Reactivations of emotional memory in the hippocampus amygdala system during sleep

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1 Rectivtions of emotionl memory in the hippocmpus mygdl system during sleep Grielle Girrdeu, Ingrid Inem, & György Buzsáki 7 Nture Americ, Inc., prt of Springer Nture. All rights reserved. The consolidtion of context-dependent emotionl memory requires communiction etween the hippocmpus nd the solterl mygdl (), ut the mechnisms of this process re unknown. We recorded neuronl ensemles in the hippocmpus nd while rts lerned the loction of n versive ir puff on liner trck, s well s during sleep efore nd fter trining. We found coordinted rectivtions etween the hippocmpus nd the during non-rem sleep following trining. These rectivtions peked during hippocmpl shrp wve ripples (SPW-Rs) nd involved sugroup of cells positively modulted during hippocmpl SPW-Rs. Notly, rectivtion ws stronger for the hippocmpus correltion ptterns representing the run direction tht involved the ir puff thn for the sfe direction. These findings suggest tht consolidtion of contextul emotionl memory occurs during ripple-rectivtion of hippocmpus mygdl circuits. Coopertion etween mygdl, prticulrly the, nd hippocmpus is criticl for contextul emotionl memory. It is elieved tht the emotionl nd sptil components of n experience re processed y the mygdl nd dorsl hippocmpl circuits, respectively,. Lesion nd other experiments hve shown tht fine sptil representtion in the dorsl hippocmpus, is required for sptil nd contextul memory, including context thret ssocitions, with limited contriution from the ventrl hippocmpus,. Becuse only the ventrl hippocmpus nd ssocited entorhinl outputs project directly to the mygdl, including the nd centrl mygdl,,, it remins to e determined how emotionl nd contextul stimuli re comined nd consolidted to form stle, integrted representtion of the context nd ssocited emotionl vlence. Sleep reply of wke sequences of plce cells during hippocmpl SPW-Rs re instrumentl for sptil memory consolidtion nd the stiliztion of newly formed sptil representtions. By comprison, the consolidtion mechnisms of mygdl-dependent memories hve only een prtilly explored,7,. We hypothesized tht contextul emotionl experience is replyed in the interconnected hippocmpus mygdl circuit during sleep. More specificlly, ecuse synchronous dischrges of neuron popultions during SPW-Rs fcilitte the comintion of neuronl informtion throughout the entire dorso-ventrl xis of the hippocmpus 9, we hypothesized tht sptil informtion from the dorsl hippocmpus my e ssocited with the thret representtion in the during SPW-Rs of non-rem () sleep. To study hippocmpus mygdl interctions, we comined clssicl sptil tsk with loction-specific versive element (ir puff). We recorded lrge neuronl ensemles simultneously in the mygdl nd dorsl hippocmpus during trining nd sleep episodes efore nd fter trining. To identify the supopultions of neurons in the mygdl tht re functionlly linked to the dorsl hippocmpus, we exmined their dischrge ptterns during SPW-Rs. We then investigted whether joint hippocmpus representtions of spce nd thret re rectivted during SPW-Rs of sleep. RESULTS Rts lern the dily loction of n versive ir puff on liner trck To study hippocmpl mygdl rectivtions, we designed tsk y comining clssicl sptil tsk with n versive component to recruit neurons. Rts (n = ) were pretrined to run ck nd forth on liner trck for wter rewrds. After stedy performnce ws chieved, we introduced n versive ir puff t the sme loction of the trck on ech lp in one of the running directions. The loction nd direction of the ir puff ws chnged dily in pseudo-rndom mnner. Previous work hs shown tht ir-puff-induced contextul fer lerning relies on oth the mygdl nd the hippocmpus. We dpted this tsk to llow dily ehviorl trining nd recordings of lrge neuronl ensemles in freely moving nimls. Ech dily recording session consisted of pre-run ehviorl test session on the trck without the ir puff, followed y pre-lerning sleep in the home cge ( pre-sleep, ctegorized s pre-rem or pre-), trining session ( run ) with the ir puff, post-lerning sleep ( post-sleep, ctegorized s post-rem or post-) nd post-run test session without the ir puff (Fig. ). Becuse rts slow down efore crossing the ir puff loction if they rememer its loction, we quntified memory performnce from the pre-run nd post-run test epochs y compring the running speed of the rt in the dnger zone of the current dy (defined s the cm preceding the ir puff loction) with the speed t the previous dy s dnger zone (Fig., nd Supplementry Fig. ). The current dnger zone, initilly neutrl during pre-run, cquires n versive vlence during trining, while the previous dnger zone loses its versive nture. Therefore, the systemtic reversl in the speed rtio (previous/current dnger zone) etween pre- nd New York University Neuroscience Institute, New York University, New York, New York, USA. Deprtment of Neurology, Medicl Center, New York University, New York, New York, USA. Center for Neurl Science, New York University, New York, New York, USA. Present ddress: Dougls Institute, McGill University, Montrel, Queec, Cnd. Correspondence should e ddressed to G.B. (gyorgy.uzski@nyumc.org). Received Ferury; ccepted August; pulished online Septemer 7; doi:./nn.7 nture NEUROSCIENCE dvnce online puliction

2 7 Nture Americ, Inc., prt of Springer Nture. All rights reserved. Pre-run Post-run Post-sleep Run (ir puff) Pre-sleep Wter min Dnger zone Current dy Air puff (current) Air puff (previous) Dnger zone Previous dy Wter Speed (cm/s) c Speed (cm/s) Previous dnger zone Current dnger zone. Pre-run Run Post-run Pre-run (no ir puff) Post-run (no ir puff) Run (ir puff) Speed rtios Run Prerun Postrun Air-puff-centered normlized position on the trck Figure Rts lern the dily loction of n versive ir puff. () Rts run ck nd forth on liner trck for wter rewrds. Gry line: one-dimensionl position of the niml on the trck over time in representtive session (one session in one niml out of sessions in nimls). An ir puff is delivered t the sme loction on the trck in one running direction during the run epoch. The ir puff loction is chnged every dy. The run epoch is flnked y two sleep epochs (pre-sleep, post-sleep) nd two test run sessions where no ir puff is delivered (pre-run, post-run). The dnger zones (DZ) re defined s the cm preceding the loction of the ir puff on the current dy (pink) nd on the previous dy (lue). () Left: speed in the current nd previous DZ cross nimls nd sessions during the three run epochs (two-wy repeted mesures ANOVA; n = sessions in nimls; significnt session effect (pre-run, run or post-run, P = 7., d.f. =, F =.), ir puff loction effect (current vs. previous, P =., d.f. =, F =.) nd interction, (P =., d.f. =, F =.). Post hoc pired t-tests showed significnt differences etween previous nd current DZ speed for pre-run, run nd post-run (P =., t() =.; P = 9.7 9, t() =.; P =.7, t() =.), s well s for the current DZ etween pre-run nd post-run (P =., t() =.7). Right: speed rtios cross sessions nd nimls (one-wy repeted mesure ANOVA; significnt session effect, P =., d.f. =, F =.; post hoc t-tests, pre-run vs. run: P =., t() =.9; pre-run vs. post-run P =., t() =.; run vs. post-run P =., t() =.; white line, medin; lck line, men; lck dots, outliers; oxes, first nd lst qurtiles; whiskers, minimum nd mximum vlues excluding outliers). P <., P <. with Bonferroni correction. (c) Air-puff-centered men speed (± s.e.m., pre-run: n = sessions in nimls; run nd post-run: n = sessions in nimls) curves in the ir puff direction in the pre-test (no ir puff), trining nd post-test (no ir puff) epochs. The current DZ is indicted y the shded pink r. Note the slower speed in the DZ post-run compred to pre-run. post-run indictes lerning of the new ir puff loction (Fig. nd Supplementry Fig. ). The typicl ehviorl pttern on the trck during run ws reduced speed efore the ir puff, followed y n ccelertion fter pssing through the dnger zone. A similr speed chnge ws mintined during post-run, wheres speed smoothly incresed throughout the trck during pre-run (Fig. c). This ws quntittively reflected y the significntly slower speed in the current dnger zone in post-run compred to pre-run (Fig.,c). The versive vlence of the ir puff grdully diminished with trining dys (Supplementry Fig. c). The loction of the ir puff on the trck did not correlte with the speed in the current dnger zone in pre-run, trining or post-run, ruling out systemtic is of the ir puff loction on the results. recordings nd sleep physiology We recorded ensemles of neurons from oth left nd right mygdl nd the dorsl CA hippocmpl region during the tsk (Fig.,). Eight-shnk silicon proes were moved downwrd y -µm steps etween ech ehviorl experiment. This llowed recording from lrge res of the mygdl nd the piriform cortex in ech rt (Supplementry Fig. ). Over the course of totl of sessions, we recorded 7,9 well-isolted units (rt,,; rt,,9; rt dvnce online puliction nture NEUROSCIENCE

3 r t ic l e s REM sleep No. cells REM rte (Hz) rte (Hz) Wke rte (Hz) No. cells Time (h) Wke REM. Time (h) Firing rte rtio Normlized cell count.... Wke rte (Hz) No. cells Normlized cell count Frequency Frequency 7 Nture Americ, Inc., prt of Springer Nture. All rights reserved..... c No. cells.... Units Hpc LFPs Right Left Hpc sleep Firing rte rtio Figure Physiologicl chrcteriztion of. () Silicon proe recordings from the dorsl hippocmpl CA (four-shnk proe) nd ilterl mygdl (eight-shnk proes; totl chnnels) nd exmple locl field potentils (LFPs) nd units (rster plots) in the hippocmpus (Hpc; red) nd left nd right mygdl (; lue). Hippocmpl SPW-R times re indicted y gry lines in sleep. () CA nd spectrogrms for n exmple session (out of 9 sessions in rts with simultneous nd hippocmpus recordings). Spectrogrms were used to define rin sttes (wke, or REM sleep; colors represent power in ritrry units, from green (low) to red (high)). (c) Distriutions of firing rtes for monosynpticlly identified pyrmidl cells (ornge, n = 7 cells; see Online Methods nd Supplementry Fig. ), interneurons (lue, n = 7 cells) nd other cells (gry, n =, cells) in during vs. wke (top left) nd REM vs. wke (top right). The distriution of REM/wke nd /wke firing rte rtios (ottom) is skewed towrd for pyrmidl cells (ornge, monosynpticlly identified pyrmidl cells; n = 7; /wke: P =.7, z =.; REM/wke: P =, z =.; Wilcoxon signed rnk tests), indicting n increse in firing rte during oth sleep stges compred to wke. Interneurons do not chnge firing rtes etween REM nd wke (lue, monosynpticlly identified interneurons, n = 7; REM/wke: P =., z =.) nd slightly decrese firing rtes during compred to wke (/wke: P =.97 9, z =.; Wilcoxon signed rnk tests; dotted lines: medins).,,; rt,,). On the sis of histologicl reconstruction of proe plcement nd proe movement record,, of these were in the, 7 in the centrl nuclei,, in the piriform cortex nd, in the hippocmpus (Supplementry Tle ). Units were further chrcterized s puttive pyrmidl cells nd interneurons y wveform nd physiologicl criteri (Supplementry Fig. nd Online Methods). The firing rtes of pyrmidl cells followed skewed distriution during sleep ( nd REM) nd wkefulness (Fig. c). In ddition, we found specific increse in the firing rte of pyrmidl cells, ut not interneurons, during REM sleep nd, to lesser extent, sleep reltive to wkefulness, s shown y the distriutions of REM/wke or /wke firing rte rtios for the two cell types (Fig. c nd Online Methods). hippocmpus coordinted rectivtions during sleep Rectivtions cross the hippocmpus mygdl network s well s within-structure networks (Supplementry Fig. ) were quntified nture NEUROSCIENCE dvnce online puliction using the explined vrince (EV). EV is the percentge of vrince in the popultion of pirwise correltions during post-sleep (REM or ) tht cn e explined y run correltions ( rectivtion ) while tking into ccount pre-existing correltions during pre-sleep (REM or ; Fig., nd Online Methods). The reverse explined vrince (REV), clculted y switching the pre-sleep nd post-sleep epochs, is used s control vlue. The EV nd REV, clculted using hippocmpus pyrmidl cell pirs, showed significnt rectivtions etween the hippocmpus nd during post- (Fig. c). The grdul decy in rectivtions over the first hour of sleep (Fig. c; men differences etween EV nd REV, ± s.e.m.: min,.9 ±.7%; min,.7 ±.%; min,. ±.%; n = 9 sessions, P =., d.f. =, χ =.; Kruskl-Wllis test) prlleled the previously descried decy for pirs of hippocmpl neurons,. Rectivtions were mintined when oth pyrmidl cells nd interneurons were included in the EV clcultion (Supplementry Fig. ). Neurons in the piriform cortex showed weker experience-induced

4 Pre- Run Post-. Hippocmpl cells.. Correltion EV REV (%) cells cells cells. 7 Nture Americ, Inc., prt of Springer Nture. All rights reserved. c d e EV REV (%) REM Hpc min min min Figure Hippocmpus ensemles rectivte during sleep. () Correltion mtrices were clculted for hippocmpus cell pirs for the pre-, run nd post- epochs in -ms time ins. These mtrices were used to clculte the explined vrince (EV) nd its control vlue, reverse explined vrince (REV). Red rrows: strong coctivtions of single pyrmidl neuron with multiple hippocmpl pyrmidl (pyr) cells in n exmple session ( niml nd session out of nimls nd sessions for sleep). () EV nd REV for the exmple session shown in. (c) Left: EV nd REV cross ll sessions for (n =, P =.9, z =.7; n = rts) nd REM sleep (n =, P =.77, z =.; n = rts; pyr pyr pirs). Right: EV nd REV for successive min epochs of for sessions where EV > REV in the first -min epoch (pyr pyr pirs; n = 9 sessions in rts, min: P =., z =.; min: P =., z =.; min: P =., z =.). (d) Hippocmpus piriform cortex EV nd REV (: n = 7 sessions, P =.; REM: n = sessions in rts, P =.; pyr pyr pirs). (e) Sme s d ut for centrl nuclei (CeN) (: n = 9 sessions, P =.77, z =. nd REM: n = 9 sessions, P =.7, z =.; ll cell pirs; n = rts). All tests re Wilcoxon signed rnk tests, P <., P <., P <.. All ox plots show the medin (red line), first nd lst qurtiles (ox), nd minimum nd mximum vlues excluding outliers (whiskers), outliers (lck dots). EV REV (%) Hpc REM P ir EV REV (%) Hpc CeN REM rectivtion with their CA prtner neurons (Fig. d; Wilcoxon onetiled rnk sum test on EV REV for hippocmpus (n = sessions, men EV REV. ±.7%, s.e.m.) vs. hippocmpus piriform cortex (n =, men EV REV. ±.%, s.e.m.), P =.9, z =.), while there were no rectivtions t ll etween the centrl nuclei nd the hippocmpus (Fig. e). Rectivtions during REM sleep 7,7, were not significnt in ny structure, despite the roust REMsleep-specific increse in puttive pyrmidl cells firing rtes (Figs. c nd c e nd Supplementry Fig. ). A suset of cells re modulted during hippocmpl SPW-Rs neurons receive direct input from ventrl, ut not dorsl, CA neurons. However, sptil loction is more precisely coded y dorsl CA neurons thn ventrl ones,. Becuse oth dorsl nd ventrl hippocmpl neurons fire together during lrge-mplitude SPW-Rs 9, SPW-Rs my estlish functionl connections etween the dorsl hippocmpus nd mygdl. To test this hypothesis, we exmined the functionl reltionship etween SPW-Rs nd neurons. A frction of neurons were significntly nd positively modulted ( upmodultion ; of interneurons (.%), 7 of, pyrmidl cells (.%)) or negtively modulted ( downmodultion ; of interneurons (.7%), of, pyrmidl cells (.%)) during hippocmpl SPW-Rs (Fig. ). This confirmed the indirect influence of dorsl hippocmpl SPW-Rs on cells. Moreover, rectivtions clculted using SPW-R-modulted cells were lrger thn for nonmodulted pirs (Supplementry Fig. ). dvnce online puliction nture NEUROSCIENCE

5 7 Nture Americ, Inc., prt of Springer Nture. All rights reserved. Ripple-modulted cells (%) Gin Gin Gin Gin 9 7 Hpc Up-mod Down-mod Pyr Int Pyr Int.. c No. ripple-modulted pyr No. ripple-modulted int.. Ripple-modulted cells re preferentilly involved in rectivtions In further ttempt to chrcterize the rectivtion dynmics during sleep, we used two complementry pproches. In the first pproch, we defined the firing properties of individul neurons reltive to ripples nd then exmined how such properties influenced rectivtions. The second pproch worked from the opposite direction. First, we quntified rectivtions for ech cell irrespective of their LFP ripple correltes nd exmined how their rectivtion vlues were relted to ripples. During run, frction of hippocmpl pyrmidl neuron pirs showed significntly positively correlted spike trins (, of 7,;.9%). Another smll percentge (, of 7,;.%) ws negtively correlted, while the remining mjority (, of 7,; 9.9%) ws not relily correlted (Online Methods; totl numer of pyrmidl pyrmidl hippocmpus pirs 7,: rt,,; rt,,; rt, 7,7). To test whether selective sugroup of pirs ws preferentilly involved in sleep rectivtions, we seprted pirs into nine sugroups sed on comintion of run correltion nd SPW-R modultion of the prtner (Fig., Supplementry Fig. nd Supplementry Tle ). Hippocmpus pirs with significnt, positive run correltions nd SPW-R upmodultion of the prtner showed the lrgest pre- to post- chnge (one-wy ANOVA, P =., d.f. =, F = 9.9; post hoc comprisons, P <.) Figure Susets of cells re up- or downmodulted during hippocmpl SPW-Rs. () Percentges of hippocmpl (Hpc) nd puttive pyrmidl cells (pyr; red) nd interneurons (int; lue) tht re significntly upmodulted (up-mod; drk red or drk lue) or downmodulted (down-mod; light red or light lue) during SPW-Rs (Hpc: n = sessions, n = rts; : n = 9 sessions, n = rts). () Perievent gin for two puttive pyrmidl cells (red: top, upmodulted; ottom, downmodulted) nd interneurons (lue: top, upmodulted; ottom, downmodulted). These neurons re indicted y the respective colored sterisks in c. (c) Normlized peri-event gin for ll upmodulted (top left) nd downmodulted (top right) puttive pyrmidl cells (top pnels) nd interneurons (ottom pnels). Top lck trces: exmple hippocmpl ripples (LFP).. Normlized gin The dominnt contriution of this specific sugroup of cell pirs to rectivtions ws confirmed y clculting the EVs cross the nine sugroups (pirs grouped cross nimls nd sessions; Supplementry Fig. ). In the second pproch, we evluted the contriution of ech cell pir to the overll EV (clculted with ll pirs cross nimls nd sessions) y removing hippocmpus pirs one y one. The chnge in EV (EV ll EV minus one pir ) indictes the individul contriution of the removed pir (the lrger the decrese in EV, the lrger the contriution of the pir; Supplementry Fig. 7). To otin per-cell contriution mesure, contriutions were verged over ll the pirs tht the cell prticipted in. We then divided cells into qurtiles ccording to the mgnitude of their individul contriutions. We found tht upmodulted neurons in the most strongly contriuting qurtile showed specific increse in SPW-R gin (tht is, firing rte during versus outside SPW-R) from pre- to post- compred to the upmodulted cells of the remining three, low-contriution qurtiles (Fig. nd Supplementry Fig. ; Wilcoxon one-til sign-rnk test on gin verged in -ms window round ripple pek; high-contriution qurtile: P =.7, z =.9; low-contriution qurtiles: P =.99, z =.). To control for the effects of firing rtes, we clculted EVs nd REVs for pirs pooled ccording to their firing rtes (individul cell firing rte, hippocmpl cell firing rte or comined firing rte). This control showed tht EV did not depend on firing rtes (Supplementry Fig. 9). The versive trjectory is rectivted during SPW-Rs cells tht re upmodulted during hippocmpl ripples show preferentil involvement in coordinted rectivtions, through n incresed gin of their modultion fter trining. However, these oservtions offer only indirect support for rectivtions during hippocmpl SPW-Rs. Furthermore, these findings lone do not directly ddress the criticl role of thret in sleep rectivtions. To otin more direct support, we used rectivtion strength (R) mesure (Supplementry Fig. nd Online Methods) nd nlyzed firing ptterns seprtely during the two directions of trvel. Since the ir puff ws presented during only one direction of run on the trck (ir puff or dnger trjectory) on given dy, the opposite run cn e considered sfe. Therefore, we compred the rectivtion strengths of the hippocmpus pirwise correltion ptterns of the ir puff vs. the sfe direction. We found tht the reinsttement of the joint hippocmpus neuron representtion ws significntly enhnced during post- SPW-R compred to pre- SPW-Rs for the ir puff direction ut not for the sfe direction (Fig., nd Supplementry Fig.,c). Becuse the firing rtes of cells did not significntly differ etween sfe nd ir puff trjectories (pyrmidl cells: P =.7, z =.; ll cells: P =.7, z =., Wilcoxon signed rnk tests), the rectivtion results cnnot e explined y ir-puff-induced firing rte increse. The occurrence rte of SPW-R ws lso not significntly different etween the pre- nd post- epochs (P =.9; z =.9, n = sessions; Wilcoxon signed rnk test; Supplementry Fig. d). These oservtions thus confirm tht sleep SPW-Rs re specific time windows within which the plce thret ssocition is reinstted during sleep. contriutes to reinsttement of new plce thret representtions Finlly, we exmined how sleep rectivtions re linked to the plce thret representtion during wkefulness. Rts lerned new ir puff loction every dy during the trining session (Fig.,). nture NEUROSCIENCE dvnce online puliction

6 . SPW-Rs, Post Correltion difference Top contriution qurtile Remining qurtiles Pre- Post- Significntly positive Nonsignificnt Significntly negtive Up-mod 7 Nture Americ, Inc., prt of Springer Nture. All rights reserved.. SPW-Rs, Pre Trining corr Men pre/post correltion difference Normlized gin Men gin Men gin Proportion of cells r t ic l e s No mod Down-mod -cell ripple modultion Figure Rectivtions rely on pirs with correlted ctivity during run nd prtner tht is upmodulted during hippocmpl SPW-Rs. Strongly contriuting modulted cells selectively increse their gin during SPW-Rs following trining. () Men differences etween pre- nd post correltions for ech group of cell pirs clssified ccording to (i) the ripple-modultion type of the cell upmodultion (up-mod), no modultion (no mod) or downmodultion (down-mod) nd (ii) the correltion during the run significntly positive, nonsignificnt or significntly negtive. Inset shows the normlized cumultive distriutions of the post- pre- correltion difference for the upmodulted, significntly positive run correltion group (red) nd downmodulted, significntly negtive run correltion group (lue; error rs: s.e.m.; n = 7, pirs, one-wy ANOVA P =., d.f. =, F = 9.9, n = rts). The upmodulted, significntly positive correltion group is the only one to e significntly different from ll others (post hoc Tukey Krmer multiple comprison test with P <.). Detils of ll distriutions re shown in Supplementry Figure. () Left: peri-ripple gins for upmodulted cells of the highly contriuting qurtile for pre- SPW-Rs nd post- SPW-Rs (color: normlized gin per cell; cells re sorted y timing of the in with highest gin during the pre-sleep epoch). Right: men (± s.e.m.) gin during pre- (lue) nd post- (red) SPW-Rs for upmodulted cells of the strongly contriuting qurtile (top) nd for the remining qurtiles (ottom). There is significnt increse in gin etween pre- nd post- SPWRs for strongly contriuting upmodulted cells (Wilcoxon one-til signed rnk tests on gin verged in -ms window round ripple pek; high contriution qurtiles: n = cells, P =.7, z =.9; low contriution qurtiles: n = 7 cells, P =.99, z =.; n = rts). The joint representtion of spce nd thret is thus expected to e different etween the pre-run test, when the new loction hs not een experienced yet, nd the post-run test, when it hs een experienced nd replyed during sleep. Figure 7 shows exmples of highly contriuting hippocmpus cell pirs tht showed irpuff-relted ctivity () nd ir-puff-relted plce fields (hippocmpus). These coordinted ptterns developed during trining nd were mintined in the post-run test in the sence of n ir puff. To quntify this reltionship, we exmined seprtely the most strongly contriuting pirs (represented y the highest.th percentile of the contriution distriution) nd the lest strongly contriuting pirs (the.th percentile of lowest contriution; Supplementry Fig. 7). We found tht for strongly contriuting, ut not for wekly contriuting, pirs the increse etween pre-run nd post-run coctivity ws significntly correlted with the increse in coctivity etween pre- nd post- (Fig. 7). This result ws mintined when the most strongly nd the most wekly contriuting qurtiles (insted of.th percentiles) of the distriution were compred (Fig. 7c). These coordinted chnges indicte tht rectivtions during sleep ply role in the stiliztion of the new spce thret representtion. DISCUSSION We found tht correlted neuronl ctivity etween neurons of the dorsl hippocmpus nd ws strengthened during sleep following experience in sptilly nchored thret model9,. Rectivtions involved sugroup of hippocmpus-responsive neurons in nd occurred in ssocition with hippocmpl SPW-Rs. Notly, the rectivtion of hippocmpus coctivity during post-experience sleep ws stronger for the ptterns of pirwise correltions dominting during the trvel through the dnger zone, compred to rectivtions of the pirwise ptterns representing the sfe direction. Previous works hve shown tht in oth sptil memory tsks nd contextul thret lerning only smll set of neurons is ctive in the hippocmpus nd mygdl7 9,. Identifying mygdl neurons tht receive hippocmpl inputs required recording from n unprecedentedly lrge numer of individul neurons simultneously in these two structures. We chieved this y using multi-shnk silicon proes nd n experimentl design tht llowed us to generte new plce thret ssocitions every dy so tht we could slowly dvnce our proes through the full structure of the mygdl nd smple new sets of neurons dily. Of the lrge numer of cross-structure neuron pirs, we identified the relevnt suset whose coctivtion incresed significntly from pre-experience to post-experience sleep nd thus contriuted to the cross-structure rectivtions. We further chrcterized the mygdl memers of these pirs s hippocmpusresponding ecuse they incresed firing rtes during hippocmpl SPW-Rs. In contrst, neurons tht decresed or did not chnge their ctivity during SPW-Rs did not show significnt chnge in their correltion with hippocmpl neuron prtners from pre-experience to post-experience sleep. Furthermore, neuron pirs cross the hippocmpus nd tht showed the strongest increse in correltion from pre-experience sleep to post-experience sleep were those tht lso showed the strongest correltions during lerning of the plce thret ssocition. For the rectivted pirs only, the chnges in the strength of coctivtion during sleep induced y trining were correlted with the chnges in the coctivtions on the test sessions on dvnce online puliction nture NEUROSCIENCE

7 7 Nture Americ, Inc., prt of Springer Nture. All rights reserved. Air puff trjectory Men rectivtion strength Sfe trjectory Men rectivtion strength Pre- Post- Time from ripple pek (s). Time from ripple pek (s). the trck. Moreover, experience-induced rectivtions were stronger for hippocmpus pirs correlted during trvel tht involved the versive ir puff, compred to trvel in the sfe direction. Overll, our findings suggest tht trining on the plce thret ssocition cretes novel joint representtion etween the hippocmpus nd the mygdl tht is susequently consolidted or reconsolidted during sleep nd is reinstted on the trck during the test session in sence of the thret. The dorsl hippocmpus is crucilly involved in sptil memory, including clssicl context thret ssocitions 9,, in line with the oservtions tht neurons in the dorsl hippocmpus crry highly specific sptil informtion. More specificlly, it hs een shown tht the indirect suppression of sleep hippocmpl SPW-Rs, known to consolidte sptil memories, impirs contextul fer conditioning. By comprison, the importnce of the ventrl hippocmpus for contextul processing is deted, gin in line with the corser sptil representtion of ventrl hippocmpl neurons,. We show tht despite the lck of direct connections from dorsl hippocmpus to, frction of pyrmidl cells nd interneurons were effectively entrined y SPW-Rs of the dorsl hippocmpus. We hypothesize tht this is possile ecuse during lrge mplitude SPW-Rs popultions of the dorsl nd ventrl hippocmpus, likely involving ventrl neurons projecting to the mygdl, roustly synchronize. Indeed, one postulted role of SPW-Rs is to comine neuronl ctivity Men rectivtion in to + ms window round ripple pek Men rectivtion in to + ms window round ripple pek Post Pre RS difference in ripple pek Sfe Air puff Figure Hippocmpus rectivtions of the ir puff trjectory pek during hippocmpl SPW-Rs. () Men (± s.e.m.) z-scored periripple rectivtion strength of the ir puff (top) versus sfe (ottom) trjectories over nimls (n = ) nd sessions (n = ) for pre- (lue) nd post- (red) hippocmpl SPW-Rs. Right: rectivtion strength in -ms window round ripple peks (gry rs) ws significntly higher in post- compred to pre- for ir puff trjectory (P = 7., z =.79), ut not for sfe trjectory (P =.7, z =.7; Wilcoxon one-til signed rnk tests). Gry lines from pre- to post- indicte single sessions. () The pre- vs. post- difference in rectivtion strength (RS) t the ripple pek is significntly higher for the ir puff trjectory compred to sfe trjectory (Wilcoxon one-til signed rnk-test; P =.7, z =.79, n = sessions; ox plots show the medin (red line), first nd lst qurtiles (ox), nd minimum nd mximum vlues (whiskers) excluding outliers). cross different segments of the hippocmpus 9, tht send nd receive projections to different prts of the neocortex, mygdl nd sucorticl structures. Alterntive explntions should lso e considered. In ddition to the direct ventrl hippocmpus mygdl projections, hippocmpus entorhinl cortex mygdl route my lso e involved, given tht entorhinl connections hve een implicted in the cquisition of contextul thret conditioning nd given tht deep-lyer entorhinl neurons lso respond roustly to SPW-Rs 7. Another potentil explntion for the hippocmpus mygdl sleep reply is tht oth neuronl popultions re simply responding to third prty, such s slow oscilltions. However, severl findings rgue ginst this possiility. First, neuronl responses in to hippocmpl SPW-Rs were immedite nd short. If firing correltions were driven y UP-DOWN sttes of sleep or spindles, more prolonged firing rte responses would e expected. Second, DOWN-UP shift induces increses ut not decreses in firing rtes. In contrst, lrge frction of the mygdl neurons responded with suppression of firing rtes during hippocmpl SPW- R. Third, only SPW-R-excited neurons showed significnt chnge from pre-experience to post-experience sleep. Conversely, we found tht neurons with high contriution to rectivtions were more likely to increse their ssocition with SPW-R during sleep fter lerning compred to sleep efore lerning. Overll, our findings suggest tht SPW-Rs re instrumentl for estlishing functionl connections etween dorsl hippocmpus nd to consolidte plce thret ssocitions. Previous findings in humns nd other nimls hve suggested tht REM sleep is criticl for the consolidtion of emotionl informtion 7,,7,,9. Our results showing n elevted firing rte of pyrmidl cells during REM sleep re in line with this hypothesis. However, we did not find significnt rectivtions during REM sleep. The short durtion of REM sleep episodes nd the consequently low numer of spikes ville for the nlyses we performed my contriute to the lck of significnt rectivtions during REM sleep. It is lso possile tht REM sleep plys different or complementry role in the consolidtion of emotionl memories tht does not involve n offline reinsttement of the joint hippocmpus representtion. Finlly, our work did not ddress the potentil role of sucorticl neurotrnsmitters in memory reply nd consolidtion or the role of the entorhinl cortex s possile meditor of informtion exchnge etween hippocmpus nd mygdl. These questions remin to e nswered y future investigtions. In summry, we identified smll suset of hippocmpus neuronl pirs tht re rectivted during sleep SPW-Rs following trining in plce thret ssocition tsk. The prtners of these pirs re preferentilly upmodulted during SPW-Rs nd selectively increse their firing rte during SPW-Rs fter trining. This finding suggests tht SPW-R reply provides physiologicl mechnism to integrte plce cell ctivity in the dorsl hippocmpus nd thret-responsive neurons in the mygdl. We hypothesize tht concerted ctivtion of hippocmpl nd cells during SPW-Rs is responsile for comining sptil/contextul nd emotionl representtions during sleep nd thus for the consolidtion of contextul fer. Direct support for this hypothesis will require further experiments, such s dynmic perturtion of neurons specificlly during SPW-Rs. Methods Methods, including sttements of dt vilility nd ny ssocited ccession codes nd references, re ville in the online version of the pper. nture NEUROSCIENCE dvnce online puliction

8 Pirwise corr. Pre- Run Post- Pirwise corr.. Pre- Run Post- Pre-run Rte 7 Nture Americ, Inc., prt of Springer Nture. All rights reserved. Run Post-run Rte Rte Rte All trck Pre/post-run corr diff.. Position on trck Note: Any Supplementry Informtion nd Source Dt files re ville in the online version of the pper. Acknowledgments We thnk J. LeDoux, C. Lén, E. Strk, A. Peyrche nd L. Roux for comments nd discussions on the nlyses nd mnuscript nd ll the memers of the Buzsáki lortory for their support. This work ws supported y the Fondtion pour l Recherche Médicle (FRM), the Fyssen Foundtion, Chrles H. Revson Senior Fellowship in Biomedicl Science (G.G.), NIH MH7 nd MH79, NS 9 nd the Simons Foundtions (G.B.). AUTHOR CONTRIBUTIONS G.G. nd G.B. designed the study, G.G. nd I.I. performed the experiments, G.G. nlyzed the dt nd G.B. nd G.G. wrote the mnuscript. COMPETING FINANCIAL INTERESTS The uthors declre no competing finncil interests. Reprints nd permissions informtion is ville online t reprints/index.html. Pulisher s note: Springer Nture remins neutrl with regrd to jurisdictionl clims in pulished mps nd institutionl ffilitions... Position on trck Pre/post-run corr diff.... Pre/post- Pre/post- Pre/post- corr diff corr diff corr diff c.. Position on trck.. Pre/post- corr diff. Vzdrjnov, A. & McGugh, J.L. Bsolterl mygdl is involved in modulting consolidtion of memory for clssicl fer conditioning. J. Neurosci. 9, (999).. Pré, D., Collins, D.R. & Pelletier, J.G. Amygdl oscilltions nd the consolidtion of emotionl memories. Trends Cogn. Sci., ().. Mren, S. & Fnselow, M.S. Synptic plsticity in the solterl mygdl induced y hippocmpl formtion stimultion in vivo. J. Neurosci., 7 7 (99).. Ikegy, Y., Sito, H. & Ae, K. Attenuted hippocmpl long-term potentition in solterl mygdl-lesioned rts. Brin Res., 7 (99).. Ikegy, Y., Sito, H. & Ae, K. High-frequency stimultion of the solterl mygdl fcilittes the induction of long-term potentition in the dentte gyrus in vivo. Neurosci. Res., 7 (99).. Goshen, I. et l. Dynmics of retrievl strtegies for remote memories. Cell 7, 7 9 (). 7. Reijmers, L.G., Perkins, B.L., Mtsuo, N. & Myford, M. Locliztion of stle neurl correlte of ssocitive memory. Science 7, (7).. Redondo, R.L. et l. Bidirectionl switch of the vlence ssocited with hippocmpl contextul memory engrm. Nture, (). 9. Hsing, H.-L.L. et l. Mnipulting cocine engrm in mice. J. Neurosci., 7 ().. Xu, C. et l. Distinct hippocmpl pthwys medite dissocile roles of context in memory retrievl. Cell 7, 9 97 ().. Phillips, R.G. & LeDoux, J.E. Differentil contriution of mygdl nd hippocmpus to cued nd contextul fer conditioning. Behv. Neurosci., 7 (99)..... Figure 7 Contriuting pirs form representtion during the run tht is rectivted during sleep nd reinstted during post-run without the ir puff. () Behviorl correltes of n exmple pyrmidl cell (left; red tringle) tht forms highly contriuting pirs with two hippocmpl plce cells (center nd right; lue nd green tringles). Firing mps re shown for pre-run, run nd post-run epochs (top to ottom) nd firing rte histogrm for run. Red line: ir puff loction. Blck rrows: running direction in which the ir puff is pplied ( ir puff trjectory ). Color rs re in hertz. () Correltion etween chnges in pre vs. post- correltions nd pre- vs. post-run correltions for strongly contriuting pirs (red,.% highest contriutions) nd wekly contriuting pirs (lue,.% lowest contriutions). nd run chnges re correlted for strongly contriuting pirs (Person r =.7, n = pirs, P =. ; n = rts), ut not for wekly contriuting pirs (r =., n = pirs, P =.; n = rts). Corr diff, correltion difference. (c) Sme s ut for strong (n = 9, pirs, r =., P =.7 ; n = rts) nd wek (n = 9, pirs, r =., P =.; n = rts) contriuting qurtiles (% highest nd lowest contriutions). dvnce online puliction nture NEUROSCIENCE

9 7 Nture Americ, Inc., prt of Springer Nture. All rights reserved.. Zelikowsky, M., Hersmn, S., Chwl, M.K., Brnes, C.A. & Fnselow, M.S. Neuronl ensemles in mygdl, hippocmpus, nd prefrontl cortex trck differentil components of contextul fer. J. Neurosci., ().. O Keefe, J. & Ndel, L. The Hippocmpus s Cognitive Mp (Oxford Univ. Press, 97).. Kjelstrup, K.G. et l. Reduced fer expression fter lesions of the ventrl hippocmpus. Proc. Ntl. Acd. Sci. USA 99, ().. Royer, S., Sirot, A., Ptel, J. & Buzsáki, G. Distinct representtions nd thet dynmics in dorsl nd ventrl hippocmpus. J. Neurosci., ().. Moser, M.B., Moser, E.I., Forrest, E., Andersen, P. & Morris, R.G. Sptil lerning with minisl in the dorsl hippocmpus. Proc. Ntl. Acd. Sci. USA 9, (99). 7. Moser, E.I.E., Kroert, K.A., Moser, M.B. & Morris, R.G. Impired sptil lerning fter sturtion of long-term potentition. Science, (99).. Smll, S.A. The longitudinl xis of the hippocmpl formtion: its ntomy, circuitry, nd role in cognitive function. Rev. Neurosci., 9 (). 9. Kim, J.J. & Fnselow, M.S. Modlity-specific retrogrde mnesi of fer. Science, 7 77 (99).. Corcorn, K.A., Desmond, T.J., Frey, K.A. & Mren, S. Hippocmpl inctivtion disrupts the cquisition nd contextul encoding of fer extinction. J. Neurosci., ().. Mren, S. & Quirk, G.J. Neuronl signlling of fer memory. Nt. Rev. Neurosci., ().. Pitkänen, A., Pikkrinen, M., Nurminen, N. & Ylinen, A. Reciprocl connections etween the mygdl nd the hippocmpl formtion, perirhinl cortex, nd postrhinl cortex in rt. A review. Ann. NY Acd. Sci. 9, 9 9 ().. Herry, C. et l. Switching on nd off fer y distinct neuronl circuits. Nture, ().. Girrdeu, G. & Zugro, M. Hippocmpl ripples nd memory consolidtion. Curr. Opin. Neuroiol., 9 ().. Wng, D.V. et l. Mesopontine medin rphe regultes hippocmpl ripple oscilltion nd memory consolidtion. Nt. Neurosci., 7 7 ().. vn de Ven, G.M., Trouche, S., McNmr, C.G., Allen, K. & Dupret, D. Hippocmpl offline rectivtion consolidtes recently formed cell ssemly ptterns during shrp wve-ripples. Neuron 9, 9 97 (). 7. Pop, D., Duvrci, S., Popescu, A.T., Lén, C. & Pré, D. Coherent mygdlocorticl thet promotes fer memory consolidtion during prdoxicl sleep. Proc. Ntl. Acd. Sci. USA 7, 9 ().. Huff, M.L., Miller, R.L., Deisseroth, K., Moormn, D.E. & LLumiere, R.T. Posttrining optogenetic mnipultions of solterl mygdl ctivity modulte consolidtion of inhiitory voidnce memory in rts. Proc. Ntl. Acd. Sci. USA, 97 (). 9. Ptel, J., Schomurg, E.W., Berényi, A., Fujisw, S. & Buzsáki, G. Locl genertion nd propgtion of ripples long the septotemporl xis of the hippocmpus. J. Neurosci., 79 7 ().. Lovett-Brron, M. et l. Dendritic inhiition in the hippocmpus supports fer lerning. Science, 7 ().. Mizuseki, K., Royer, S., Di, K. & Buzsáki, G. Activity dynmics nd ehviorl correltes of CA nd CA hippocmpl pyrmidl neurons. Hippocmpus, 9 ().. Kudrimoti, H.S., Brnes, C.A. & McNughton, B.L. Rectivtion of hippocmpl cell ssemlies: effects of ehviorl stte, experience, nd EEG dynmics. J. Neurosci. 9, 9 (999).. Lnsink, C.S., Goltstein, P.M., Lnkelm, J.V., McNughton, B.L. & Pennrtz, C.M.A. Hippocmpus leds ventrl stritum in reply of plce-rewrd informtion. PLoS Biol. 7, e7 (9).. Hoffmn, K.L. & McNughton, B.L. Coordinted rectivtion of distriuted memory trces in primte neocortex. Science 97, 7 7 ().. Wilson, M. & McNughton, B. Rectivtion of hippocmpl ensemle memories during sleep. Science (- ), 7 (99).. Shen, J., Kudrimoti, H.S., McNughton, B.L. & Brnes, C.A. Rectivtion of neuronl ensemles in hippocmpl dentte gyrus during sleep fter sptil experience. J. Sleep Res. 7 (Suppl. ), (99). 7. Genzel, L., Spoormker, V.I., Konrd, B.N. & Dresler, M. The role of rpid eye movement sleep for mygdl-relted memory processing. Neuroiol. Lern. Mem., ().. Hutchison, I.C. & Rthore, S. The role of REM sleep thet ctivity in emotionl memory. Front. Psychol., 9 (). 9. LeDoux, J.E. Coming to terms with fer. Proc. Ntl. Acd. Sci. USA, 7 7 ().. Moit, M.A., Rosis, S., Zhou, Y., LeDoux, J.E. & Blir, H.T. Putting fer in its plce: rempping of hippocmpl plce cells during fer conditioning. J. Neurosci., 7 7 ().. Wng, M.E. et l. Differentil roles of the dorsl nd ventrl hippocmpus in predtor odor contextul fer conditioning. Hippocmpus, ().. Bnnermn, D.M. et l. Regionl dissocitions within the hippocmpus memory nd nxiety. Neurosci. Bioehv. Rev., 7 ().. Ciocchi, S., Pssecker, J., Mlgon-Vin, H., Mikus, N. & Kluserger, T. Brin computtion. Selective informtion routing y ventrl hippocmpl CA projection neurons. Science, ().. Buzsáki, G. Hippocmpl shrp wve-ripple: cognitive iomrker for episodic memory nd plnning. Hippocmpus, 7 ().. Logothetis, N.K. et l. Hippocmpl corticl interction during periods of sucorticl silence. Nture 9, 7 ().. Sprt, D.R. et l. Inhiition of projections from the solterl mygdl to the entorhinl cortex disrupts the cquisition of contextul fer. Front. Behv. Neurosci., 9 (). 7. Chrok, J.J. & Buzsáki, G. High-frequency oscilltions in the output networks of the hippocmpl-entorhinl xis of the freely ehving rt. J. Neurosci., (99).. Boyce, R., Glsgow, S.D., Willims, S. & Admntidis, A. Cusl evidence for the role of REM sleep thet rhythm in contextul memory consolidtion. Science (- ), (). 9. Mquet, P. et l. Experience-dependent chnges in cererl ctivtion during humn REM sleep. Nt. Neurosci., ().. Atherton, L.A., Dupret, D. & Mellor, J.R. Memory trce reply: the shping of memory consolidtion y neuromodultion. Trends Neurosci., 7 (). nture NEUROSCIENCE dvnce online puliction

10 7 Nture Americ, Inc., prt of Springer Nture. All rights reserved. ONLINE METHODS Sujects nd electrode implnttion. All experiments were pproved y the Institutionl Animl Cre nd Use Committee (IACUC) t New York University Medicl Center. Four individully housed mle Long-Evns rts were used in this experiment, nd mintined on h:h light-drk cycle (lights on t 7.m.) throughout the study. Animls ( g, months old t time of surgery) were deeply nesthetized with isoflurne. Three silicon proes ( with shnks, with shnks, recording chnnels totl, NeuroNexus H nd H, A-style, Buzski nd lyout) mounted on individul movle microdrives were implnted ove the mygdle ilterlly (AP. mm ML ±. to. mm from regm) nd in the dorsl hippocmpus (left or right, CA, AP. mm, ML ±. mm). The drives were secured to the skull using dentl cement. Skull screws ove the cereellum were used s ground nd reference. The drives nd proes were protected y cement-covered copper-mesh Frdy cge on which the proe connectors were ttched. Animls were llowed to recover for t lest d with d liitum food nd wter. In one niml, the hippocmpus proe filed during the course of the experiment. This niml ws hence not used for nlysis out hippocmpus coordintion, ut ws used for intr-mygdl nd intr-piriform cortex physiology nd rectivtion nlysis. Dt collection nd nlysis we not performed lind to the conditions of the experiments. Recordings nd ehvior. All nimls were free from prior mnipultion efore eing included in the study. After week of dily hndling, nimls were plced on wter restriction nd trined to run ck nd forth on liner trck for wter rewrds (Fig. ). All experiments were performed during the dy (light cycle). Three dys efore surgery, they regined ccess to d liitum food nd wter. After the recovery period, the proes were slowly lowered in the rin nd the recordings strted when reching hippocmpl CA pyrmidl lyer nd the superior limit of, respectively. During this period, the rts were plced ck on wter restriction to >% of their norml weight nd re-exposed to the liner trck. The position of the niml ws trcked using cmer mounted on the ceiling nd red LED ttched to the hed of the niml. Signls were recorded t khz using n Amplipex recording system (Amplipex Inc., Szeged, Hungry) nd the ssocited Amplirec softwre. The mygdl electrodes were lowered y µm t the end of ech recording session to ensure complete spnning of the mygdl region over the course of the experiment. The hippocmpl proe ws djusted dily to optimize ripple nd unit recording. Preprocessing. -khz signls were resmpled t, Hz to extrct LFP dt. Spikes were extrcted y high-pss filtering ( Hz) nd thresholding the signl, then clustered using Klustkwik ( followed y mnul clustering using Klusters ( Dt were visulized nd preprocessed using Neuroscope ( sourceforge.net/) nd NDMnger ( Units were clssified into puttive pyrmidl cells nd puttive interneurons using monosynptic connections (Supplementry Fig. ). The remining, unidentified cells were sorted using k-mens clustering (two clusters) on the inverse frequency (tht is, durtion; fst Fourier trnsform) nd pek-to-trough vlues (in milliseconds) of the men wveform of the spikes. Sleep stges were mnully scored through visul inspection of the hippocmpus nd mygdl spectrogrms nd ccelerometer signl using the visul scoring custom progrm TheStteEditor. Periods of were ssocited with immoility nd high thet/delt rtio (in hippocmpus) or gmm ( Hz)/rod low frequency ( Hz) nd rtio (in mygdl). REM sleep ws chrcterized y sleep posture nd regulr thet wves. Sttisticl nlysis. Non-prmetric Wilcoxon rnk sum or signed rnk sum (two-tiled, unless otherwise specified) tests were used throughout the pper. All tests used re specified in the figure legends or in the text. Smple sizes were not predetermined, ut our smple sizes re similr to (n nimls) or higher thn (n cells) those generlly employed in the field. When prmetric tests were used, the dt stisfied the criteri for normlity (Kolmogorov Smirnov test) nd equlity of vrince (Brtlett s test for equl vrince). For multiple comprisons in the post hoc tests, the originl P-vlues re shown ut the significnce thresholds P <., P <., P <. re indicted with either Bonferroni corrections or Tukey Krmer test for multiple comprisons. All dt re represented with ox plots showing the medin with centrl nd dispersion sttistics. Some extreme dt points re not shown in the figures for clrity ut ll dt points were included in the nlyses. Br plots (Fig. ) re shown only in comintion with the full distriutions (Supplementry Fig. ). P-vlues for Person s correltions re computed using Student s t distriution for trnsformtion of the correltion (Mtl corr function). A Life Sciences Reporting Summry is ville for n overview of ethics nd sttistics. Anlysis. All nlyses were performed using Chronux ( the FMAToolox ( nd Mtl (The MthWorks, Inc., Ntick, MA, USA) uilt-in functions nd custom-written scripts. For ehviorl mesures, speed rtios were clculted s (pdz speed cdz speed)/(pdz speed + cdz speed), with pdz the previous dnger zone ( cm preceding the ir puff loction of the previous trining dy) nd cdz the current dnger zone ( cm preceding the ir puff loction of the current trining dy). Becuse rts run more nd fster lps when hituted to the ir puff, hitution index ws clculted for ech trining session nd niml s the totl numer of ck-nd-forth lps divided y the totl time spent on the mze. To otin the ir-puff-centered speed curves, the trck positions were normlized nd ligned to the ir puff loction for ech session. In this plot (Fig. c), two sessions re missing due to corruption of the niml position dt. Firing rte (FR) chnges etween wkefulness nd REM sleep were evluted using the REM/wke rtios, clculted s (REM FR wke FR)/(REM FR + wke FR). Positive rtios indicte REM FR > wke FR. Ripple detection ws performed y nd-pss filtering (~ Hz), squring nd normlizing, followed y thresholding of the field potentil recorded in CA pyrmidl lyer. SPW-Rs were defined s events strting t s.d., peking t > s.d., nd remining t > s.d. for < ms nd > ms round the pek. A control detection ws performed on nonhippocmpl chnnel nd ll events simultneously recorded from the hippocmpl nd control chnnels (for exmple, musculr noise) were removed. Ripple modultion ws ssessed using Poisson test with P <.. This pproch tests whether the prmeters for the Poisson cumultive distriution function of spikes outside SPW-Rs (seline) re the sme s for the Poisson cumultive function during SPW-Rs (custom progrm clling the poiscdf Mtl function). The seline (inter-ripple) firing rte ws computed during sleep epochs excluding SPW-Rs. To void contmintion of rte chnges round SPW-Rs, the -ms periods efore nd fter ech ripple were lso excluded. Explined vrince (EV) nd reverse explined vrince (REV) were clculted per session using susets of cell pirs selected from the structures of interest. Only sessions with minimum of shnk nd cells in ech structure were included in the nlysis. This criterion ccounts for the vrition in the numer of sessions depending on the suset of cells the EV nd REV re clculted for (pyrmidl cell only vs. ll cells). For REM sleep, only sessions with minimum of min of totl REM sleep (ll REM sleep epochs were pooled together) in oth pre- nd post-sleep were included. Pirwise correltions for EV nd REV were clculted using the Person correltion coefficient on -ms-inned spike trins. The coefficients were seprtely clculted for pre-sleep ( or REM), trining, nd post-sleep ( or REM) periods nd ssemled into correltion mtrices. The correltions etween ll comintions of these three mtrices were then clculted nd were used to ssess the percentge of vrince in the post-sleep period tht could e explined y the ptterns estlished during trining while controlling for pre-existing correltions in the pre-sleep session (EV): EV = = RT, S RT, S RS, S R T, S S ( RT, S) ( RS, S) where R vriles re the correltion coefficients etween trining (T), presleep (S) nd post-sleep (S) pirwise correltion mtrices. The control vlue (REV) is otined y switching the temporl order of the pre- nd post-sleep session 7,. Only sessions with EV > REV for the first min epoch were used to clculte the decy of rectivtions (EV/REV in first nd susequent -min epochs; sessions with EV > REV were excluded). Rectivtions were considered significnt when EV ws significntly different from REV (Wilcoxon sign rnk test). Comprisons of rectivtion cross time or structures were performed on the difference EV REV (Wilcoxon rnk sum tests). nture NEUROSCIENCE doi:./nn.7