNeurociencias,CSIC-UMH,SanJuandeAlicante,Spain12

and133DepartmentofAnatomy,14

HokkaidoUniversityGraduateSchoolofMedicine,15Sapporo,Hokkaido,060-8638,Japan16

17

*Co-firstauthorship18

19

CorrespondingAuthorandLeadContact:RichardGMMorris20

(r.g.m.morris@ed.ac.uk);NowatCentreforDiscoveryBrainSciences(Edinburgh)21

22

23

24

Rossato et al - 2

SUMMARY 25

26

We introduce the concept of 'silent learning' - the capacity to learn despite27

neuronalcell-firingbeinglargelyabsent.Thisideaemergedfromthinkingabout28

dendriticcomputation[1,2]andexaminingwhethertheencoding,expressionand29

retrievalofhippocampal-dependentmemorycouldbedissociatedusingtheintra-30

hippocampal infusion of pharmacological compounds. We observed that very31

modest enhancement of GABAergic inhibition with low-dosemuscimol blocked32

bothcell-firingandtheretrievalofanalreadyformedmemory,butleftinduction33

of long-termpotentiation(LTP)andnewspatialmemoryencodingintact(silent34

learning). In contrast, blockade of hippocampal NMDA receptors by35

intrahippocampalD-AP5impairedboththeinductionofLTPandencoding,buthad36

noeffect onmemory retrieval. BlockadeofAMPA receptorsbyCNQX impaired37

excitatorysynaptictransmissionandcell-firing,andbothmemoryencodingand38

retrieval.Thus,inkeepingwiththesynapticplasticityandmemoryhypothesis[3],39

thehippocampalnetworkcanmediatenewmemoryencodingwhenLTPinduction40

isintactevenunderconditionsinwhichsomaticcell-firingisblocked.41

42

RESULTS 43

44

Encodingand retrievalofdeclarativememoryare the twosidesof a coinwith45

respect to the neural mechanisms of learning and memory. Encoding refers to the46

acquisitionofnewinformation,whereasretrievalinvolvesthereactivationofpreviously47

learnedmemorytraces.Identifyingtheneuralactivityassociatedwithspecificmemory48

processessuchastheseisanecessarysteptounderstandofhowinformation-processing49

circuitsoperate. The present study testswhether (a)memory retrieval requires cell-50

firing, enabling information transfer within and between networks; and (b) memory51

encodingmayminimallyrequiretheinductionandexpressionofsynapticplasticity,with52

littleornosomaticcell-firing.Theoccurrenceoflearningwouldnotbeobservable,but53

wearguethatrecentadvancesinthephysiologyofdendriticcomputationpredictsuch54

'silentlearning'couldoccur.55

56

Rossato et al - 3

Loss-of-function manipulations such as lesions, drugs and molecular57

interventionshavelongbeendeployedtolookfor‘learningimpairments’inspecifictasks.58

Manystudiesfocusonmisleadinglearningcurvesduringanintervention[4,5]thatlikely59

reflectimpactsonencoding,storage,consolidationand/orretrievalwithoutdissociating60

theirrelativecontributions.Lesionscanalsocause‘performance’effects(i.e.deleterious61

effects upon sensorimotor processes or motivation). Specific protocols to dissociate62

encodingandretrievaldefinitivelyinclude:forretrieval-monitoringperformanceonthe63

firsttrialofnewtrainingbeforeanynewlearningcantakeplace[6];andforencoding-64

applicationof the interventionduring thenewtraining,but testing later in itsabsence65

whenretrievalshouldbeoperatingnormally[5].Asuitableprotocolforthewatermazeis66

thedelayed-matchingtoplace(DMP)or'everydaymemory'procedurewhichinvolves67

learninganewdailyspatiallocationoftheescapeplatformduringeachsession[7].The68

principleisthat,oneachsession,theanimalsfirstretrieveamemoryofwhereescape69

waspossibleduring theprevious session, and thenupdate theirmemorybyencoding70

wheretheescapeplatformisnowlocatedduringthecurrentsession.Theeffectiveness71

ofthismemoryencodingistestedonasubsequentsessioninwhichtheanimalsshould72

againdemonstratememoryoftheprecedingsession.73

74

Themainstudyweexaminedthe impactofdrugsover5successiveseriesof375

linkedsessions(hereaftercalleds1,s2ands3)inawithin-subjectmanner(Figure1A).It76

followedanimalhandling,bilateraldrugcannulaimplantationandinitialtrainingover8–77

10sessions,duringwhichthe16animalslearnedtheDMPtaskwelleachdaywithanew78

platformlocationchosenforeachsessionof4learningtrials(Figures1A,B,E).Ananimal79

mightbetrainedtoencodethattheescapeplatformisintheNW(North-West)quadrant80

onall4 trialsof session1 (s1,Figure1B).On thenext session (s2),usinganAtlantis81

Platformprocedure[8]inwhichmemoryretrievalisassessedduringthefirst60softhe82

firsttrialbeforeanynewlearningtakesplace(Figures1B,C,DandS1),theanimalshould83

rememberthisNWlocation(reddottedcircle)bysearchingthere(duringthefirst60s)84

beforelearningthatthehiddenplatformhadbeenmovedtoSSW(South-South-West).85

Encodingof thisnewlocationoccursduringthe fourescapetrialsofs2,updatingand86

over-writing the memory acquired in s1. The platform is moved again for s3, again87

allowing an analogous test ofmemory during the first 60 s. In this protocol,memory88

retrievalisprocedurallydissociatedfromnewmemoryencoding.89

Rossato et al - 4

90

Thisprocedureenabledeffectivememoryencodingofeachdaily location,with91

performance typically characterised by a stable monotonic decline in first-crossing92

latencyacrosstrialswithineachsession(Figure1E;DataS1).Theswim-latencyontrial93

1 (≃50-70 sec) was stable across sessions, as was the ‘savings’ in latency of94

approximately30sbetweentrials1and2ofeachsession.Asymptoticperformanceover95

trials2-4reflectstheeffectivenessofmemoryupdating.96

97

InvivoHippocampalPhysiology98

Previous studies have established that blocking hippocampal N-methyl-D-99

aspartate(NMDA)receptors,viapharmacological [D-2-amino-5-phosphonopentanoate100

(D-AP5)]ormolecular-geneticinterventions,limitsmemoryencodingwithouteffecton101

retrieval[7,9-12].Thisisobservedwith1-2μlbilateralinfusionsintodorsalhippocampus,102

withautoradiographyindicatingsubstantialspreadalongthelongitudinalaxisfollowing103

a 2μl infusion[13]. This behavioural pattern is mechanistically linked to blockade of104

NMDA receptor-mediated activity-dependent synaptic plasticity[3, 14]. Blocking a-105

amino-3-hydroxy-5-methyl-4-isoxazolepropionate(AMPA)receptorspharmacologically106

limits encoding, consolidation and retrieval [15]. Prior focus on AMPA and NMDA107

receptors left uninvestigated the possible contribution ofg-aminobutyric acid (GABA-108

ergic)inhibitionwhichisknowntoregulatelong-termpotentiation(LTP)induction[14].109

The importance of dynamic patterns of inhibitory activity is now recognised as110

functionally important[16, 17], along with learning-associated changes in inhibitory111

circuitrythatcanaffectthefidelityofmemory[18,19].112

113

Using in vivo electrophysiology in male Lister Hooded rats (n=20) to identify114

appropriatedrugconcentrationsusingandthetimecourseoftheireffects(Figure2),we115

sought drug doses that would differentially affect (a) cell-firing, (b) fast synaptic116

transmission,and/or(c)activity-dependentsynapticplasticityinvivointhehippocampal117

formation. We chose to monitor the dentate gyrus electrophysiologically, while118

recognisingthatan infusiontargetingtheoutermolecularof thedorsaldentategyrus119

woulddiffusethroughoutdorsalCA1andCA3aswell.Inthecaseofmuscimol,itshould120

incur reasonably widespread binding to somatic and dendritically located GABAA121

receptors.122

Rossato et al - 5

123

A key new finding is that low-dose muscimol blocked cell-firing but not LTP124

induction.Infusionof0.38nanomolesofmuscimolcausedamodest35%decreaseofthe125

fieldexcitatorypostsynapticpotential(fEPSP)(Figures2A,B;DataS2),butthedentate126

populationspikeceasedalmostcompletelyfrom30minpost-infusionfor2hr(measured127

at 1.0 to 1.5 mm from the infusion cannula; Figures 2C,D). Strikingly, low-dose of128

muscimol infusion left induction of LTP intact (Figures 2E,F; Data S2). Enhanced129

GABAergic inhibitionnormallyblocks the inductionofLTP[14],butourdose titration130

downto0.38nanomolesachievedasituationinwhichLTPinductionwasintactdespite131

theabsenceofpre-inductioncell-firing.Thislowdoseofmuscimoldidnotpreventavery132

small population spike post-LTP (Figure S3; <3 mV), a change that is unlikely to be133

relevant to themoredistributedpatternsoflearning-associateddendriticandsomatic134

neuralactivityinthefreely-movinganimal(seebelow).135

136

In contrast,D-AP5 (60nanomoles) causeda transitorydisruptionof the fEPSP137

beforeareturntobaselinewithin30min,andapartialalbeitmoresustainedinhibition138

ofthepopulationspike(Figures2A–D;DataS2).However,asexpected,itblockedLTP139

induction(Figures2E,F;DataS2).CNQX(6nanomoles)causedthefEPSPtobecompletely140

inhibitedwithin30minwithrespect tobothsynapticactivationandcell-firing(>90%141

decreaseforover1hr,Figures2A–D).IntheabsenceofameasureablefEPSP,CNQXwas142

nottestedwithrespecttoLTP.143

144

SilentLearning145

Thestagewasnowsettoconductthecompanionbehaviouralstudyusingmale146

ListerHoodedrats(n=16)trainedinthetaskandnowsubjecttointrahippocampaldrug147

infusions. Eachsessionwasconducted ‘blind’withrespect todrug-assignment[7,20],148

usingafullycounterbalancedrepeated-measureswithin-subjectsdesign,suchthateach149

animalservedasitsowncontrolacrosssuccessive‘linked’sessions,consistingofapre-150

drugsession(s1),drugsession(s2)andpost-drugsession(s3)withineachblock(Figure151

1A).152

153

Our second key finding was ‘silent learning’ with low-dose muscimol.154

Representative swim-paths show Rat-G7207 treated with muscimol searching155

Rossato et al - 6

appropriatelyon s1,but swimmingall over thepoolons2withoutmemoryof the s1156

platformlocationuntil iteventually foundthenewescape locationafter>60s;onthe157

followingdrug-free s3, thisrat searchedsuccessfully ina focusedzonearound the s2158

location (Figure 3A, panel with pool shaded blue, note multiple crossings of the s2159

locationons3).Thispatternofsearchingbehaviourimpliesthatmemoryencodingwas160

intact during s2 under muscimol, despite memory expression being blocked.161

Representativepathsofotherratsshowrespectively:goodmemoryforeachprevious162

session for aCSF; goodmemory retrieval but no new learning under D-AP5; and no163

memory retrievalornew learningunderCNQX.Quantitatively,weobserved adouble164

dissociationbetweentheimpactofthethreedrugsonmemoryencodingandretrieval165

(Figure3B; two-wayANOVA: significantDrug× Sessions interaction: F6,90 = 3.65, p =166

0.003). This statistical interaction justified separate analyses of each drug condition167

comparedtoaCSFvehicle,aswellasplannedcomparisonstochance-levelperformance.168

169

Vehiclesessions(aCSF)showedgoodabovechancememory(chance=4%,dotted170

line)acrossallthreesessions(Figure3B,blackbars;DataS3).Withlow-dosemuscimol,171

the animalswere at chance on s2with the animals failing to remember the previous172

session, but above chance for the location trained under the drug on s2when tested173

during trial1of s3 (noteU-shaped function inFigure3B;DataS3).That is, low-dose174

muscimolwaspermissivefornewencodingdespitecausingacompleteblockofmemory175

retrieval.TheoppositepatternprevailedwithD-AP5,withabovechanceretrievalofs1176

on trial1of s2,but chanceperformanceduring trial1ofs3.WithCNQX, theanimals’177

memoryofs1displayedontrial1ofs2andtheirmemoryofs2ontrial1ofs3wereboth178

atchance.Thus,CNQXtreatedratscouldneitherretrievenorencode.179

180

Inthisprotocol,theanimals‘update’theirmemoryduringeachsession-akinto181

theconceptof‘headedrecords’inwhichhumansubjectsoftenrememberthelastthing182

that happened but tend to overwrite earlier events[21]. Updating should only be183

observedifnewlearningoccursons2;thus,adistinctpatternofdrugeffectsisexpected184

ons3.Thespecificprediction is thatD-AP5andCNQXwouldblockmemoryupdating,185

whereasaCSFandlow-dosemuscimolwouldbothbepermissiveofmemoryoverwriting.186

Wequantifiedtheswimsearchpatternons3withrespecttotheproportionoftimespent187

inthecorrectzonefors1(i.e.2sessionsback;Figures3C,D)andcomparedthesevalues188

Rossato et al - 7

tothosefors2(1sessionback).TheoverallANOVAwasthusa2×4analysisforthetime189

spentsearchingdurings3 inthe locationsusedons1andons2asa functionof the4190

drugs,revealingahighlysignificantSession-Memory×Druginteraction(F3,45=7.35,p=191

0.001).Unpacking this triple interactionusingplannedcomparisonsrevealed that the192

averagelevelofmemorymeasuredons3afterCNQXorD-AP5durings2washigherfor193

thes1locationthanfors2(i.e.minimalupdating);whereas,withaCSFandmuscimol,the194

oppositepatternprevailed.Theorthogonalcomparisonforthiscontrastwassignificant,195

butagraphicallysimpleranalysisistolookattheabsolutelevelofmemoryforthes1196

locationthatisexpresseddurings3(Figure3C).TheANOVAforthesedataalsoshowed197

asignificantdrugeffect(F3,45=3.64,p<0.05).Aspredicted,memoryforthes1location198

afteraCSFormuscimolhadbeen infusedons2was lowerthanwhenD-AP5orCNQX199

wereinfused(F1,45=8.65,p<0.01;Figure3C).Thissuccessfulupdatingundermuscimol200

ons2arguesagainsttheretrievaldeficitdisplayedunderthedrugons2beingamere201

‘performance’, ‘off-target’ or 'state-dependent' effect, as it is unclear howsuch effects202

couldselectivelyaffectretrievalbutnotmemoryencoding.Illustrativepathsareshown203

followingtheadministrationofaCSF(successfulupdating)andCNQX(noupdating)on204

differents2sessions(Figure3D).205

206

A concernwas that intactmemory encodingwith low-dosemuscimol is some207

artefact of differential spread of the drug, the most likely possibility being that the208

infusionwasrestrictedtoasmallregionofthedorsalHPC.Thismighthavebeensufficient209

todisrupt cell-firingduringretrieval andpattern completion,but insufficient toaffect210

newlearningwithinalargervolumeofunaffecteddorsalandintermediatehippocampus.211

TheproblemwiththisinterpretationisthatthediffusionofD-AP5andCNQXislikelyto212

havebeensimilartothatofmuscimol(MWs=197,232and114respectively),andthus213

newencodingshouldalsohaveoccurredforthesedrugs-whichitdidnot.Thissuggests214

thatourdeliberatechoiceofa2µlinfusionvolumeachievedsubstantialspreadalongthe215

longitudinalaxis.Anticipatingthis,weconductedadditionalelectrophysiologicalstudies216

withrecordingintheintermediatezoneofthelongitudinalaxisofhippocampusfollowing217

infusionofmuscimolintothedorsal/septalregion(infusion2.5mmfromtherecording218

electrode,FigureS2B).InhibitionofthefEPSP(FiguresS2C,E,circa23%))wasslightly219

lessthanatthemoreproximalrecordingsite(35%)but,importantly,cell-firingremained220

almostcompletelyblocked(circa86%,FiguresS2D,E).Wealsoattemptedtolookatdrug221

Rossato et al - 8

diffusiondirectlyusingfluorescentmuscimol[22].Theanalysisof3animalssubjectto222

bilateral2μlinfusionsof0.19mMfluorescentmuscimolbodipy,indicateddiffusionalong223

the longitudinal axis of up to 3.5 mm (Figure S2F,G), with minimal spread into224

retrosplenialcortex(RSC)oroverlyingparietalcortex.Butthismeasureisconservative225

asthemolecularweightoffluorescentmuscimol(MW=607)ismuchlargerthanthatof226

muscimolitself(MW=114),anditismorelipophilicbyvirtueofthefluorescentlabel.227

228

Athirdbutunlikelypossibilityisthatdrugdiffusionreachesbeyondhippocampus229

toretrosplenialcortex,longimplicatedinspatialmemory[23].Onthisview,muscimolin230

RSCmaybecontributingtotheimpairedmemoryretrieval.Wehadhopedthatdataon231

diffusionoffluorescentmuscimolcoulddefinitivelyaddressthisissue,butitisunclear232

howthisaccountwouldcouldexplaineffectivenewmemoryencodinginRSC.Extensive233

diffusionofmuscimolitselfissurelyunlikelyasthecloselypacked,myelinatedfibresof234

theoverlyingalveusandcorpuscallosumwouldrestrictthisfromhappening.Asinour235

earlierautoradiographicandregionalcerebralbloodflowstudiesofglutamatereceptor236

antagonists [15, 24], there were non-spherical ‘rugby-ball’ shaped diffusion volumes237

withinhippocampus,alsoobservedwithfluorescentmuscimol[22].Somedisruptionto238

cell-firinginRSCmightnonethelesscontributetotheretrievaldeficitseenwithmuscimol239

and CNQX, possibly by affecting the translation of memory representations from240

allocentrictoegocentrictoenableaccurateheadingtotherememberedescapelocation.241

242

DISCUSSION243

The present findings point to a new conceptwhichwe shall refer to as ‘silent244

learning’ - new memory encoding in the absence of cell-firing. Silent learning245

corresponds behaviourally to new episodic-like memory encoding in the absence of246

memoryretrieval. WesuggestthatthiscansometimesoccurifLTPinductionisintact247

during cellular silence, allowing activity-dependent synaptic potentiation to encode a248

newspatialmemoryasadistributedpatternofpotentiatedsynapsesinthehippocampus249

(dentate,CA3and/orCA1).Cell-firingwouldnotalwaysbenecessary.250

251

Fromabehaviouralperspective,theconceptof‘silentlearning’isdistinctfromthe252

classicalconceptof‘latentlearning’whichreferstosuccessfullearningintheabsenceof253

reward[25].LatentlearningwasachallengeforHull’sdrivereductiontheory[26]which254

Rossato et al - 9

supposedthatanimalshadtobemotivatedtolearn(‘drive’)andthatstimulus-response255

connectionswere‘stamped-in’by‘drive-reduction’followingreward.Silentlearningis256

different, being more consonant with Tolman’s ‘cognitive map’ theory[25] which257

assertedthatlearningcouldoccurintheabsenceofreward,resurrectedinO’Keefeand258

Nadel’stheoryofhippocampalfunction[27].259

260

The distributed associative neural circuit of the hippocampus[28] has been261

proposedtooperateindistinctencodingandretrievalmodesatdifferentphasesofthe262

thetarhythm[29].Wereasonedthatitmightbepossibletorealiseadoubledissociation263

of the processes of memory encoding and retrieval as a function of task demands.264

Blocking NMDA receptor activation is permissive for memory retrieval but prevents265

encoding[9, 12],whereasAMPA receptor inhibition blocks both[30]. Ournew finding266

indicates that, even though memory retrieval fails to occur when cell-firing in the267

hippocampus is blocked by low-dose muscimol, new encoding can occur provided268

synaptic plasticity is intact and fast synaptic transmission only modestly affected.269

Although not accompanied by electrophysiological analysis, a previous behavioural270

pharmacology studywas also suggestive of such a possibility,with spared ‘extinction271

learning’beingobservedinaninhibitoryavoidancetaskduringintrahippocampallow-272

dosemuscimol (approximately twice as high aswe used[31]). Impaired retrieval has273

beenshowntooccurwithhigherdosesofmuscimol[32],butthepossibilityofintactor274

impairedlearningwasnotinvestigated.275

276

Thequalificationisthepossibilityofacute‘off-target’alterationofneuralcircuits277

(e.g.RSC)thatwerenotdirectlyenvelopedbythemuscimolinfusion.Alterationsinthe278

level of learning-associated immediate early-gene expression in RSC are seen as a279

networkeffectfollowinglesionsofthehippocampalformation[23].Wesuspectinstead280

that enhanced inhibition targets the complexity of inhibitory circuitry in281

hippocampus[16] coupled to dynamic changes of parvalbumin-positive GABAergic282

inhibitionassociatedwith learning[19].Our findings suggest that 'on-target' effectsof283

muscimolareamorelikelyexplanation.284

285

From a physiological perspective, this interpretation requires that synaptic286

plasticity can sometimesoccur in theabsenceof cell-firing. Indeed, thismaybemore287

Rossato et al - 10

frequent thangenerallyrealisedandhasbeenconsideredsincetheoriginalpaperson288

LTP[33,34].Theconceptuallyinterestingideaisthatanetworkmaysometimesbeable289

tochangethepatternofsynapticweightsonitsinputsynapses'secretly'fromneurons290

furtherdownstream.Ourelectrophysiologyfocusedontheperforantpathinputtothe291

dentategyrus,butasimilar 'silent learning'effectmayoccurontheentorhinalcortex292

layer III input toCA1andontheentorhinal cortex layer II input toCA3; itwouldnot293

necessarilyrequireactivationofrecurrentcircuitryinCA3.Understandingthedetailed294

physiologicalmechanismwasnottheprimarypurposeofthisinitialstudy,butcomments295

relevant to such a future project are appropriate. First, the complexities of dendritic296

inhibitioninthehippocampusmightallowforafailureofmemoryretrievaltobecaused297

byablockofcell-firingduetomuscimolactivationofGABAAreceptorsexpressedonthe298

cell soma of hippocampal excitatory neurons innervated by parvalbumin-positive299

GABAergicinterneurons[16,17,35].AnimpactofGABAAreceptorsinthedendritesmay,300

however,reflectadifferentialeffectontonicratherthanphasicinhibition[36,37],arising301

because low-dosemuscimol acts preferentially (but not exclusively) at extra-synaptic302

GABAA sitesmediating tonic inhibition. Amodest increase in tonic inhibitionmay be303

permissive for postsynaptic backpropagating dendritic spikes[1, 2, 38]. A further304

possibilityisthataugmentedGABAAmediatedinhibitionindendritesmayleaveintrinsic305

changes indendriticmembranepotentialunaffected, and thesearenowknown tobe306

permissive for place-cell formation[39] and behavioural time-scale synaptic307

plasticity[40].Addressing thesedistinctpossibilitieswillnotbeeasy in freely-moving308

animals. The retrieval/encoding dissociation might be examined optogenetically or309

chemogenetically[41,42]usingappropriatepromoterlinesthatwouldallowdifferential310

targeting of distinct GABAergic neurons. Interestingly, the possibility that changes in311

excitation-inhibitionbalanceisrelevanttounmaskinglatentmemoryhasalsorecently312

beenstudiedinhumans[43].313

314

To summarise, the phenomenon of ‘silent learning’ in the awake animal is315

compatible with dendritic computation and the complexity of inhibitory network316

connectivityinthehippocampus.Itsuggeststhatsynapticplasticitycanlurkcryptically317

under conditions inwhich thenetworkexpressionofnewmemorytrace formation is318

preventedbysomatic inhibition. Ithasnotescapedournoticethatsuch learningmay319

Rossato et al - 11

occurmoreoftenthanisgenerallyappreciatedand,indeed,beacharacteristicofseveral320

aspectsofcognitivelearning.321

322

Rossato et al - 12

ACKNOWLEDGEMENTS323

324

Wethanktheanonymousreviewersfortheirvaluableinput,especiallyontheissueof325

drug diffusion. Jane Tulloch and Patrick Spooner provided technical assistance, and326

StuartCobband IrisOrenofferedcommentsonearlierdraftsof themanuscript.This327

work was supported by an ICT-FET grant from the European Union (GRIDMAP), a328

European Research Council Advanced Investigator Award (NEUROSCHEMA-268800),329

andtheBrazilianNationalCouncilforScientificandTechnologicalDevelopment(CNPq),330

and the Spanish State Research Agency (MINECO) and FEDER funds under grants331

BFU2015-64380-C2-1-Randthe“SeveroOchoa”ProgrammeforCentresofExcellencein332

R&D(ref.SEV-2013-0317).JanineRossatoisnowatUniversidadeFederaldoRioGrande333

do Norte and the Brain Institute, Natal, RN, Brazil. Lisa Genzel is now at Radboud334

University in Nijmegen, Holland. AndreaMoreno and Tomonori Takeuchi are now at335

AarhusUniversity,Denmark.TheCentreforCognitiveandNeuralSciencesinEdinburgh336

isnowassimilated intotheCentre forDiscoveryBrainScienceswhichisRM'scurrent337

affiliation.338

339

340

AUTHORCONTRIBUTIONS341

342

RM conceived the study. JR conducted the behavioural studies, AM the343

electrophysiological studies, and MY the drug diffusion studies; LG, TT, SC and RM344

prepared the manuscript. We acknowledge the considerable contribution of three345

anonymousreviewers.346

347

348

DECLARATIONOFINTERESTS349

350

Theauthorsdeclarenocompetinginterests.351

352

353

Rossato et al - 13

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482

Rossato et al - 16

FIGURE LEGENDS 483

484

Figure1.ANovelWatermazeProtocoltoDissociateEncodingandRetrieval485

(A) The experimental design with animal handling, drug-cannula implantation, DMP486

training for 8–10 sessions, followed by the series of 3 linked sessions for the drug487

infusionsinterspersedwithanadditionalinterleavedtraining.488

(B)Aerialdrawingsofthewatermazeacross3linkedsessions(repeatedacrosssessions489

fordifferentdrugconditions),withexemplardailylocationsofthesingleescapeplatform490

(blackcircles;seeFigureS1).Thelocationonaspecificsession1(s1;blackcontinuous491

circleinNWquadrant)andthatonthenextsession2(s2;blackcircleinSWquadrant)492

areeachshown,withthedottedlineinblackreflectingwheretheplatformwasavailable493

after60s,andthecontinuous lineshowing itbeingavailable(60–120s;itwasalways494

hidden below thewater). The black filled parts of the cartoons are the platforms (in495

proportion)andthesurroundblackcircleistheassociatedanalysiszone(20cmdiameter,496

centredoneachlocation,4%ofareaofpool).Thelocationusedons1isshownfors2as497

areddottedline(the'memory'locationofs1,butnotactuallyusedons2;likewisefors3498

withrespecttos2).Notetheplatformalwaysstayedinthesamelocationforall4trials,499

andwasavailableimmediatelyforescapeontrials2–4.500

(C)AtlantisPlatformthatisunavailableforthefirst60softrial1ofeachsession,but501

thenrisestonearthewatersurface.Cartoonsbelow(dottedandcontinuousline)areas502

inpanelB.503

(D)Aseriesof3linkedsessionswhendrug(oraCSF)isadministered30min(CNQX)or504

40min(muscimolandD-AP5)befores2.Thesamelocationsaredisplayedasin(B).Note505

thatthefirst60softrial1ofs2offeredtheopportunitytoretrievethememoryofs1(red506

continuousline),whiletrials1-4ofs2aretheopportunitiesfornewmemoryencoding[of507

black continuous circle in SW (south-west) quadrant]. When encoding was blocked508

durings2byadrug,preventingupdating,s3mayhaveofferedtheopportunitytoretrieve509

thelocationons1(reddottedline).510

(E)Meanfirst-crossinglatenciesontrials1-4ofmultipleinterleavedtrainingsessions.511

Thesetrainingsessionsareinterspersedbetweenthesuccessive3linkedsessions(grey512

bars).Means±SEM.513

514

Rossato et al - 17

Figure2.ImpactandTime-CourseofDistinctDrugsonHippocampalPhysiologyin515

vivo516

(A)Differentialimpactof2μlinfusionsofmuscimol(0.38nmoles,orange),D-AP5(60517

nmoles,pink)andCNQX(6nmoles,blue)ontheearly-risingslopeofevokedfEPSPsin518

thedentategyrustoperforantpathstimulation,normalisedtothepre-drugbaseline(n=519

5forallconditions).Arrowpointstothedruginfusion;yellowshadingindicateswhen520

thebehaviouraltestisapplied(40–70minlater).521

(B)NormalisedaveragedfEPSPvaluesforbaseline,endoftestperiod(aftertest),and3522

hrpost-infusion(end),normalizationtoaCSFbaseline.523

(CandD)Normalizedchangesinthepopulationspike(PS).524

(E)ImpactofaCSF,muscimolandD-AP5ontheinductionofLTP(yellowlightningmarks),525

withthefEPSPslopere-normalisedwithrespecttothepre-tetanusbaseline(10min)and526

comparedtothecorrespondingdrug-treatedgroupthatwasnotsubjecttotetanisation.527

AkeynewfindingisintactLTPinductionunderlow-dosemuscimolaswellasaCSF,but528

not D-AP5. Yellow shading reflects the daily timing and duration of the behavioural529

experiment.LTPdataplottedforthattime.ForabsolutePSdata,seeFigureS3.530

(F)AveragedLTPdata.531

(G)Schematicofstimulatingandrecordingelectrodes,anddrugcannulalocationsofrat532

braininvivo.DetailinFigureS2.Pairedtwo-tailedttest(versuschance):**p<0.01,****p533

<0.0001.Means±SEM.534

535

Figure 3. Impact of Distinct Pharmacological Manipulations on Encoding and536

Retrieval,andonMemoryUpdating537

(A) Illustrativepathsofrepresentativeratsduringtrial1of3 linkedsessions forall4538

drugconditions.Blackplatform=locationthatsession;continuousredline=locationon539

previoussession;dottedredline=platformlocationtwosessionsback;smallgreencircle540

=startofswimpath;smallbluecircle=endofswim-path;dottedblueline=latterpart541

oftheswimpath,notcalculatedinthememoryretrievaldata,aftertheAtlantisPlatform542

became available. The key finding of silent learning is shown for the representative543

muscimoltreatedanimal.Noterandomsearchalloverthewatermazeons2(duringthe544

drug;middle), but focused search at the s2 location on s3 (right).With aCSF, the rat545

alwayssearchesatorveryclosetotheprevioussessionlocation;withD-AP5,searching546

Rossato et al - 18

isoptimalon s2butat chanceons3;withCNQX, the rat fails toshoweithermemory547

retrievalornewencoding.548

(B)Impactofhippocampaldruginfusiononencodingandretrieval.Quantitativemeasure549

ofsearchinthezonearoundaplatformlocationoneachtrial1of3linkedsessions,with550

drugadministeredons2(drugconditionswerecounterbalancedandgivenblind).Search551

time is plotted for the platform location of the preceding session. Note stable above-552

chanceperformancefors1–s3foraCSFcondition(blackbars),butadifferentpatternin553

eachof the threedrugs.Following theoverallDrugs×Sessions interaction (see text),554

separateANOVAswereconductedforeachdrugovers1–s3(muscimol:F1,45=4.49,p=555

0.029;D-AP5:F1,45=4.53,p=0.007;CNQX:F1,45=18.37,p=0.001).Thekeyfindingwas556

chanceperformanceinmuscimolconditionons2butrecoveryduringretrievalons3.557

(C)Impactofhippocampaldruginfusiononmemoryupdating.Dissociableimpactofthe558

drugsonmemoryupdatingasmeasuredons3. In theaCSFandmuscimol conditions,559

memoryofs1durings3wasatchance(successfulupdating)andsignificantlybelowthat560

observedforD-AP5andCNQXwhicharebothabovechance(noupdating).561

(D)Representativesearchpathsontrial1ofs3reflectingupdating(aCSF:pathfrequently562

crosses continuous red lineofs2)ornoupdating (CNQX:path frequently crossed the563

dottedredlineofs1).Pairedtwo-tailedttest(versuschance):**p<0.01.4%chance=564

ratioofsurfaceareasofsearchzoneandpoolarea.Means±SEM.565

566

567

STAR§METHODS568

569

Silentlearning570

JanineIRossato*,AndreaMoreno*,LisaGenzel,MiwakoYamasaki,TomonoriTakeuchi,571

SantiagoCanalsandRichardGMMorris572

573

CONTACTFORREAGENTANDRESOURCESHARING574

Furtherinformationandrequestsforresourcesandreagentsshouldbedirectedtoand575

willbefulfilledbytheCorrespondingAuthor,RichardMorris(r.g.m.morris@ed.ac.uk).576

577

EXPERIMENTALMODELANDSUBJECTDETAILS578579

Rossato et al - 19

Rats580

AllexperimentswereperformedonadultmaleListerHoodedRats(200–300gonarrival;581

Charles River, UK) in accordance with the United Kingdom Home Office Animal582

ProceduresAct(1986)conductedunderaProjectLicence(PPL60/4566)heldbyRGM583

Morris. They were kept in a vivarium in the same building on a 12 hr lights on/off584

schedule,ingroupcagesof4ratspercage,withfreeaccesstofoodandwater.585

586

METHODDETAILS587588

BehaviouralapparatusandtheAtlantisPlatform589

Thewatermazeisanapparatusinwhichavarietyofdistinctbehaviouralprotocolscan590

betrained[44].Itconsistsofalargepoolofwater(2mindiameter)fromwhichrodents591

learntoescapeontoahiddenplatformwhosetopsurfaceis1.5cmbeneaththewater592

surface(SupplementaryFigureS1A).Latexsolutionisaddedtorenderthewatercloudy593

andtherebyhidetheescapeplatform.Watertemperatureisregulatedat25°Csuchthat594

escapeisdesirable,buttheprocedureisnotstressfulasthisisonly12–13°Clowerthan595

bodytemperature.596

597

Whilemanystudieshavedeployedareferencememoryprocedureinwhichtheescape598

platformremainsinthesamelocationacrossdays,analternativeistheso-called‘delayed599

matching to place’ (DMP) protocol [7] inwhich the escape platformmoves fromone600

locationtoanother.This‘everydaymemory’protocol,withrepeatedtrainingacrossdays601

and weeks, requires the integrity of the septal (dorsal) and/or intermediate602

hippocampus[45]. Lesions placed at different positions along the longitudinal axis603

damagingupto90%+orlessthan20%+ofthehippocampushaveindicatedthatlearning604

ismediatedviadorsalandintermediatehippocampus,withintactventralhippocampal605

tissuebeing insufficient foreffectiveday-to-day learning.Akeyanalytic featureof the606

DMP protocol is that it allows a clean dissociation between memory encoding and607

memoryretrieval.Performanceistypicallycharacterisedbyalongescapelatencyontrial608

1 as the animal searches for the platform whose location that day is still unknown,609

searchinginitiallyattheprevioussession’slocation,followedbyrapidmemoryencoding610

duringthe30speriodoutofthewaterfollowedbyrelativelydirectpathstothatday’s611

locationontrials2–4.Fourtrialsperdayareusedtoensurethataneffectivememoryis612

Rossato et al - 20

formedofthedailylocationthatcanberecalledduringtrial1ofthenextsession.The613

intervalsbetweentrialscanbevariedsystematically,withthetrial1totrial2interval614

being20mininthisstudy,whiletrial2totrial3,andtrial3totrial4waskeptshortat615

nomorethan5safterthe30speriodontheplatform.Inthiswayalso,therapidlylearned616

strategyoflearningwheretogoineachsession(onesessionperday)ismaintained,even617

inthefaceofinterventionssuchastheapplicationofdrugs.Alargenumberofdifferent618

platformlocationscanbeusedacrosssessions,someonanoutervirtualringandthe619

others on the inner ring (Supplementary Figure S1B). This distribution of possible620

locations(n=24inthisstudy)encourageswidespreadsearching.621

622

Memoryretrievalisdisplayedaspreferentialsearchingontrial1ofeachsessioninthe623

location that the escape platformhad occupied during theprevious session. Thiswas624

quantified by running trial 1 of each sessionas a rewarded probe test, using an ‘on-625

demand’ or ‘Atlantis’ platform [8, 46] (Supplementary Figure S1C) which remained626

inaccessiblefor60s.Thisconsistsofa12cmdiameterescapeplatformmountedona627

stainless-steelspindle,initiallyatthebottomofthepoolandthusunavailable,butwhich628

canbecomputercontrolledtorisetowithin1.5cmofthewatersurfaceon-demand.By629

makingthisplatformunavailablefor60sontrial1,thetrialservesasamemory‘probe’630

fortheprevioussession;theplatformthenrises,theanimalsfinditandthenclimbonto631

itintheusualway.632

633

An overhead camera, associated DVD recorder and on-line analysis software co-634

developed byWatermaze Software (Edinburgh) and Actimetrics (Evanston, USA) are635

usedtomonitorthepathtaken,andmeasurelatency,path-lengthetc.Duringtheinitial636

periodof60softrial1,twoseparatemeasuresofperformancewerecomputed:637

638

• First-crossing latency: is the time in seconds until the animal first crosses the correct 639location (12 cm diameter) where the platform will become available. As the platform is 640not actually available until 60 s has passed, this is not strictly-speaking an ‘escape 641latency’ (the animal keeps swimming), but it serves as a ‘surrogate’ of escape latency. 642This measure was also computed for all 4 trials of the session. 643

• Memory search tendency: The second measure is computed from the time spent 644swimming in a virtual zone of 40 cm diameter centred on the location of the platform 645during the previous session. This time is normalised with respect to the full 60 s of the 646

Rossato et al - 21

trial, and represented as a percentage relative to the 4% area of the pool that the zone 647occupies. This 4% level represents ‘chance’ if the animal were to be swimming around 648the pool randomly. 649 650

ThisDMPtaskoffersanalyticadvantagesoverthereferencememoryprotocol.First,the651

learningofseveraldifferentplatformlocationscanbestudied.Second,oncetheanimals652

havereceivedanumberof initial trainingsessions(8–10sessions),memoryencoding653

andstoragetakesplaceinonesession.Third,reversibleinterventionscanbeintroduced654

(such as intrahippocampal drug infusions, conducted blind) using a within-subjects,655

repeatedmeasuresdesign–withalltheassociatedadvantagesofbothreducedvariability656

andreduceduseofanimals.657

658

Stereotacticsurgery659

Anaesthesiawasinducedusingisoflurane(induction,5%;maintenance,1–2%;air-flow,660

1 l/min)(Zoetis,USA).Theanimalswereplaced inthestereotactic frame(DavidKopf661

Instruments,USA).Infusionguidecannulae(26gauge,4.4mmlength,C315,PlasticsOne,662

USA)withstylets(33gauge)thatprotruded0.5mmbelowtheendofthecannulawere663

inserted into the hippocampus bilaterally through small holes drilled into the skull.664

Cannulaimplantationcoordinateforthehippocampusisasfollows:anterior-posterior665

(AP) frombregma, –4.00mm;mediolateral (ML), ±3.00mm; and dorsal-ventral (DV)666

from the dura, –2.66 mm (Paxinos G,Watson C (2007) The Rat Brain in Stereotaxic667

Coordinates,Ed6.Amsterdam:AcademicPress/Elsevier).Carprofen(0.08ml/kgbody668

weight; Zoetis)was administered by subcutaneous injection at the end of all surgical669

procedures.Animalsrecoveredonaheatingpaduntilnormalbehaviourresumed.Allrats670

wereallowedarecoveryperiodofatleast7daysforthemtoregaintheirpre-surgery671

weightsbeforebehaviouraltesting.672

673

Drugs674

With respect to thedrugs,phosphate-bufferedartificial cerebrospmal fluid (aCSF) (in675

mM:150Na+,3K+,1.4Ca2+,0.8Mg2+,155Cl–,0.2H2PO4–,0.8HPO42–,pH7.2)wasusedas676

theinfusionvehicleandforcontrolinfusions.Drugconcentrationsforinfusionswere:677

678

• 0.19 mM of the GABAA receptor agonist muscimol (C4H6N2O2; Tocris, UK) 679

Rossato et al - 22

• 30 mM of the competitive NMDA receptor antagonist D-AP5 (C5H12NO5P; Tocris) 680• 3 mM of the competitive AMPA/kainate receptor antagonist CNQX (disodium 681

salt; C9H2N4O4Na2.H2O; Tocris) 682 683

ThepHofthedrugsolutionswasadjustedto7.2byadditionof1MNaOHsolution(forD-684

AP5),orofconcentratedphosphoricacid(forCNQX).BothaCSFanddrugsolutionswere685

preparedinlargerquantities,dividedintosmallaliquots,andkeptfrozenat–20°Cuntil686

theyused.WefacilitatedthesolutionofCNQXbysonification.Notehigherconcentrations687

ofmuscimolwereusedinpilotstudies.688

689

Behaviouraltraining690

WeusedmaleListerHoodedrats(n=16,250+g)forthebehaviouralaspectofthisstudy.691

Theywerestereotaxicallyimplantedwithbilateralguidecannulaetargetingthedentate692

gyrusinthedorsalhippocampus.Afterrecoveryfromsurgery,theyweretrainedonthe693

DMPtaskover8–10sessionswhereupontheyshowedtheusualstrikingdeclineinescape694

latencybetweentrials1and2ofasession(DataS1)andabovechancememoryofthe695

previoussession’slocationduringtrial1.696

697

Thereafter, using a counterbalanced Latin-Square design, we examined the impact of698

aCSF,muscimol,D-AP5andCNQXonperformanceintheDMPtask.The16animalswere699

allusedastheirowncontrols(i.e.everyanimalreceivedeachdrugconditionondifferent700

sessions)with¼ofthegroup(i.e.4animals)beingsubjecttoanyonedrugoneachdrug701

session.702

703

Formicroinfusionofdrugs,theratswerehabituatedtotheexperimentalprocedureof704

injectionforseveraldaysbeforethedrugsessionsinordertominimisestress.Thestylets705

intheguidecannulaewerereplacedbytwosingleinfusioncannulae(33gauge,Plastics706

One) connected to two10µlmicrosyringes (Hamilton,USA) inamicroinfusionpump707

(WorldPrecision,USA)via flexibleplastic tubing filledwithFluorinert(3M,USA).The708

tipsofinfusioncannulaeprojected0.5mmbelowthetipoftheguidecannulae.Thedrugs709

werebilaterallyinfused,inavolumeof2µlperhemisphere,overa4minperiodwitha2710

minperiodafterdruginfusionbeforetheinfusioncannulaewerereplacedwithstylets.711

Rats received drug injection 30 min in the case of CNQX and 40 min in the case of712

Rossato et al - 23

muscimolandD-AP5priortothestartoftraining,andboth30and40min(fordifferent713

animals) for aCSF – this intervalwas based on data from in vivo electrophysiological714

recordings.715

716

Drug sessionsonlyoccurredevery4th sessionas the test sessionswere conductedas717

follows (Figure 1A). First, each set of sessions consisted of three successive sessions718

(called3linkedsessions)thatarereferredtoassession1,session2andsession3(s1,s2719

ands3,respectively).Thenomenclatureispotentiallyconfusing,becausethese3linked720

sessionswerethenrepeatedaccordingtorequirementsoftheLatin-Squaredesign.Each721

repetitive3 linkedsessionsalwaysconsistedofs1,s2ands3andtheresultingscores722

wereconcatenatedinExcelfileuntilwehadtestedall16animalsineachcondition.723

724

Second,ineach3linkedsessions,thedrugswereonlyadministeredons2.Inthisway,725

wecouldexaminethefollowing:726

727

• Impact of the presence of a drug (within the hippocampus) on memory retrieval 728of the location of the platform on the previous session (i.e. memory for s1). 729

• Impact of the drug on new learning (i.e. delivered during s2, but measured on s3 730in the then drug-free state). 731

• Impact of the drug on ‘updating’ by comparing, on s3, the relative memory for 732s2 (the immediately preceding session) and that of s1 (two sessions before that). 733

734

Third, these3 linked sessionswere separatedby1+ interleaved training session.The735

objectof theseadditional trainingsessionswastomaintainstabilityofthestrategyof736

memoryretrievalfollowedbynewencoding,thisbeingmonitoredbycheckingthatthe737

first-crossinglatencyremainedstablethroughouttheexperiment(whichitdid).Thedata738

plottedinFigure1Eshowsfirstcrossinglatenciesacrossthe4trialsofeachinterleaved739

trainingsession;thelongesttimewasspentsearchingontrial1withrapidescapetothe740

newlylearnedlocationontrials2–4.Representativesearchpathsoncriticalprobetrials741

areshowninFigure3A,withthegroupbehaviouraldataforthecriticalprobesessions742

showninDataS3.743

744

Rossato et al - 24

Inearlierpilotwork(behaviouralandelectrophysiological),weexaminedtheimpactof745

higherconcentrationsofmuscimol(0.5mMand1.3mM)anddifferenttime-periodsafter746

infusionfortesting.Inthesecases,encodingdurings2andlong-termpotentiation(LTP)747

inductionwereeach impaired.Thedissociationbetween impairedretrievaland intact748

encodingonlyemergesatthelow-doseofmuscimol(0.19mM).749

750

MainElectrophysiologyStudies751

Theaimoftheelectrophysiologywastoprovidedatatoguidethechoiceofdrugdoses752

formuscimol,D-AP5andCNQX,andtoexamineexcitatorysynaptictransmission,cell-753

firing and LTP induction. We sought concentrations that definitively blocked LTP754

inductionwithD-AP5,butparadoxicallysparedLTPinductionwithmuscimol.755

756

Separateanimals(maleLister-Hoodedrats,weighing250+g,n=5perdrugcondition)757

wereusedinthenon-recoveryelectrophysiologystudies.Theseanimalswereprepared758

for acute surgery in a stereotaxic apparatus (David Kopf Instruments) under non-759

recoveryurethaneanaesthesia(1.3g/kgbodyweight;Sigma-Aldrich,USA),withthefirst760

intraperitonealinjectiongivenduringbriefisofluoraneanaesthesia(4%isofluranein0.8761

l/minO2).Theelectrophysiologystudiestypicallylasted6–8hr,withtheinitial2hrbeing762

spent securing accurate placement of the stimulating and recording electrodes and763

cannula,andthesubsequent4hrmonitoringfield-potentialbaselineandtheimpactof764

intrahippocampaldruginfusions.765

766

StimulatingandrecordingelectrodepositionsareshowninFigureS2A.Thestimulating767

electrode was a twisted bipolar Teflon-coated platinum-iridium electrode (20 µm768

diameter, 400 µm coated diameter for each of the two single strands) aimed at the769

angularbundleoftheperforantpath(AP0.0mmfromlambda;ML4.2mm;DV2.15mm770

fromdura).TherecordingelectrodewasasingleTefloncoatedplatinum-iridiumwire771

targetingthehilusofthedentategyrus(AP4.08mmfrombregma;ML2.5mm;DV3.5772

mm).Thedrugcannulawasa28gaugestainlesssteeltubewhosetipwas,forthedata773

reportedinFigure2,stereotaxicallylocatedatleast0.5to1.0mm(±0.3)mmawayfrom774

therecordingelectrode(AP3.6mmfrombregma;ML2.6mm;DV3.5mm).775

776

Rossato et al - 25

Conventional field-potential recordings weremade, with stimulation every 20 s, and777

thesemonitoredandcalculatedon-lineusingEPSsoftware[47].Inresponsetobiphasic778

200µsstimuluspulsesofcirca600–800µA,wemeasuredboththeearly-risingslopeof779

theevokedpotentialbylinearregressionoverseveralpoints,andtheamplitudeofthe780

evokeddentatepopulationspike.Thestimulus intensitywasadjustedtosecure initial781

population spike amplitudes of circa 3–6mV. LTP induction was attempted in some782

studiesusingahigh-frequencystimulationprotocol.Thistetanicstimulationconsistedof783

trainsof50pulses(200Hz),andrepeated3timesatanintervalof5min[47].784

785

Once acquired using suitable electrode placements, potentials typically remained786

relativelystableoverperiodsofupto3–4hr,withasmallupwarddriftofthepopulation787

spike(butnotfEPSP)thatrarelyexceeded15%overthislongperiod.Animalsforwhich788

thepotentialswereunstablewerediscarded.Thesame longtime-periodstabilitywas789

observed when aCSF was infused into the dorsal hippocampal formation at a depth790

targetingaregionencompassingthestratummoleculareofareaCA1.Avolumeof2µl791

was infused(0.5µl/min)that,onthebasisofpreviousautoradiographicdata[24,48]792

wouldbeexpectedtodiffusethroughouttheentireCA1,CA3anddentategyrusregions793

oftheseptal(dorsal)hippocampus.794

795

Wethenexaminedtheimpactofvaryingdosesofdrugs.Weexaminedmuscimol,D-AP5796

andCNQX. Intrahippocampal infusions (2µl)of artificial cerebrospinal fluid (aCSF, as797

vehicle),muscimol(0.19mM),D-AP5(30mM)orCNQX(6-cyano-7-nitroquinoxaline-2,3-798

dione,3mM)weremadeintothedorsalhippocampusofmale,Lister-hoodedrats(n=20,799

n = 5 per drug condition, aCSF at pH7.2; experimenter blind to drug administered;800

urethaneanaesthesia;perforantpathstimulation,recordingelectrodeinthehilusofthe801

dentategyrus,drugcannula1.0mmdistance,Figures2GandS2A;DataS2).Theinfusion802

volumeanddoseswerevariedinpilotwork,settlingonaprotocolof2μlforallthree803

drugsthatwould,onthebasisofautoradiographicdata[13,15,48],likelyaffecttheentire804

dorsal (septal) hippocampal formation, and extend to the intermediate region (to805

minimisethechancesofafalsenegativeinthebehaviouralstudy[45,49]).While2μlis806

high,suchavolumeshouldstilldisplayminimalspreadbeyondhippocampus.Theaim807

wastoachieve:808

809

Rossato et al - 26

• Shutting down cell-firing but with minimal effect on fast synaptic transmission 810and LTP induction (with low-dose muscimol – 0.38 nanomoles) 811

• Blocking the induction of LTP with minimal effect on fast synaptic transmission 812or cell-firing (D-AP5 – 60 nanomoles). 813

• Shutting down both fast synaptic transmission and cell-firing (CNQX – 6 814nanomoles) 815

816

EffectivedosesforD-AP5andCNQXwereknownfrompreviouswork[9,10],butchecked817

to realize internal consistency. Concentrations of 1.3, 0.5 and eventually 0.19mM of818

muscimolwereexamineduntil,atthelowestdose,thedissociationwewereseekingwas819

securedwithaninfusionvolumeof2µl.Figure2showstheprimaryresults(lowestdose820

ofmuscimol–0.38nanomoles).821

822

Diffusionofmuscimol823

Critically, we sought to measure the diffusion of low-dose of muscimol along the824

longitudinal axis of the hippocampal formation in two ways: electrophysiology and825

fluorescentimaging.Asoutlinedinthemaintext,wewonderedif"silentlearning"inthe826

presence ofmuscimol could be an artefact ofminimaldrug diffusion from the site of827

dorsalinfusiontotheintermediateregionofthehippocampalformation.Thiscouldleave828

intacthippocampaltissuetomediatelearning.Accordingly,furtherelectrophysiological829

experimentswereconductedwiththesamelow-doseofmuscimol(0.38nanomoles),but830

withtherecordingelectrodelocationintheintermediatehippocampus(AP,–5.52mm;831

ML,3.8mm;DV;4.1mm)-adistanceof2.0to2.5mmfromtheinfusioncannula(Figure832

S2B). Drug concentration and volume remained unchanged. Note that cell-firing833

remainedsubstantiallyinhibitedinthisintermediatezoneofthehippocampus(Figures834

S2C–S2E).835

836

Whilethesedatareflecttheimpactofthedrug,wealsosoughtdirectevidenceofdrug837

diffusion.Thisistrickytodowithsomestudiesconductingradiographyusingtritiated838

(C14)drugs,othersusing fluorescently labelledcompounds.Muscimol isavailableasa839

fluorescently labelled compound that can be visualisedmicroscopically, but it suffers840

fromthedifficultythatthemolecularweightismuchhigher(607insteadof114)andmay841

bemore"sticky"withrespecttodiffusioninaCSF.Accordingly,itislikelyaconservative842

Rossato et al - 27

estimateofdiffusionbut is at least adirectmeasure.Fluorescently-labeledmuscimol-843

bodipydissolvedinaCSF(0.19mM;HelloBio,UK)wasusedtoanalyzethedistribution844

profile of fluorescent muscimol 40 min after its bilaterally infusion into the dorsal845

hippocampus.Cannulaepositionsandthedruginfusionprocedureweresameasinthe846

mainbehaviouralexperiment.Brainswereremovedandshock-frozenonpowdereddry847

ice, and 50-μm-thick coronal sections were acquired with a cryostat (CM1900, Leica848

Biosystems, Germany), and mounted on Silane-coated glass slides. Bright field and849

fluorescentimagesofserialsectionsequallyspaced100μmweretakenwithaBZ-X700850

Microscope(Keyence,Japan).Fluorescentimagesingrayscalewereshownasarbitrarily851

assignedcolordisplaymode(pseudocolor)accordingtotheirgraylevelswithinarange852

of0–90[arbitraryunits (AU)]anda3D image showing thedistributionof fluorescent853

signalwasreconstructed fromserialsectionsusingaMetamorphsoftware(Molecular854

Devices,USA).Overlaidimagesofbright-fieldandpseudocolorimagesweremadeusing855

aPhotoshop(Adobe,USA).856

857

Theproblemofpopulationspikepotentiation858

Aseparatecomplicationwithlow-dosemuscimolwasthatnotonlywasitpermissivefor859

the induction of LTPmeasured using the early rising slope of the fEPSP, it was also860

permissive forspikepotentiation.Thus,whileminimalcell-firingwasobservedbefore861

LTP,somecell-firingunderlow-dosemuscimolwasobservedafterLTPinduction(Figure862

S3).Thiswasmodestandso,ratherthanplotnormaliseddatatoanear-zeropre-tetanus863

baseline, absolutedataareplotted.This raises thepossibility that, in thebehavioural864

study,itmayhavebeenpossiblefortheanimalstoretrieveinformationaboutaprevious865

session(s1)underlow-dosemusicimolaftertheyhadstartedencodingnewinformation866

about platform location in the current session (s2) (because cell-firingmight then be867

possible). In practice, we suspect such cell-firing is very unlikely in the behavioural868

situation. This is because LTP induction using 3 trains of 50 pulses at 250 Hz is an869

artificial tetanisation protocol designed to investigate activity-dependent synaptic870

plasticity in vivo but a firing pattern that does not occur during normal behavioural871

learning. Activity-dependent synaptic potentiation in vivo depends on more subtle872

patternsofneuronalactivation,suchasspike-timingdependentplasticityinasubsetof873

neuronswhichisunlikelytocausemuchpost-LTPcell-firing.874

875

Rossato et al - 28

QUANTIFICATIONANDSTATISTICALANALYSIS876

877

DataAnalysis878

879

BehaviouralDataAnalysis880

Themeasuresofperformanceusedwere(1)firstcrossinglatency(s)duringtrainingand881

(2)percenttimespentswimminginatargetzoneduringDMPprobetests.Theretrieval882

testswerealwaysonthefirsttrialofeachsessionand,asdescribedabove,involvedthe883

hiddenplatformbeingraisedtowithin1.5cmofthewatersurfaceonlyafter60shad884

passed.885

Repeated-measures analysis of variance (ANOVA)was used to examine the impact of886

withinsubjectsvariablesonbehavioralmeasureswithwithinsubjectfactorssessionand887

condition. Orthogonal comparisonswere used to further examinemain effects of the888

ANOVA.Two-tailedone-samplettestswereusedtocomparesearchpreferencemeasures889

tothevalueexpectedbychance(4%).ThelevelofsignificancewassetP<0.05.Dataare890

presentedasmean±SEM.ThestatisticalanalysiswasmadeusingIBMSPSSStatistics891

(IBM,USA).892

893

ElectrophysiologicalDataAnalysis894

TheanalysesweredoneusingroutinesimplementedinSpike2,version6.03(Cambridge895

Electronic Design, UK). Quantitative measurements reflecting the fEPSP and the896

populationspike(PS)activityweredonefollowingstandardcriteria.ThePSrecordedin897

theDentateGyrusismeasuredasthedifferencebetweenthemaximalnegativityofthe898

spikeandthemaximalpointoftheprecedentpositivecrest.ThefEPSPismeasuredasthe899

steepestslopeinthenegativegoingpotentialinmV/ms.900

901

AllstatisticswereperformedandplottedusingGraphPadPrism5.04software(GraphPad902

Software,USA).Foranystatisticalanalysisshown,two-tailedrepeated-measuresttest903

(fortwogroups,oronegroupvs.afixednumber)or1-wayANOVA(formorethantwo904

groups)forasignificancelevelofP<0.05wasused.When2factorsconcur,2-wayANOVA905

is utilizedwith the same significance threshold. For repeatedmeasures experiments,906

repeated-measuresANOVAareutilized.Post-hocBonferronimultiple-comparisonstest907

isusedtodescribetheoriginofsignificance.AllgraphsrepresentMean±SEM.908

Rossato et al - 29

909

HistologicalDataAnalysis910

For analysis of diffusion for fluorescent muscimol, areas in the dorsal/intermediate911

hippocampus, retrosplenial cortex and parietal association/secondary visual cortices912

were equally divided into grid squares (200 × 200 µm) and the averaged fluorescent913

intensitywasmeasured.Also,wedividedtheentirecorticalareaattheinjectionpoint914

(i.e., –4.00 mm from the Bregma) into ten equal regions, calculated the averaged915

fluorescent intensity, and set the thresholdas themean+2SD.Themeasurementsof916

fluorescentintensityandareawasmadeusingaMetaMorphsoftware.917

918

DATAANDSOFTWAREAVAILABILITY919

Electrophysiologicalandbehaviouraldataareavailableuponrequestbycontactingthe920

CorrespondingAuthors,RichardMorris(r.g.m.morris@ed.ac.uk)andon-line.921

922

SUPPLEMENTALINFORMATION923

Data S1: First-crossing latency for behavioural task during initial training displaying924

withinday learningtoapproachthecorrectescape locationacrosseachdailysetof4925

trials.RelatedtoFigure1E.926

927

DataS2:FullelectrophysiologydataforFigure2.928

929

DataS3: Probetestdatashowingpatternsofrecallandnewlearningacrossdifferent930

drugconditions.931

932

Figure S1. The Watermaze and Atlantis Platform. Related to Figure 1. (A) Photograph of the pool and associated 3D extramaze cues in the laboratory in Edinburgh. (B) The possible platform locations (n=24), with only one used per session, located on virtual outer and inner rings as viewed by the overhead camera. (C) The ‘on-demand’ or ‘Atlantis Platform’ is unavailable at the bottom of the pool until it rises on a spindle until its top surface is 1.5 cm below the water surface, and thus available for escape.

Figure S2. Electrophysiological analysis of effect drug diffusion through longitudinal axis of hippocampus, and of diffusion of a fluorescent labelled muscimol. Related to Figure 2. (A) Electrode

placements for the main study in which the recording electrode was within 0.5 mm from the AP location of the 2 ul drug infusion (i.e. region of maximal effect). Data shown in main Figure 2. (B) Electrode placements for the subsidiary study in which the recording electrode was 2 mm from the AP location of the 2 ul drug infusion. (C, D, E) fEPSP, Population Spike and averaged data (n=5) of the animals in the subsidiary study. The drug continues to have a substantial impact at 2 mm distance, suggesting a likely spread over 4 mm in the AP direction of muscimol. (F, G) Image and quantitation of diffusion of fluorescent muscimol bodipy from an infusion site at AP 4.0. One animal (H0205) showed diffusion of muscimol bodipy over approximately 3.0 mm, the other 2 animals showed less diffusion. There is no diffusion in the retrosplenial cortex, but some is detected in the parietal area immediately above the tip of infusion cannula reflecting "leakage" up the cannula track.

Figure S3. Population spike LTP shown in mV. The usual way in which LTP data is plotted is normalised to a pre-tetanus baseline. However, this is not sensible with a near zero baseline for the muscimol treated animals. Related to Figure 2. (A) Clear potentiation of the population spike (PS) occurred after aCSF infusion, with the mean population spike reaching circa 8 mV. (B) Potentiation of the spike also occurred with low-dose muscimol (0.38 nanomoles), but the absolute magnitude was very small and a mean pop spike of circa 2 mV. (C) D-AP5 blocked spike potentiation, when judged against the steadily rising control condition (see Figure 2). However, some spiking is observed as with muscimol.