Emotional enhancement of memory via amygdaladriven facilitation of rhinal interactions

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1 6 Nture Pulishing Group Emotionl enhncement of memory vi mygdldriven fcilittion of rhinl interctions Rony Pz, Joe Guillume Pelletier, Elizeth P Buer & Denis Pré Emotions generlly fcilitte memory, n effect medited y the solterl mygdl (BLA). To study the underlying mechnisms, we recorded BLA, perirhinl nd entorhinl neurons during n ppetitive trce-conditioning tsk. We focused on the rhinl cortices ecuse they constitute the interfce etween the hippocmpus, meditor of memory consolidtion, nd the neocortex, the storge site of declrtive memories. We found tht, fter unexpected rewrds, BLA ctivity incresed impulse trnsmission from perirhinl to entorhinl neurons nd tht this effect decyed s the ssocition etween conditioned stimuli nd rewrds ws lerned. At this lte phse of lerning, the BLA effect occurred when the nimls were nticipting the rewrd. By enhncing the processing of sensory cues, the BLA-medited fcilittion of rhinl interctions my explin how the mygdl promotes memory formtion in emotionl conditions. Humns generlly form more vivid memories of emotionlly chrged events thn of mundne experiences. How do emotions fcilitte memory? It is known tht the fcilittion of memory y emotions requires n intct BLA in humns 4 nd nimls 5,6. Moreover, there is evidence tht the mygdl fcilittes memory y enhncing ttention during encoding nd y modulting consolidtion nd storge fter lerning 5,7. In humns, ttention is enhnced y emotionl stimuli nd this effect is sent in suject with mygdl lesions 8. Consistent with these results, functionl imging studies indicte tht the mygdl is ctivted y emotionl stimuli 9. In ddition, there is strong positive correltion etween mygdl ctivity t encoding nd the long-term recll of emotionl mteril,. In nimls, evidence shows tht the mygdl is criticl site of plsticity when using clssicl fer-conditioning protocols. However, for mny other types of emotionl memories, including stritl- nd hippocmpl-dependent ones, the mygdl does not seem to e storge site ut rther modultes memory consolidtion in its trgets. Indeed, post-trining injections of drugs tht presumly enhnce or reduce BLA ctivity respectively fcilitte or impir retention, even when memory ws tested long fter the effects of these drugs hs dissipted 3. In contrst, injections of lidocine into the BLA dys fter trining, just efore testing retention, do not ffect performnce on stritl- nd hippocmpl-dependent memory tsks 4. Although emotionl rousl recruits the BLA y mens of stress hormones 5 7, cusing long-lsting increse in BLA firing rtes 8,the impct of this incresed ctivity on trget structures hs received little ttention so fr. Here, we investigted how the presenttion of iologiclly significnt nd rousing stimuli ffects neuronl interctions etween the BLA nd rhinl cortices, y simultneously recording neurons of the BLA, perirhinl res 35 nd 36, nd entorhinl cortex in cts performing trce-conditioning tsk known to e dependent on the mygdl, rhinl cortices nd hippocmpus 9 3. Indeed, the rhinl cortices receive mssive BLA inputs 4,5 nd form the interfce 6,7 etween the hippocmpus, criticl meditor of memory consolidtion, nd the neocortex, thought to e long-term repository of declrtive memories 8 3.Wefoundtht BLA ctivity enhnces the processing of sensory cues during ehviorlly slient events y fcilitting impulse trnsmission from the perirhinl to the entorhinl cortex. Furthermore, this effect ws tightly linked to lerning. RESULTS To test the possiility tht mygdl projections to the rhinl cortices fcilitte memory y promoting impulse trnsfer etween the neocortex nd hippocmpus, we simultneously recorded from BLA (n ¼ 39), perirhinl (n ¼ 8) nd entorhinl (n ¼ 3) neurons in cts, y mens of 4 microelectrodes (Fig.,). We nlyzed BLArelted chnges in rhinl ctivity in four conditions: under nesthesi, during wking periods where no rewrds were dministered, fter unexpected rewrds nd during trce-conditioning tsk. The following nlyses include only neurons whose loction ws confirmed y post-hoc histologicl reconstructions of microelectrode trcks (Fig. c f). Impct of BLA ctivity on spontneous rhinl interctions To study how BLA ctivity ffects impulse trffic in the rhinl cortices, we first computed cross-correltions of spontneous firing generted y perirhinl nd entorhinl neurons locted in the sme coronl plne (Fig. g i nd Supplementry Fig. online). Despite the strong reciprocl connections etween the perirhinl nd entorhinl cortices, Center for Moleculr nd Behviorl Neuroscience, Rutgers, The Stte University of New Jersey, 97 University Avenue, Newrk, New Jersey 7, USA. Correspondence should e ddressed to D.P. (pre@xon.rutgers.edu) or R.P. (rony.pz@gmil.com). Received 6 June; ccepted August; pulished online Septemer 6; doi:.38/nn77 NATURE NEUROSCIENCE VOLUME 9 [ NUMBER [ OCTOBER 6 3

2 6 Nture Pulishing Group c ec e OB M BLA EC R C rh LA L d ec rh V LA f Amygdl Perirhinl Entorhinl impulse propgtion occurs with low proility in the trnsverse xis of the rhinl cortices 3. In keeping with this, we oserved low proportion of significnt perirhinl-entorhinl cross-correltions (Fig. g; % of 445 cell couples, P o.5, Methods) under nesthesi nd during quiet wking. Similrly, entorhinl cells were generlly not coctive with BLA cells (Fig. h; 6% of 45). However, when this nlysis ws restricted to perirhinl spikes tht occurred within 3 ms of BLA spike, n intervl corresponding to the verge ltency of synpticlly evoked dischrges in the BLA-rhinl network 3,theproportion of significnt perirhinl-entorhinl cross-correltions nerly douled (Fig. i; 38% of 445; w test, P o.). However, cross-correltions hve severe limittions ecuse they relte the ctivity of only two cells, forcing one to choose ritrry intervls when studying how third neuron ffects the reltion etween them. To overcome this difficulty, we studied triplets of perirhinl, entorhinl nd BLA cells (n ¼ 445) using spike-triggered joint histogrms (STJH), n dpttion of the joint peristimulus time histogrm (JPSTH) method 33, in which BLA spikes were used s temporl reference 34 to study correlted perirhinl nd entorhinl firing. STJHs (Fig. ) re computed y tking segments (± 5 ms) of rhinl ctivity round BLA spikes (time in x nd y). The spikes (red ticks) generted y the perirhinl nd entorhinl cells re plotted on the x- nd y-xes, respectively. STJH ins contining coincidence of spikes (squres) re incremented. Thus, if round given BLA spike, CE BL rh V BM ME OT AHA H g h i Entorhinl spikes per s Spike numer Entorhinl spikes per s Spike numer Entorhinl spikes per s Spike numer Time from PRH spike (ms) PRH Time from BLA spike (ms) BLA Time from PRH spike (ms) PRH (BLA) Figure Simultneous recordings of mygdl, perirhinl, nd entorhinl neurons. () Ventrl view of ct rin showing position of microelectrodes (dots). Cross indictes orienttion. EC, entorhinl cortex; OB, olfctory ul; rh, rhinl sulcus. () Spontneous ctivity. (c f) Histologicl verifiction of recording sites. Coronl rin sections. Arrows, electrolytic lesions performed t the end of the experiments to mrk the tip of electrodes tht coursed through the lterl mygdl (LA; c), sl mygdloid nuclei (BL, BM; d), re 35 (e), nd re 36 nd the entorhinl cortex (f). AHA, mygdlohippocmpl re; CE, centrl nucleus; ec, externl cpsule; H, hippocmpus; ME, medil nucleus; OT, optic trct; V, ventricle. (g i) Activity of n entorhinl neuron round perirhinl (g), BLA (h) or perirhinl nd BLA (± 3ms, i) firing s illustrted in crosscorreltions (top) or rster plots (ottom). PRH, perirhinl. Scle rs: 3 mm in, sin, mminc. there ws spike from the perirhinl cell t time x nd one from the entorhinl cell t time y, one count is dded to the mtrix in (x,y). Repeting this process for ech BLA spike grdully produces the rw STJH (Fig. ). Becuse we were interested in BLA-relted rhinl correltion, we tested the rw STJHs ginst two null hypotheses (Methods): (i) tht the oserved correltion is similr to tht expected independently of BLA ctivity, nd (ii) tht the correltion merely reflects independent responses of rhinl neurons to BLA ctivity. To test the first possiility, we compred the rw STJH to the verge of 5 STJHs computed fter shuffling the BLA spike trin (Fig. c). This is equivlent to computing the STJH round rndom times. To test the possiility tht the correltions evidenced in the STJHs reflect independent responses of rhinl neurons to BLA ctivity, we computed STJHs y shuffling the BLA spike trin of one of the two rhinl cells with respect to the other 5 times nd verging the result (Fig. d). This technique corresponds to the shift predictor 33.Wethenperformed in-to-in comprisons of significnce etween the rw nd the two rndomized sets of STJHs, using Poisson distriution with threshold P-vlue corrected for multiple comprisons (.5 divided y the numer of ins: 9; Fig. e). Finlly, we verified tht most significnt ins clustered round one dominnt pek (Methods nd Supplementry Fig. online). Another importnt property disclosed y the STJH is the reltive timing of perirhinl nd entorhinl firing in reltion to BLA spikes. Bins ove or elow the min digonl of the STJH represent correlted ctivity where entorhinl firing respectively follows or precedes perirhinl spikes (Fig. f). We used this informtion to compute directionlity index (Methods). We will return to the directionlity index in the section descriing the impct of rewrds on BLA-rhinl interctions. We computed cross-correltions of perirhinl-entorhinl cell couples (Fig. 3 d), long with the corresponding STJH conditioned on BLA spikes (Fig. 3e h) nd control STJH (Fig. 3i l) produced y rndom shuffling of the BLA spike trin. These STJHs (Fig. 3, middle) showed tht, in reltion to BLA spikes, there ws high degree of 3 VOLUME 9 [ NUMBER [ OCTOBER 6 NATURE NEUROSCIENCE

3 6 Nture Pulishing Group Figure Method used to compute STJHs nd determine whether they re sttisticlly significnt. () Segments of entorhinl (y-xis) nd perirhinl (x-xis) ctivity centered on BLA spikes were isolted to identify ins tht contined coincidence (squres) of entorhinl nd perirhinl spikes (red ticks). The mtrix ins contining coincident rhinl spikes were incremented nd this process ws repeted for ll BLA spikes, grdully producing the STJH. ( e) Method used to ssess whether STJHs were sttisticlly significnt. To determine whether the oserved rhinl correltion () ws dependent on the timing of BLA ctivity, the BLA spike trins were shuffled 5 times nd the result verged (c). To test whether the correltion in the STJH reflects independent responses of rhinl neurons to BLA ctivity, the BLA spike trin of one of the two rhinl cells ws shuffled 5 times nd the result verged (d). STJH ins were considered significnt when they differed from oth of the rndomly generted sets of vlues, t significnce level of P o.5/9 (the numer of ins, e). (f) The loction of significnt ins in the STJHs indictes the prevlent direction of impulse trffic in the rhinl cortices. A concentrtion of significnt ins ove the min digonl of the STJH indictes tht perirhinl cells tended to fire efore entorhinl neurons in reltion of BLA spikes. A concentrtion of significnt ins elow the min digonl indictes tht the opposite firing sequence previled. correlted perirhinl-entorhinl ctivity, even when the corresponding cross-correltions showed little (Fig. 3) or no(fig. 3,c) evidence of correlted firing. Moreover, even high cross-correltion (Fig. 3d) could ctully e centered on nd relted to BLA spikes (Fig. 3h i). Thus, the STJHs revel tht uried in the cross-correltions re periods of enhnced rhinl interctions tht prevlently occur when BLA cells re ctive. Using the criteri mentioned ove, we found tht 34% nd 8%, respectively, of STJHs were deemed significnt in the nesthetized nd quiet wking conditions (higher thn expected y chnce, P o., Fisher exct test). These results suggest tht in reltion to BLA ctivity, impulse trnsfer is fcilitted in the rhinl cortices. Impct of BLA ctivity during ehviorlly slient events To exmine whether the BLA-relted fcilittion of rhinl interctions is influenced y ehviorlly slient events, we studied the impct of unexpected liquid rewrds on the incidence of significnt STJHs. These rewrds were slient ecuse they were delivered t rndom intervls (3 9 s) nd the cts (n ¼ 3) were fed only during the recordings. The proportion of significnt STJHs (computed in 5-ms windows) incresed mrkedly fter rewrd (from 3% to 44% of 5; P o., w ; Fig. 4 nd Supplementry Fig. 3 online). The grnd verge of significnt post-rewrd STJHs (Fig. 4) reveled tht correlted rhinl ctivity (red ptch) tended to occur fter BLA firing (t time ). These findings indicte tht, fter ehviorlly slient events, rhinl interctions re fcilitted in reltion to BLA ctivity. But does this effect prticipte in memory formtion? f Lterl to medil (PR -> ER) (ER -> PR) Medil to lterl Figure 3 Enhnced neuronl interctions within rhinl cortices round BLA ctivity. Four pirs (rows) of simultneously recorded perirhinl nd entorhinl neurons. ( d) Cross-correltions of spontneous ctivity. (e h) STJHs for sme cell couples (perirhinl, x-xis; entorhinl, y-xis) conditioned on BLA firing (t time ). STJHs were normlized to the men of the BLA-shuffled mtrix nd the counts were color-coded. (i l) Control STJHs computed round rndom times (verge of 5 shuffles). In pnels e nd h, STJHs revel tht BLA spikes tend to e followed y correlted rhinl firing, which is smered cross the digonl in the shuffled STJHs (i,l). This suggests tht BLA ctivity is mjor contriuting fctor to the peks of the cross-correltions. 5 5 STJH conditioned on BLA firing 5 5 c STJH (shuffled BLA spikes) d 5 Shift predictor BLA-relted rhinl interctions in lerning context To ddress this question, cts (n ¼ 3) were trined on trceconditioning tsk in which visul conditioned stimulus (CS) predicted rewrd delivery 3 s lter. The CS ws glol chnge in the illumintion of n LCD screen plced ft in front of the cts. Lerning in this tsk is dependent on the mygdl, rhinl cortices nd hippocmpus 9 3. The predictive vlue of the CS ws lerned e i Normlized counts Normlized counts Normlized counts Normlized counts Time (ms) Time (ms) Time (ms) Time (ms) f c g k d h l j e 5 P-vlue of significnt ins Time from rndom event (ms) Time from rndom event (ms) NATURE NEUROSCIENCE VOLUME 9 [ NUMBER [ OCTOBER 6 33

4 6 Nture Pulishing Group Rtio of significnt STJHs Rewrd grdully, s evidenced y grdul increse in CS-evoked nticiptory (pre-rewrd) licking over trining sessions (Fig. 5, dshed line; r ¼.6; P o.). To determine if there were lerning-ssocited chnges in the reltion etween BLA ctivity nd rhinl interctions, the results otined in erly (dys 3) nd lte (dys 8 ) phses of lerning were considered seprtely. In the erly phse (Fig. 5), the proportion of significnt STJHs incresed mrkedly fter rewrd delivery (y 6%, n ¼ 8; P o., w ) ut not in reltion to the CS or the dely. In contrst, in the lte lerning phse (Fig. 5c), the proportion of significnt STJHs incresed during the lte prt of the CS nd dely (y 67%, n ¼ 8; P o., w ) ut not fter rewrd. Consistent with the ide tht the enhncement of rhinl interctions seen in reltion to BLA ctivity fcilittes memory formtion, nlysis of dy-to-dy fluctutions in the proportion of significnt post-rewrd Figure 4 BLA-relted modifictions of rhinl interctions during the presenttion of unexpected liquid rewrds. () Proportion of significnt STJHs (y-xis; ± s.e.m.) s function of time (x-xis) round unexpected rewrds. () Averge of ll significnt STJHs fter rewrd delivery. Note tht correlted rhinl ctivity (red ptch) tended to occur fter BLA firing (time ) nd tht this ctivity ws higher ove the min digonl, indicting tht perirhinl cells generlly fired efore entorhinl neurons. Similr results were otined whether we considered rewrd-relted ctivity within or outside the trining context. STJHs reveled tht s lerning progressed (Fig. 5, dshed line), the BLA-relted fcilittion of rhinl interctions grdully decresed (Fig. 5, continuous line). Moreover, the rtio of significnt postrewrd STJHs ws tightly nd inversely correlted (r ¼.79, P o.) with memory strength s inferred from ehvior (nticiptory licking) in ll tested cts, considered individully or s group (Fig. 5d). In contrst, s lerning progressed, the rtio of significnt STJHs during the dely incresed (Fig. 5e, continuous line) nd ws positively correlted with ehvior (r ¼.48, P o.; Fig. 5f). Notly, these effects were proly relted to lerning, s in the previous experiments, where rewrds remined unexpected, there ws no time-dependent decrese in the proportion of significnt STJHs fter rewrd (r ¼., P ¼.4). Overll, these findings indicte tht in reltion to BLA dischrges, rhinl interctions re fcilitted in lerning context nd this phenomenon is tightly relted to the lerning phse. Directionlity of fcilitted rhinl interctions An importnt question for the mechnisms of memory encoding nd retrievl is whether, in reltion to BLA ctivity, there is fcilittion of impulse trnsmission from perirhinl to entorhinl neurons (lterl to medil) 35 or in the reverse direction. In the STJHs, predominnce of counts ove the min digonl indicte tht BLA firing is preferentilly ssocited with lteromedil rhinl interctions, wheres counts elow the min digonl indicte the opposite (Fig. f). Thus, we clculted directionlity index (DI) for ll cell triplets (Methods). The DI rnges from to, with positive vlues indicting preference for lterl (perirhinl) to medil (entorhinl) communiction. In periods ssocited with low proportion of significnt STJHs (quiet wking, pre-cs nd pre-rewrd, outside nd within the lerning context), the DI hd n verge ner (Fig. 6, lue curve,. ±.7, P 4.). In contrst, fter rewrds, the DI distriution ws skewed to the right (Fig. 6, red curve) with men significntly higher thn (. ±.4, P o., t-test), consistent with the verge of significnt post-rewrd STJHs (Fig. 4) where significnt ins were concentrted ove the min digonl. Anlyzing the time course of fluctutions in Anticiptory licks per s STJHs (post-rewrd) Anticiptory licking Trining session.4.3. Rtio of significnt STJHs.5.5 CS on Erly trining phse CS off Rewrd Figure 5 Time-dependent chnges in proportion of significnt STJHs during lerning in trce-conditioning protocol. () Dshed lines, rte of nticiptory (pre-rewrd) licking (left y-xis; ± s.e.m.) s function of trining session (x-xis). Continuous lines, proportion of significnt STJHs (right y-xis; ± s.e.m.) fter rewrd delivery s function of trining session (x-xis). (,c) Normlized rtio of significnt STJHs (y-xis; ± s.e.m.) during trce-conditioning trils in erly () nd lte (c) phses of lerning. (d) Correltion etween numer of nticiptory licks (x-xis) nd proportion of significnt STJHs (y-xis) seen in reltion to rewrd delivery. Ech dt point represents one trining session e Anticiptory licks per s c Rtio of significnt STJHs Lte trining phse CS on CS off Rewrd STJHs (dely) Anticiptory licking Trining session.3.. d frtio of significnt STJHs (dely) Rtio of significnt STJHs Post-rewrd STJHs vs. ehvior r =.79 P = Anticiptory (dely) licks per s Pre-rewrd STJHs vs. ehvior r =.48 P = Anticiptory (dely) licks per s (verge cross cts). (e) Dshed lines, rte of nticiptory (pre-rewrd) licking (left y-xis; ± s.e.m.) s function of trining session (x-xis). Continuous lines, proportion of significnt STJHs (right y-xis; ± s.e.m.) during dely period s function of trining sessions (x-xis). (f) Correltion etween numer of nticiptory licks (x-xis) nd proportion of significnt STJHs (y-xis) seen during the dely period. Ech dt point represents one trining session (verge cross cts). 34 VOLUME 9 [ NUMBER [ OCTOBER 6 NATURE NEUROSCIENCE

5 6 Nture Pulishing Group Proportion of STJHs.. Pre-rewrd ( 5 to ms) Post-rewrd (5 to 75 ms).5.5 M -> L Directionlity index L -> M..5.5 Time from rewrd (s) the verge DI round rewrd delivery (Fig. 6) reveled shift in directionlity tht peked round 5 ms fter rewrd delivery (similr results were otined when seprtely considering the unexpected rewrds nd those dministered during the erly lerning phse). Thus, these results suggest tht, in reltion to BLA ctivity, there is n enhnced impulse propgtion from the perirhinl to entorhinl cortex. Mechnisms of BLA fcilittion of rhinl interctions We further nlyzed the temporl dynmics of the impct of ech BLA spike on rhinl correltions y clculting normlized proilities of entorhinl firing s function of the timing of perirhinl nd BLA spikes. These nlyses reveled tht the proility of n entorhinl spike ws gretest when oth BLA spike nd perirhinl spike occurred within r5 ms (Fig. 7, lue curve). This effect ws significntly higher thn predicted (Fig. 7, lck curve, P o., t-tests) from the proility of entorhinl firing ssocited with isolted perirhinl or BLA spikes (Fig. 7, green nd red curves, respectively; Supplementry Fig. 4 online). Thus, ech BLA spike hs fcilitting effect (lsting B5 ms) on impulse trnsfer in the rhinl cortices. This timescle is consistent with the monosynptic glutmtergic projections from the BLA to the rhinl cortices 4. Fluctutions in the proportion of significnt STJHs were not simply tied to vritions in firing rtes. In BLA neurons for instnce (Fig. 7), rewrd-relted increse in firing rte occurred t erly nd lte stges of lerning. Yet, the proportion of significnt STJHs only incresed in the initil lerning phse. We oserved similr dissocitions in rhinl neurons (Supplementry Fig. 5 online). To further ssess the impct of BLA firing rtes, we compred the proportion of significnt STJHs Directionlity index. Rewrd Figure 6 Directionlity of rhinl interctions fcilitted y BLA ctivity. () Frequency distriution of directionlity index (DI) for sttisticlly significnt STJHs. () DI(y-xis; men ± s.e.m.) s function of time (x-xis) round rewrd delivery (rrow) in the erly lerning phse. sed on BLA cells with versus without post-rewrd increses in firing rte; it ws 4% for neurons with nd 8% for neurons without increses in firing rte (oth proportions were significntly higher thn chnce, P o., Fisher exct test). Thus, chnges in firing rtes, lthough they contriute, do not ccount for ll cses of significnt STJHs. Next, we considered the possiility tht more synchronized BLA output underlies the fcilittion of rhinl interctions, s such synchrony would enhnce the summtion of inputs. Hence we clculted cross-correltions for ll pirs of simultneously recorded BLA neurons nd found tht the overll cross-correltion ws higher fter thn efore unexpected rewrds (Fig. 7c, n ¼ 543, P o., t-test). To correct for firing-rte effects, we rndomly shuffled the trils of one neuron in reltion to those of the other nd compred the verged shuffled cross-correltions to the originl one. The rtio of significnt post-rewrd cross-correltions significntly decresed from the erly (n ¼ 89) to the lte (n ¼ 35) phse of lerning (Fig. 7d, P o., w ). The opposite ws seen in reltion to the dely (Fig. 7d, P o.5, w ). Overll, these nlyses suggest tht the BLA-relted fcilittion of rhinl interctions involves higher nd more synchronized BLA output. DISCUSSION The present study ws undertken to exmine how the presenttion of iologiclly significnt nd rousing stimuli ffects neuronl interctions etween the BLA nd rhinl cortices. The interest of this question stems from previous work showing tht the BLA fcilittes memory for emotionlly rousing events. Here we focused on the impct of BLA ctivity on perirhinl nd entorhinl neurons ecuse the rhinl cortices constitute the min route for impulse trffic into nd out of the hippocmpus. Our findings suggest tht BLA ctivity is ssocited with fcilitted neuronl interctions in the rhinl cortices, phenomenon tht ws most pronounced in reltion to ehviorlly slient events. Moreover, in trce-conditioning tsk, we oserved tht the BLA-medited fcilittion of rhinl interctions ws tightly linked to memory formtion. In the following ccount, we consider the significnce of these findings for the fcilittion of memory y emotions nd for the role of mygdl ctivity in enhncing the ssociility of unexpected cues. The rhinl cortices occupy pivotl position Multiple lines of evidence suggest tht the rhinl cortices hve criticl role in vrious spects of lerning nd memory, including the formtion of sptil representtions 36,37, the encoding of the configurtion of visul stimuli 38,39, the representtion of stimulus fmilirity 4,4 s well Proility of entorhinl spike.7 AP...E Pro(E (A,P) t ).6 A(not P)...E Pro(E (A,~P) t ) (not A)P...E Pro(E (~A,P) t ).5 Expected Time to entorhinl spike (ms) BLA firing rte (s.d. of seline) 3 CS on CS off Rewrd 3 4 Erly Lte c Normlized counts.5.5 Before rewrd After rewrd BLA-BLA CCs.... d Rtio of significnt BLA CCs Rewrd Dely Erly Lte Lerning phse Figure 7 Mechnisms underlying BLA modultion of rhinl interctions. () Plot of proility of entorhinl firing (y-xis) s function of the timing of perirhinl nd BLA spikes. See Supplementry Figure 5. () BLA firing rtes (± s.e.m.) in the trce-conditioning protocol, t erly (top, lue) nd lte (ottom, red) phses of lerning. (c) Averge cross-correltion etween couples of BLA neurons. (d) Proportion of significnt cross-correltions s function of the lerning phse (erly versus lte) in reltion to rewrd (solid line) nd during the dely (dshed line). CC, cross-correltion. NATURE NEUROSCIENCE VOLUME 9 [ NUMBER [ OCTOBER 6 35

6 6 Nture Pulishing Group s the cquisition, consolidtion nd retrievl of declrtive memories 8. Furthermore, trct-trcing dt indicte tht the rhinl cortices receive multimodl sensory inputs from the neocortex nd tht they constitute the interfce etween the neocortex nd the hippocmpus 4. Despite the fct tht the perirhinl cortex forms strong reciprocl connections with the temporl neocortex nd the entorhinl cortex, perirhinl trnsmission of neocorticl nd entorhinl inputs occurs with low proility 43. For instnce, electricl stimultion of the lterl olfctory trct in the whole rin in vitro evokes lrge field potentils in the entorhinl cortex ut no responses in re 36 (ref. 44). Similrly, stimultion of the temporl neocortex or re 36 evokes no locl field responses in the entorhinl cortex 44,ndrecentin vivo studies reched the sme conclusions 3,3. However, the discrepncy etween ntomicl nd physiologicl dt out this network is only pprent ecuse recent ultrstructurl oservtions suggest tht n importnt proportion of the connectivity etween the perirhinl nd entorhinl cortices involves excittory inputs to GABAergic neurons s well s long-rnge GABAergic projections to principl neurons 45. As result, perirhinl trnsfer of neocorticl nd entorhinl impulses is sujected to strong inhiitory pressures 43. BLA-relted fcilittion of rhinl interctions Consistent with the ove, cross-correlting the ctivity of perirhinl nd entorhinl neurons generlly yielded little evidence of correlted ctivity in the present study. However, restricting the nlyses to rhinl spikes tht occurred in close temporl proximity to BLA ctivity reveled tht uried in the cross-correltions re periods of enhnced rhinl interctions tht prevlently occur when BLA cells re ctive. Thus it seems tht the strong inhiitory pressures regulting perirhinlentorhinl interctions 43 cn e countercted y BLA inputs. This contention is supported y phrmco-ehviorl studies indicting tht BLA ctivity is required for the fcilittion of memory consolidtion produced y immedite post-trining mnipultions of entorhinl excitility 46,47. Notly, the BLA-relted fcilittion of rhinl interctions ws most pronounced in response to unexpected rewrds, oth outside nd within lerning context. Anlyzing the ehvior of BLA neurons reveled tht lthough incresed rhinl interctions were more likely to occur when BLA neurons incresed their firing rte, firing rtes lone could not ccount for ll cses of incresed rhinl interctions. In fct, sensory events tht cused n increse in the firing rte of BLA neurons were not necessrily ssocited with fcilitted rhinl interctions. Insted, it seemed tht this effect depended criticlly on more synchronized BLA output. This contention is sed on the fct tht in ll BLA-relted periods of fcilitted rhinl interctions, crosscorrelting the ctivity of simultneously recorded BLA neurons reveled tht they fired in more synchronized mnner, even fter controlling for chnges in firing rte. Overll, these oservtions suggest tht in reltion to ehviorlly slient events, fferents to the medil temporl loe produce conditions tht fvor the emergence of higher nd more synchronized BLA output. In turn, vi the mssive glutmtergic BLA projections to the rhinl cortices 4, BLA ctivity produces depolriztion of trget neurons, fcilitting communiction in the rhinl cortices nd thus enhncing the processing of sensory cues. Although it is likely tht other rhinl nd BLA fferents prticipte in this effect, the precise locking of correlted rhinl ctivity to BLA spikes oserved in the STJHs suggest tht BLA inputs to the rhinl cortices re key contriutors. In fct, the higher nd more synchronized BLA outputs to the rhinl cortices proly enhnce the likelihood tht these other fferents will trigger orthodromic spikes in rhinl neurons. The presenttion of unexpected rewrds cused short ( s long) BLA-medited fcilittion of rhinl interctions, nd our conditionl proility nlyses reveled tht ech BLA spike hd rief effect on rhinl interctions (5 ms). However, this is only the effect of one spike. When BLA firing rtes increse for long periods of time, s fter highly rousing events, the BLA-driven fcilittion of rhinl interctions should lso hve long durtion, s ech BLA spike will produce fcilittion of rhinl interctions. Consistent with this, stimuli cusing more intense emotionl response (unexpected electric shock to the pws) produces long-lsting increse (round h) in the firing rte of BLA cells coupled to more synchronized BLA output 8. In this context, it is importnt to relize tht ecuse BLA neurons re glutmtergic, once stress hormones hve recruited them (either directly in the cse of glucocorticoids or indirectly vi nordrenline in the cse of drenline), they depend on short-lived glutmtergic effects for the fcilittion of memory. Although ech spike hs rief impct, the influence of long-lsting increses in firing rtes, s seen fter intense emotionl rousl, will lso hve prolonged time course. By enhncing the processing of sensory cues, the BLA-relted enhncement of rhinl interctions proly prticiptes in the fcilittion of memory formtion y emotions. This contention is supported y the close temporl prllel etween the BLA effect nd lerning in the trce-conditioning tsk. At erly stges of lerning, when the rewrd ws still unexpected, the BLA-relted fcilittion of rhinl trnsmission incresed in connection with the rewrd. However, this effect grdully decresed s lerning progressed nd the rewrd cme to e predicted y the CS. At these lte stges, the BLA-relted fcilittion occurred erlier, when the cts were nticipting the rewrd. Notly, BLA ctivity did not fcilitte rhinl interctions rndomly ut in specific direction, from perirhinl to entorhinl neurons. In light of studies indicting tht the rhinl cortices form the interfce etween the neocortex nd hippocmpus 6,7,thissuggeststhtBLA ctivity preferentilly enhnces impulse trnsfer from neocorticl ssocition res towrd the hippocmpus, s would e expected for fcilittion of memory encoding. Finlly, our dt is lso consistent with the extensive literture linking the mygdl nd rewrd to multiple ssocitive processes. In prticulr, it ws shown tht mygdl ctivity enhnces the ssociility of unexpected cues nd is required for the cquisition nd representtion of reinforcement vlue 3,48,49. Our findings support nd extend these findings y reveling tht BLA ctivity might fcilitte the ssociility of rousing nd unexpected cues t the level of the rhinl cortices. Although this effect might depend on enhnced interctions etween the neocortex nd the hippocmpus, the evidence showing tht the rhinl cortices cn store ssocitive representtions in the sence of the hippocmpus 5 rises the possiility tht the enhnced rewrd-relted BLA ctivity might fcilitte storge in the rhinl cortices themselves. A chllenge for future experiments will e to test these two possiilities. METHODS BLA, perirhinl nd entorhinl neurons were recorded simultneously y mens of microelectrode rry in nesthetized (n ¼ 4) or unnesthetized (n ¼ 6) cts ( kg ody weight). Procedures were pproved y the Institutionl Animl Cre nd Use Committee of Rutgers Stte University, in complince with the Guide for the Cre nd Use of Lortory Animls (Deprtment of Helth nd Humn Services). Acute surgeries. In cute experiments, cts were prenesthetized with mixture of ketmine (5 mg per kg ody weight) nd xylzine ( mg per kg ody weight) intrmusculrly (i.m.), nd rtificilly ventilted with mixture of mient ir, oxygen nd isoflurne. Atropine (.5 mg per kg ody weight, i.m.) 36 VOLUME 9 [ NUMBER [ OCTOBER 6 NATURE NEUROSCIENCE

7 6 Nture Pulishing Group ws dministered to prevent secretions. The end-tidl CO concentrtion ws mintined t 3.7 ±.% nd the ody temperture t C (using heting pd). The one overlying the mygdl nd rhinl cortices ws removed nd the dur mter opened. Then n rry of tungsten electrodes (FHC) ws stereotxiclly lowered into the rin until the electrodes reched the deep lyers of the rhinl cortices. This rry ws constructed y drilling smll holes in Teflon lock nd inserting the electrodes into the lock. The Teflon lock ws then inserted into tightly fitting Delrin sleeve, which ws cemented to the skull. During the recording sessions, the electrodes could e lowered s group y mens of micrometric screw. The lengths of the electrodes were djusted so tht unit recordings could e otined simultneously from the BLA nd the perirhinl nd entorhinl cortices. Survivl surgeries. Survivl surgeries were performed s ove with the following exceptions. First, the cts were dministered penicillin (, UI per kg ody weight, i.m.) nd n nlgesic (Ketophen, mg per kg, sucutneously, dily for 3 d). In ddition, electrodes were implnted in the suproritl cvity to monitor eye movements. The electro-oculogrphic mesurements were performed to distinguish wking from prdoxicl sleep, s oth sttes re chrcterized y desynchronized electroencephlogrm (EEG) ut rpid eye movements only occur in prdoxicl sleep. Finlly, four screws were cemented to the skull so tht the the ct s hed could e fixed lter without pin or pressure. Recording sessions egn 8 d fter the surgery. Recordings. During the cute nd survivl experiments, neuronl ctivity ws smpled t Z mm intervls. Ech time the electrodes were moved to new recording site, we llowed 3 min to elpse efore dt were cquired, to ensure mechnicl stility. The signls picked up y the electrodes (. Hz to khz) were oserved on n oscilloscope, digitized nd stored on hrd disk. Spike sorting ws performed offline using clustering lgorithm sed on principl component nlysis nd K-mens. Behvior. In survivl experiments, the cts were fed only during recording sessions. They were given liquid rewrds (Gerer s pureed y food, Sweet pottoes nd turkey ) either outside (n ¼ 3) or within (n ¼ 3) ehviorl trining context. In the former cse, rewrds ( ml per tril) were delivered t unexpected times (intervls of 3 9 s). In the ltter cse, the cts were trined on n ppetitive trce-conditioning protocol:.5-s-long visul CS ws followed y.5-s-long dely period, fter which the sme liquid rewrd ws dministered. These trils occurred t rndom intervls (3 9 s). We monitored ehvior y mens of switch tht detected when the ct s tongue contcted the receptcle where the food rewrd ws dministered. The visul CS ws glol chnge in the illumintion (from lck to white) of LCD screen plced ft in front of the cts. Detection of the visul CS did not necessitte tht ct mintin fixed gze t the center of the LCD screen for the following resons: (i) ecuse the monitor ws plced ft from the ct s hed, the screen encompssed most of its visul field; nd (ii) the difference in illumintion ws so pronounced tht even with eyes closed, the CS could e esily detected. However, it should e mentioned tht ecuse the cts were hungry, they were highly roused nd remined wke t ll times (s ssessed y EEG recordings) with their eyes opened. Previous experiments hve shown tht the hippocmpus, rhinl cortices nd BLA re involved in the cquisition of trce-conditioning tsks using lesions or drug injections 9. However, the exct prmeters of these studies vried. One study, implicting the hippocmpus in the cquisition of n ppetitive trceconditioning tsk 9, used wter rther thn liquid food s the unconditioned stimulus. A second study lso implicting the hippocmpus in this form of lerning used trce eye-link conditioning tsk with n versive unconditioned stimulus (ir puff to the eye). Both these studies used shorter durtion dely period (round.5 s) thn the present study (.5 s) nd n uditory rther thn visul CS. The study implicting the rhinl cortices in this form of lerning lso involved trce eye-link conditioning, n uditory CS nd shorter dely period (75 ms). Lst, the study implicting the BLA in this form of lerning involved n olfctory trce-conditioning tsk with n olfctory CS, longer dely period nd n versive unconditioned stimulus (sickness). Histology. At the end of the experiments, recording sites were mrked with electrolytic lesions (.5 ma, 5 s). The cts were then given n intrvenous (i.v.) overdose of sodium pentoritl (5 mg per kg ody weight) nd perfusedfixed. The rins were lter sectioned on virting microtome (t mm) nd stined with cresyl violet to verify the position of recording electrodes. Microelectrode trcks were reconstructed y comining micrometer redings with the histology. Computtion of STJHs. The STJH is n dpttion of the joint peristimulus time histogrm (JPSTH) method 33 : insted of externl stimuli, BLA spikes re used s temporl reference (time in Fig. ; see lso ref. 34) to study correlted perirhinl nd entorhinl firing. Thus, like JPSTHs, STJHs re trilsed. Only insted of stimuli, s in the stndrd JPSTHs, our STJHs were BLA spike sed. The method used to construct the STJHs is descried in the results (Fig. ). STJH counts re color-coded s in Figure. TherwSTJHswere then normlized to the men of the BLA-shuffled mtrix. For presenttion purposes only, the STJHs were smoothed using two-dimensionl gussin with vrince equl to 5 ms (when the ins re ms). Sttisticl significnce of the STJHs. Sttisticl significnce ws ssessed using unsmoothed dt y performing in-y-in comprisons with two control STJH mtrices. The first control tests tht peks in the STJH re indeed locked to BLA firing, nd the second tests tht they re not merely due to independent rhinl responses to the BLA. To otin the first control STJH, we rndomized the BLA spike trin nd recomputed the STJH; repeting this process 5 times nd verging the result produced the BLA-shuffled control STJH. The second control STJH ws otined y computing STJHs fter shuffling the BLA spike sed trin of one of the two rhinl cells with respect to the other, repeting this 5 times nd then verging to produce the second control STJH. Note tht this technique corresponds to the shift predictor 33. We then performed in-to-in comprisons of significnce etween the rw nd the two rndomized STJHs, using Poisson distriution with threshold P-vlue corrected for multiple comprisons (.5 divided y the numer of ins: 9). For in to e considered significnt, it hd to meet this criterion when compred to oth rndomly generted sets of vlues. Interprettion of the STJHs. The reson why the STJHs revel correltions tht re invisile in rw cross-correltions is tht cross-correltions etween perirhinl nd entorhinl neurons include ll the spikes these cells generte, wheres the STJHs consider only rhinl spikes tht occur in close temporl proximity to BLA spikes. Becuse BLA neurons fire t very low rtes compred to rhinl neurons, rw cross-correltions re dominted y BLA-independent interctions etween perirhinl nd entorhinl neurons. As the rw crosscorreltions show, BLA-independent rhinl interctions re typiclly uncorrelted (the cross-correltions re usully flt). However, when one considers only the few rhinl spikes tht occurred just fter BLA firing, evidence of correlted ctivity is oserved. Test for unimodlity of STJHs. Visul inspection of the STJHs indicted tht most of them hd single dominnt pek. This ws further quntified y clculting Hrtign s dip sttisticl test for unimodlity. Using this pproch, we found tht s mny s 83% of the STJHs were indeed unimodl. For the remining, we fitted mixture of gussins to the dt (using stndrd expecttion mximiztion (EM) lgorithm) nd found tht in most of them (73%), one weight ws more thn twice the other, indicting tht this gussin ws much more dominnt. Clcultion of the rtio of significnt STJHs. For ech ct, we computed the rtio of significnt STJHs in nonoverlpping 5-ms-long time windows nd normlized the dt to the pre-cs period. We then verged the results otined in ll cts. Directionlity index. The DI ws otined y seprtely dding ll ins ove the min digonl () versus those elow the min digonl () in the STJHs nd then computing (( / + )) for ech STJH. The DI rnges from to ; positive vlues indicte tht the perirhinl neuron tended to fire efore the entorhinl cell, negtive vlues indicte the opposite. Assessing the significnce of cross-correlogrms. For cross-correlogrms of spontneous ctivity, we compred the centrl ins ( 5 to 5 ms) to the vlues of peripherl ins ( 5 to ms, nd to 5 ms). This computtion ws NATURE NEUROSCIENCE VOLUME 9 [ NUMBER [ OCTOBER 6 37

8 6 Nture Pulishing Group sed on Poisson distriution of peripherl in vlues (P o.5) nd we used Bonferroni correction for multiple comprisons. This pproch mounts to clculting the proility of oserving the centrl in vlues given Poisson process with men determined from the periphery of the histogrms. For event-sed cross-correltions, such s round the time of rewrd nd CS, the trils of one neuron were shuffled with respect to the other nd crosscorreltions were recomputed. This process ws repeted times. The center ins ( 5 ms to 5 ms) of the originl cross-correltion (clculted from the unshuffled trils) were then compred to the derived distriution (P o.5, corrected for multiple comprisons). Clcultion of conditionl proilities. To investigte the time course of BLA effects on rhinl interctions, we clculted direct proilities using the following equtions for ll ville spikes: p ¼ pðejða; PÞ t Þ¼ pðe; ða; PÞ t Þ pðða; PÞ t Þ ; where E, A nd P represent the occurrence of n entorhinl spike, BLA spike nd perirhinl spike, respectively. The two control conditionl proilities were clculted s nd p ¼ pðejð:a; PÞ t Þ¼ pðe; ð:a; PÞ t Þ pðð:a; PÞ t Þ p 3 ¼ pðejða; :PÞ t Þ¼ pðe; ða; :PÞ t Þ pðða; :PÞ t Þ : For comprison nd sttisticl significnce, we normlized ech to p(e), ecuse for long time delys (for exmple, those 4 ms), the event E is independent of the event (A,P) nd therefore p ¼ p ¼ p 3 ¼ p(e). The expected proility ws then clculted y the ddition rule p control ¼ p + p 3 p p 3 ; nd compred to p t ech time point using t-test. Note tht when computing conditionl proilities, we hd to choose from mny possile conditions (perirhinl efore entorhinl, entorhinl efore perirhinl, nd so on). The specific exmple depicted in Figure 7 is sed on previous nlysis (of directionlity index), where it ws found tht perirhinl cells tended to fire efore entorhinl neurons. Supplementry Figure 4 nlyzes other conditions ut shows the sme time course. We lso investigted the condition where entorhinl firing precedes perirhinl ctivity (dt not shown); in ccordnce with the directionlity index, the effect ws of much lower mgnitude nd shorter durtion (B ms). Note: Supplementry informtion is ville on the Nture Neuroscience wesite. ACKNOWLEDGMENTS Thnks re due to memers of the Pré l for comments on n erlier version of this pper. This work ws supported y RO grnts MH-736 nd MH from the US Ntionl Institutes of Helth to D.P. R.P. ws supported y Fulright postdoctorl fellowship. COMPETING INTERESTS STATEMENT The uthors declre tht they hve no competing finncil interests. Pulished online t Reprints nd permissions informtion is ville online t reprintsndpermissions/. Christinson, S.A. 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