edinburgh research explorer · rossato et al - 2 summary25 26 27 we introduce the concept of...
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Edinburgh Research Explorer
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
2
Silent learning 3
4
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
([email protected]);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
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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
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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([email protected]).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([email protected])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.