Two different lateral amygdala cell populations contribute to the initiation and storage of memory

Transcription

1 rticles Two different lterl mygdl cell popultions contriute to the initition nd storge of memory J. Christopher Rep, Jeff Muller, John Apergis, Theres M. Desrochers, Yu Zhou nd Joseph E. LeDoux W.M. Keck Foundtion Lortory of Neuroiology, Center for Neurl Science, New York University, 4 Wshington Plce, Room 809, New York, New York 10003, USA Correspondence should e ddressed to J.C.R. (jcrep@cns.nyu.edu) Single-cell ctivity ws recorded in the dorsl sunucleus of the lterl mygdl (LAd) of freely ehving rts during Pvlovin fer conditioning, to determine the reltionship etween neuronl ctivity nd ehviorl lerning. Neuronl responses elicited y the conditioned stimulus typiclly incresed efore ehviorl fer ws evident, supporting the hypothesis tht neurl chnges in LAd ccount for the conditioning of ehvior. Furthermore, two types of these rpidly modified cells were found. Some, locted in the dorsl tip of LAd, exhiited short-ltency responses (<20 ms) tht were only trnsiently chnged. A second clss of cells, most commonly found in ventrl regions of LAd, hd longer ltency responses, ut mintined enhnced responding throughout trining nd even through extinction. These ntomiclly distinct cells in LAd my e differentilly involved in the initition of lerning nd long-term memory storge. Pvlovin fer conditioning hs een used extensively to study how the rin lerns out stimuli ssocited with dnger. In fer conditioning, n initilly neutrl conditioned stimulus (CS), fter eing pired with n versive unconditioned stimulus (US), egins to evoke defensive fer responses. Evidence indictes tht the mygdl is centrl in fer conditioning 1 4, ut its precise contriutions hve een deted 5,6. Antomicl trcing, lesion nd electrophysiologicl recording studies suggest tht the lterl nucleus of the mygdl (LA), nd especilly the dorsl sunucleus (LAd), is the sensory gtewy to mygdl circuits 1, nd tht the processing of the CS y LA neurons is enhnced y the co-occurrence of the US Wheres these nd other findings 12,13 strongly suggest tht LAd might e n importnt site of plsticity, severl questions remin unnswered out the nture of the physiologicl chnges oserved in LAd during fer conditioning. First, no study to dte hs exmined in detil the reltionship etween the cquisition rtes of neuronl chnges in the LAd nd ehviorl lerning. Whether the neurl chnges in LAd ctully ccount for the conditioning of fer ehvior is therefore not known. In previous electrophysiologicl studies, the emphsis ws on the recording of unit ctivity rther thn on ehvior 7,9,11. This is prtly ecuse sujects exhiit ehviorl fer rections such s freezing fter the first US presenttion, mking it difficult to ccurtely ssess freezing conditioned to the CS. Here we ypssed this technicl prolem y using procedure in which fer conditioning ws superimposed on n opernt r-pressing tsk 14,15. Although rts freeze in the presence of the CS once it is ssocited with the US, they press r during the periods when the CS is not presented. As result, this tsk llows sensitive mesurement of CS-elicited ehviorl fer nd cn e used to ssess the rte of lerning of ehviorl fer responses in reltion to the cquisition of cellulr plsticity y mygdl neurons. A second unresolved issue is whether the mygdl is site of permnent storge of fer memories 5,6. Previous studies in rts found tht mygdl ctivity, fter initil increses erly in trining, resets towrd seline levels during lter trining 16. A similr finding ws otined in imging studies in humns undergoing fer conditioning 17,18. These dt hve een interpreted s evidence tht the mygdl is not site of storge 6. However, these studies hve typiclly not used concurrent mesurements of mygdl ctivity nd ehvior to evlute their reltionship. In ddition, reltively few cells were recorded in the previous rt study. Therefore, s the finding tht mygdl chnges re trnsient hs importnt implictions for the wy mygdl contriutions to fer lerning should e viewed, we re-evluted neurl ctivity in LAd using the procedures descried ove. RESULTS Behvior Fer conditioning ws ssessed using two mesures, freezing nd suppression of r-pressing. Although oth sensitively ssess fer conditioning 15, they reflect dissocile spects of ehviorl fer medited y different neurl systems 19. The use of the r-pressing procedure mkes it possile to ssess oth CS-elicited freezing nd suppression, s the motivtion to press the lever for food overcomes the tendency to remin immoile fter shock presenttion. The 13 rts receiving pired CS US trils exhiited incresed fer levels, s mesured y oth conditioned suppression nd freezing, during conditioning trils nd erly extinction trils (Fig. 1 nd ). In contrst, the nine rts receiving unpired CS nd US presenttions showed no evidence of CS-elicited fer t ny point. Unit ctivity A totl of 170 LAd cells from 22 rts were included in the nlyses. Of these, 100 cells were from 13 rts in the pired group, 724 nture neuroscience volume 4 no 7 july 2001

2 rticles nd 70 were from 9 rts in the unpired group (exmple of recording site, Fig. 2). Consistent with previous studies 7, the spontneous firing rtes of LAd neurons were low. The verge firing rte ws 2.7 Hz; however, 61% of the cells hd rtes less thn 1 Hz, nd the geometric men (± s.e.m.) ws 0.4 ± 1.2 Hz. The prepondernce of low rte nd wide spike-width cells suggests tht, like in the hippocmpus 20, mny of the cells in our smple were pyrmidltype projection cells s opposed to interneurons 21. Conditioning did not hve ny systemtic effect on spontneous firing rtes (p > 0.25, repeted-mesures nlysis of vrince), mesured t vrious time points throughout the experiment (see Methods). Seventy-three of the 100 cells in the conditioned group were clssified s CS-responsive. This ws determined y comining peri-event time histogrms (PETHs) for ll phses of the experiment (see Methods). The verge response ltency, defined s the first 10-ms in following stimulus onset tht hd significntly greter firing thn pre-cs levels, ws 42 ± 6 ms; this verge ws skewed y 7 cells with response ltencies tht exceeded 100 ms. Of the 66 cells with ltencies under 100 ms, the verge ws 28 ± 2 ms. Nineteen cells responded with ltencies under 20 ms. Conditioned responding CS-evoked ctivity n hour fter conditioning (during erly extinction trils) ws compred to pre-conditioning (hitution) levels to determine if trining-induced chnges in neuronl responsivity were evident n hour fter trining for the entire neuronl popultion. For ech cell, the CS-evoked ctivity from ms following CS-onset ws quntified s n verge Z- score (see Methods) during extinction nd hitution. The difference etween these two Z-scores provided mesure of the chnge in cell s responsivity, nd is depicted for ech of the 100 Fig. 1. Behviorl mesures of conditioned fer. () Men (± s.e.m.) suppression rtio for oth the pired nd unpired groups throughout the three phses of the experiment, hitution, conditioning nd extinction. Positive suppression rtios indicte r-press suppression during the CS, wheres suppression rtio of 0 indictes no chnge in press rtes during the CS. The suppression rtios re derived from the rw r-press rtes during the pre-cs period (60 s) nd during the CS (20 s), which re depicted on the right for the pired (top) nd unpired (ottom) groups (rtes re given in r-presses per second). () Men (± s.e.m.) CS-elicited freezing levels (freezing during the 20-s CS minus freezing during the previous 20 s) for oth the pired nd unpired groups throughout the three phses of the experiment. For () nd (), dt points re four tril locks verged together, except the first point, which is the verge of the finl 6 trils of hitution. LAd cells in the pired group in Fig. 3 (left). The popultion ws skewed towrd positive Z difference scores, indicting tendency for LAd cells to exhiit greter CS-elicited responses during erly extinction trils thn during hitution trils. In contrst, the Z difference scores in the unpired group (Fig. 3, right) were reltively flt, nd centered on difference of zero, suggesting little to no chnge from pre-trining levels. In support of this oservtion, the men Z difference score for the pired group ws 0.66 ± 0.15, significntly greter thn the unpired group s men of 0.05 ± 0.06 (p < 0.001, one-tiled t-test), which did not differ significntly from zero. To test for chnges in neuronl responsivity occurring during the conditioning phse of the experiment, the ove nlysis ws lso pplied to dt recorded during conditioning. For this nlysis, the 16 conditioning trils were divided into four 4-tril locks, nd the Z difference score ws clculted for ech lock reltive to hitution response levels. To cpture ny incresed responsivity tht my hve een trnsient in some cells, the mximum Z difference score of the four conditioning locks ws determined for ech cell (Fig. 4). This nlysis reveled tht the pired group (men ± s.e.m., 0.90 ± 0.11) hd significntly greter Z difference scores thn the unpired group (0.48 ± 0.10; p < 0.01). To further explore the cquisition of conditioned responding reltive to ehvior on cell-y-cell sis in the pired group, individul cells showing plsticity t vrious time points during trining were identified s follows: for ech cell, neuronl CS-elicited firing during conditioning trils ws compred, in 4-tril locks, to hitution levels (p < 0.05, t-test). Twenty-four cells from this pired group pssed this criterion for cell-y-cell plsticity during t lest one of the conditioning locks. With respect to CS-onset times, increses in firing were seen s erly s ms fter the onset of the stimulus (Fig. 5). The chnge Fig. 2. Photomicrogrph of thionin-stined rin section from representtive rt, showing electrode trct nd lesion site in LAd. LAd, dorsl sunucleus of the LA; LAvm, medil division of the ventrl LA; LAvl, lterl division of the ventrl LA; BL, solterl mygdl. nture neuroscience volume 4 no 7 july

3 rticles Fig. 3. Neuronl plsticity during erly extinction, for ll cells. The distriution of Z difference scores is depicted, showing the verge CS-elicited response from ms fter stimulus onset, expressed s Z score, during erly extinction minus hitution levels, for ech of the 100 LAd cells in the pired group nd 70 LAd cells in the unpired group. Vlues re orgnized long the sciss in rnk order, from the smllest to lrgest Z difference score. Ech r represents exctly one cell. Note the shift of the pired popultion towrd positive Z difference scores, indicting tendency for these LAd cells to exhiit greter CS-elicited responses during erly extinction trils thn during hitution trils. in neurl response pproched its mximum level during the first two locks of conditioning trils (Fig. 5). In contrst, conditioned fer ehvior ws more grdully cquired, reching mximl levels towrd the end of conditioning (Fig. 1). To etter determine whether the chnges in neurl response preceded chnges in ehviorl response, single-tril nlysis ws performed on the plstic cells. For ech of these cells, the first conditioning tril during which the CS response significntly exceeded hitution levels (p < 0.05, one-tiled) ws compred to the first tril on which ehviorl evidence of fer conditioning ws oserved. To compre neurl chnges to the erliest possile evidence of ehviorl lerning in our protocol, the first tril of ehviorl lerning ws defined s the first tril on which either significnt freezing or suppression occurred (p < 0.05, onetiled), whichever cme first. For significnt mjority of the cells (17 of 24; p < 0.05, inomil proility test), the chnges in the neurl response occurred erlier or on the sme tril s the chnges in the ehvior (Fig. 6 nd ). This oservtion enhnced LAd neurl ctivity preceding fer ehvior is lso evident in the group dt, s shown in Fig. 6c, where the verge CS response is plotted on tril-y-tril sis reltive to the tril on which ehvior ws lerned. Note tht the verged neurl response egn to increse efore fer ehvior ws evident (tht is, it incresed from trils 5 to 1), nd, in fct, peked t the tril t which fer ehvior ws first detected (tril 0). Even for the seven cells tht first showed evidence of incresed neurl responsivity on the sme tril s ehviorl lerning (Fig. 6 nd ), the incresed firing of these cells likely preceded the ehviorl response. Tht is, the men ltency of the incresed firing of these 7 cells ws within ms of the onset (the first uditory pip) of the CS on which ehviorl fer ws first evident. However, the shortest ltency ehviorl response elicited y CS itself is electromyogrphic (EMG) neck muscle ctivity, which is not expressed until ms following CS onset 22. In generl, ehviorl rection times in response to wrning stimulus, even for prcticed humn sujects, re of similr ltency 23. Therefore, neurl responses occurring ms fter CS onset very likely precede ehviorl conditioned fer responses such s freezing. Persistence of conditioned response The verged neurl response of plstic cells reched mximum levels during the first two 4-tril locks of conditioning, nd then tended to diminish during lter conditioning trils (Fig. 5). To determine how individul cells contriuted to this pttern, persistence vlue ws quntified for ech cell y dividing the increse in CS responses (over hitution levels) during lte conditioning (finl 8 trils) y the incresed CS response during erly conditioning (first 8 trils). Therefore, persistence vlue of zero indictes tht responses returned to seline (pre-conditioning) levels, wheres vlues of one or greter indicte tht responses remined elevted in lte conditioning. The distriution of persistence vlues suggests tht there re two types of cells (Fig. 7). One group hs persistence vlues clustered round 0, indicting tht their incresed responses returned to seline levels, wheres second group of cells hs vlues clustered ove 1.0, indicting tht their incresed responses were mintined throughout lter conditioning trils. Therefore, cutoff persistence level of 0.75, which isects this dul distriution, ws used to clssify these cells s either trnsiently plstic (12 cells) or long-lsting plstic (12 cells). As expected sed on their clssifiction, the response of trnsiently plstic cells fell ck to pre-trining levels during lter conditioning trils, wheres long-lsting plstic cells mintined their elevted firing levels throughout conditioning (Fig. 7). Both cell types (9 of 12 trnsiently plstic nd 8 of 12 long-lsting plstic cells) tended to exhiit conditioned chnges efore ehviorl fer ws evident, though the long-lsting plstic cells took longer to rech their mximl firing rtes (Fig. 7). These two clsses of cells could lso e distinguished sed on other chrcteristics. First, the long-lsting plstic cells hd smller CS response efore trining, during hitution (for exmple, Figs. 7, nd 8 nd ). In ddition, uditory response ltencies of 20 ms or less, which suggest direct ctivtion from thlmic efferents 24, were only seen in the trnsiently plstic group (Fig. 8 nd ). Furthermore, plsticity ws gretest t these short ltencies in the trnsiently plstic cells, wheres incresed firing in very long ltency ins (over 100 ms) ws much more common in the long-lsting plstic cells. Finlly, the trnsiently plstic cells were exclusively found ner the dorsl tip of LAd, wheres the long- Fig. 4. Neuronl plsticity during conditioning, for ll cells. The distriution of Z difference scores is depicted, showing the verge CS-elicited response from ms fter stimulus onset, expressed s the mximum Z-score of four conditioning 4-tril locks minus hitution levels (see text for detils), for ech of the 100 LAd cells in the pired group nd 70 LAd cells in the unpired group. Vlues re orgnized long the sciss in rnk order, from the smllest to lrgest Z difference score, nd ech r represents exctly one cell. 726 nture neuroscience volume 4 no 7 july 2001

4 rticles lsting plstic cells were found throughout LAd, nd were most prevlent in the ventrl hlf of LAd (Fig. 8c). Extinction of conditioned responding Both trnsiently plstic nd long-lsting plstic cells showed enhnced CS-evoked ctivity n hour fter the conditioning phse, tht is, during erly extinction trils (Fig. 7). The trnsiently plstic cells, fter returning to pre-trining response levels during lte conditioning, gin exhiited incresed responses during erly extinction trils (p < 0.05, one-tiled t-test), only to fll ck to seline levels lte in extinction. Long-lsting plstic cells, on the other hnd, mintin their incresed CS-evoked firing rtes not only in lte conditioning trils, ut lso in oth erly nd lte extinction trils (ll such time points re significntly elevted from hitution levels, p < 0.02). DISCUSSION We recorded from LAd neurons while simultneously monitoring ehvior during fer conditioning, primrily to ddress two previously unresolved issues regrding lerning-induced neurl ctivity. Specificlly, do the chnges in LAd neurl ctivity precede ehviorl chnges during lerning, nd do the neurl chnges persist? Antomicl considertions Recordings were mde in LA ecuse this region hs een implicted in fer conditioning using vriety of different experimentl pproches 1,4,5. However, LA is composed of severl sudivisions. We focused on LAd ecuse trct trcing nd physiologicl studies show tht this region is the primry trget of pthwys tht trnsmit the uditory CS to the mygdl nd is site of CS nd US convergence 28, nd ecuse previous studies hve shown shortltency conditioned chnges in neurl ctivity in this region 7,9,11. Distinct neurl responses were found in the dorsl versus the ventrl portions of LAd dorsl LAd cells were trnsiently plstic wheres plsticity in ventrl LAd cells persisted even through extinction. This ntomicl distinction is supported y the results of trct trcing studies 25,26 s well s studies tht hve mpped the locliztion of certin moleculr chnges in LA during fer conditioning 29. These findings pinpoint the locus of plsticity Fig. 5. Ltency nd time course of neuronl plsticity. () Men PETHs (spikes normlized to the pre-cs seline; 10-ms ins) for two phses of the experiment (hitution trils 1 8 nd verge of conditioning trils 1 8 nd 9 16) for ll conditioned cells from the pired group (n = 24 cells). () Time course of the verge CS response of plstic cells. Men (± s.e.m.) CS response for ll conditioned cells from the pired group (n = 24 cells) throughout the three phses of the experiment. Dt points re four tril locks verged together, except the first point, which is the verge of the finl 6 trils of hitution. during fer conditioning to reltively smll popultions of neurons tht my differentilly ccount for the initil lerning nd susequent mintennce of the long-term memory of the trining experience, s discussed in more detil elow. LAd neurl plsticity is ssocitive One hour fter conditioning, only cells from nimls receiving pired CS US presenttions showed evidence of enhnced responding to the CS. Cells from nimls receiving explicitly unpired CS US presenttions (which resulted in no oservle ehviorl fer response to the CS itself) exhiited little to no chnge in responsivity to the CS. Similrly, during the conditioning phse, neuronl CS-elicited firing in the pired group showed greter increses thn in the unpired group. Therefore, chnges in the pired group re likely due to the coding of the CS US ssocition rther thn to sensitiztion or other non-ssocitive effects cused y exposure to the US. LAd neurl chnges predict ehvior A previous study of LAd plsticity during fer conditioning found tht the chnges in neurl ctivity occur within the first three or four trining trils 16. However, tht study did not hve concurrent mesure of ehviorl lerning. Becuse fer conditioning cn e lerned in s little s one tril 30, the possiility remins tht ehviorl chnges my in fct hve preceded the chnges noted in LAd ctivity, which would suggest tht chnges in LAd processing re not criticl to the genertion of ehviorl fer responses. In the present study, three fctors llowed the comprison of neurl nd ehviorl conditioning rtes on tril-y-tril sis. First, we used reltively low shock intensity (0.4 ma) for the US to slow down the rte of ehviorl lerning. Second, greter sttisticl reliility of neuronl dt ws chieved y presenting 20 seprte uditory presenttions s single CS during ech tril 8. Third, the use of the food-motivted r-pressing tsk ensured tht the nimls would e ctive rther thn immoile in the intertril period, nd thus llowed sensitive mesurement of CS-elicited ehviorl fer responses on ech tril. With this pproch, we demonstrted tht the neurl chnges preceded the ehviorl chnges for most of the LAd cells tht showed evidence of plsticity in their CS-evoked ctivity during the conditioning phse. These results support the hypothesis tht incresed LAd neurl ctivity leds to the initition of ehviorl fer responses. One possile cvet is tht lerning my normlly tke plce erlier thn it ws oserved vi freezing or suppression, s these ehviors my hve een msked erly in trining y the conflicting drive to r-press for food rewrds. However, it cnnot e rgued tht the incresed neurl responsivity in the LAd is merely consequence of the ehviorl conditioned responses oserved here, s the neurl chnges re oserved efore the onset of ehviorl chnges. Furthermore, the strong tie etween LA neurl ctivity nd ehviorl fer conditioning is supported oth y the present dt (the neurl ctivity peks on the sme nture neuroscience volume 4 no 7 july

5 rticles Fig. 6. Acquisition of neuronl versus ehviorl mesures of lerning. () First significnt tril for conditioned responding of neuronl units (y-xis) plotted versus the first significnt tril for conditioned responding of fer ehvior (x-xis) s determined y r-press suppression nd freezing, whichever chnged first. Ech conditioned cell from the pired group is plotted once (n = 24 cells). The outlined 45-degree re indictes cells tht first showed neuronl conditioning on the sme tril on which ehviorl conditioning ws first detected. The circled region represents cells for which the neuronl chnges were detected on the sme or erlier trils thn ehviorl chnges. () Dt summrized from (), showing the difference for ech cell, in trils, etween the first significnt neuronl tril nd the first significnt ehviorl tril. Negtive numers indicte cells for which neuronl unit chnges were detected efore ehviorl chnges, wheres zero indictes cells for which unit nd ehviorl chnges were detected on the sme tril. Drk gry rs represent ll cells for which unit chnges were detected on the sme or erlier trils thn ehviorl chnges, s summrized in the right pnel of (). (c) Men CS-response for ll conditioned cells from the pired group (n = 24 cells) throughout hitution nd conditioning. Conditioning trils re ligned sed on the tril of ehviorl lerning (tril 0). tril s the onset of the ehvior), nd y mny previous studies tht implicte the LA s criticl for fer conditioning (discussed in more detil elow) 5. Thus, the most prsimonious interprettion of the present results is tht the incresed LAd ctivity contriutes to the genertion of the lerned fer ehvior. Persistence of LAd plsticity Hlf the LAd cells tht incresed their CS-evoked firing rtes during conditioning mintined those incresed response levels throughout the lter trils of conditioning. Becuse ehviorl fer levels, s mesured y oth suppression nd freezing, were lso elevted throughout the lter conditioning trils, these elevted LAd response levels my reflect spects of fer memory stored within the LAd. At first glnce, the current results re t odds with some previous studies of mygdl physiology in oth rts nd humns. In rts, cells tht incresed their responses erly during trining showed generl trend towrd diminished responses lte in trining 16. In humn fmri studies, mygdl ctivtion decresed with dditionl trining trils 17,18, though this decrese ws not significnt in one of the studies 18. A numer of differences etween these previous studies nd the present design my help explin the conflicting results. First, c the conclusions from the study in rts 16 were sed on smller smple of cells showing conditioned responding, which my hve hmpered detection of the long-lsting plstic cells. Second, the nlyses in tht study focused on only the first 50 ms of the CS-elicited responses. Becuse in the present study the shortest ltency responses were found in the trnsiently plstic cells, which were the min cells reported in the previous study 16, the nlytic or recording methods used in the previous study my hve ised the smple. In contrst, in the present study, the entire envelope of the CS response ws nlyzed, up to 250 ms fter the CS onset. With regrd to the filure of the humn studies to detect incresed mygdl ctivity lte in trining, one possiility is tht mygdl ctivity during lter trils my e restricted to the persistently responding cells tht store the trce. Becuse fewer cells re involved t the end thn t the eginning of trining, the lter ctivity might go undetected in fmri studies, especilly ecuse lerning-induced mygdl ctivtion is ner the detection threshold for this technique 17. A second possiility is tht fer levels of humn sujects my ctully decrese during lter conditioning phses, especilly in light of the low intensity Fig. 7. Persistence of neuronl plsticity during conditioning. () Persistence vlues (see text for detils) for ll conditioned cells from the pired group (n = 24 cells). Dshed line, cutoff used to distinguish trnsiently plstic cells from long-term plstic cells. () Men (± s.e.m.) CS-response for trnsiently plstic nd long-term plstic cells (n = 12 cells per group) throughout the three phses of the experiment. Dt points re four tril locks verged together, except for the first point, which is the verge of the finl six trils of hitution. Asterisks indicte tril locks during which the response is significntly greter thn hitution levels (p < 0.05). 728 nture neuroscience volume 4 no 7 july 2001

6 rticles c d shock used s US. Consistent with this possiility, when humns fer responses were mesured using skin conductnce chnges, they virtully disppered during lter fer conditioning trils 17. Fig. 8. Brekdown of trnsiently plstic versus long-lsting plstic cells, y ltency of plsticity nd ntomicl loction. (, ) Men PETHs (spikes normlized to the pre-cs seline; 10-ms ins) for two phses of the experiment (hitution trils 1 8 nd verge of conditioning trils 1 8 nd 9 16) for trnsiently plstic cells (; n = 12 cells) nd long-term plstic cells (; n = 12 cells). (c) Antomicl recording loctions for trnsiently plstic cells (gry circles) nd long-term plstic cells (white circles). (d) Digrm of our hypothesis of how LAd cells encode fer lerning. Arevitions re s in Fig. 2. d nd v, dorsl nd ventrl portions of LAd, respectively; CE, centrl nucleus of the mygdl. Two different cell popultions in LAd A numer of differences etween the trnsiently plstic nd longlsting plstic cells, in ddition to the differentil persistence levels of their enhnced uditory responding, further suggest tht they my represent two different popultions of LAd cells. The trnsiently plstic cells tended to hve shorter ltency nd more roust uditory responses efore trining. Furthermore, the trnsiently plstic cells tended to e locted more dorslly, ner the dorsl tip of the LAd, wheres the long-lsting plstic cells were most prevlent in the ventrl regions of this sunucleus. One possiility consistent with these chrcteristics is tht the trnsiently plstic cells receive more direct projections from the thlmus, wheres the long-lsting plstic cells insted re the trgets of intr-mygdl, nd/or cortico mygdl projections. This is suggested y the short ltency responses of the trnsiently plstic cells, s ltencies less thn 20 ms re possile only through direct thlmic projections 24, wheres the longer ltencies of the long-lsting plstic cells re consistent with dditionl synpses eing involved. Further support tht two cell types exist in LAd comes from recent experiments in our l. For instnce, one study of the iochemicl mechnisms of fer conditioning found tht cells exhiiting ctivtion of mitogen-ctivted protein kinses fter lerning re much more prevlent in ventrl rther thn dorsl LAd, nd tht such chnges re criticl for the consolidtion of fer conditioned memories tht lst six hours or longer 29. In ddition, trct-trcing studies revel tht the regions of LAd contining the two types of cells my receive slightly different fferent informtion from the uditory thlmus 25,26. As the functionl roles of the thlmic regions in question re poorly understood, the significnce of these projections, nd whether they contriute to the trnsient versus long-lsting properties found in LAd cells, remins to e determined. Given tht LAd hs pproximtely 20,000 excittory neurons (sed on unpulished stereologicl cell counts) nd tht the dorsl nd ventrl LA re roughly the sme size, plsticity my e triggered nd stored within two popultions of out 10,000 cells in LAd (Fig. 8d). If so, the prolem of identifying cell iologicl correltes of fer conditioning in the mmmlin rin would ecome trctle pursuit. The CS responses of the two cell types conform well to the chnges in CS processing predicted y clssicl lerning theory, the Perce Hll model 31. In this theory, ttentionl processes rise the ssociility of CS when the discrepncy etween the expected nd received US is high on recent trils. This descries the firing ehvior of the trnsiently plstic cells, which respond most to the CS during erly conditioning nd erly extinction trils. Conditioned responding in the model is controlled y the strength of the previous conditioning to the CS, which is firly well represented y the firing of the long-lsting cells. The finding tht these cells mintin n elevted responsivity lte into extinction my reflect the mintennce of memory trce of the trining experience, which is known to persist even fter ehviorl fer hs een extinguished 32. Locus of synptic plsticity The present dt leve open the possiility tht the mesured chnges in neuronl responsivity my e due to plsticity tht occurs outside the LAd, in fferent structures such s the thlmus 33,34 nd/or uditory cortex 35,36, oth of which contin cells with responses tht cn e modified y fer conditioning. However, certin spects of thlmic nd corticl plsticity re mygdl-dependent 37,38. Although further study will help etter resolve the contriutions of thlmic nd corticl plsticity to LAd ctivity, it is likely tht some if not most of the plsticity in LAd is due to locl integrtion of the CS nd US. For exmple, the LAd is site of mssive CS US convergence 28, more so thn fferent res in the thlmus 39. Studies of long-term potentition (LTP) indicte tht LA synpses re cple of plsticity 4,12,13, nd evidence suggests tht fer conditioning induces LTP in LA 8,10,29. Fer lerning is locked y temporry disruption of mygdl function during trining, even when the mygdl is intct during lter testing 30, Furthermore, fer lerning is impeded y locl disruption of puttive lerning mechnisms in the mygdl, including mcromoleculr synthesis, s well s the intrcellulr cscdes medited y mitogen-ctivted protein kinses nd cyclic AMP-dependent protein kinse 29,44,45. Collectively, these nd other findings strongly implicte LAd s site nture neuroscience volume 4 no 7 july

7 rticles of plsticity during fer conditioning 1,4,5. The present findings suggest it my lso e involved in long-term storge. Summry The present dt dd previously missing foundtion to the hypothesis tht fer conditioning my in prt e suserved y increses in LAd responsivity to the CS fter it is pired with the US. The results show tht LAd responsivity increses rpidly ut incrementlly during erly trining trils until presumly some threshold is reched, t which time ehviorl lerning is expressed. Furthermore, it seems tht the initition of plsticity nd storge of long-term memories my e differentilly encoded y incresed CS-responsivity of cells in the dorsl versus ventrl prts of the LAd. METHODS Animls nd r-press trining. Studies were done on mle Sprgue Dwley rts weighing g efore ehviorl trining. All procedures were in ccordnce with Pulic Helth Service guidelines nd were pproved y the niml use committee of New York University. Animls were kept on restricted diet to mintin them t 95% ody weight. They were then plced in n opernt conditioning ox ( cm, MED Assocites, St. Alns, Vermont) nd were trined to press r for food rewrds (45 mg; Noyes, Lncster, New Hmpshire) until minimum of 10 responses/min t 60-s vrileintervl (VI60) reinforcement schedule ws reched, which typiclly took out 1 week of trining. Surgery. Surgicl procedures were similr to those in previous studies 7. Once the r-press response ws lerned, sujects were pretreted with tropine (0.24 mg/kg, intrperitonelly) nd were nesthetized with sodium pentoritl (50 mg/kg, intrperitonelly). Supplementl doses of nesthetic were dministered throughout surgery s needed, nd ody temperture ws regulted y gel heting pd. Burr holes were drilled ove the mygdl 46, nd ove frontl cortex nd cereellum for insertion of self-tpping set screws to nchor the implnt to the crnium. An electrode ssemly 47 of 8 10 independently movle undles of wires ws stereotxiclly implnted so tht the electrode wires were positioned in the neostritum just dorsl to LA (2.5 mm dorsl to errzero). Ech individul wire undle ws shethed in protective stinless steel tue (33 G), nd consisted of 4 or more individul nichrome wires (25 micron dimeter), tetrode, or stereotrode 48 mde from nickel/chromium lloy wires (13 micron). All wires were insulted except for the cut tip (impednce < 1 MΩ t 1 khz). The overll configurtion of wires hd dimeter of 0.7 mm. At the end of surgery, the drive ws cemented in plce nd rts were llowed five dys to recover. Unit recording. After recovery, nimls were given dditionl r-pressing sessions to ensure performnce ws t lest t pre-surgery levels. During this time, the electrode ws connected to hed stge contining unitygin opertionl mplifiers. A cle then pssed the signl through hole in the top of the conditioning chmer to multichnnel differentil mplifiers (LYNX, Tucson, Arizon) vi slip-ring commuttor (Crist Instruments, Dmscus, Mrylnd) tht llowed the rt to move freely. Signls were mplified (10,000 gin), pssively filtered (600 6,000 Hz), digitized t 25 khz/chnnel, nd displyed on digitl oscilloscopes nd on computer monitor using Experimenter s Workench 32 softwre (DtWve Technologies, Longmont, Colordo). Spike wveforms corresponding to single cells were sorted off-line on the sis of wveform prmeters using cluster isoltion methods s descried previously for single wires 7, stereotrodes nd tetrodes 48, using the DtWve softwre. Only cells tht were well isolted throughout the experiment were nlyzed. Ech wire undle ws dvnced in 40-µm steps until discriminle single units were isolted t depths elieved to e in LAd. Units were tested for uditory responses using numer of experimenter-produced stimuli such s clps, tps nd vocliztions. The undles were lowered until multiple uditory-responsive cells were isolted, t which time conditioning egn. Conditioning. Conditioning took plce in n opernt conditioning ox similr to the one where the niml hd een trined to r-press. The opernt ox ws enclosed in lrger sound-ttenuting chmer. The conditioning protocol ws modified from previous studies in our l 7,8. The CS ws 20-s series of coustic white noise pips (50-ms durtion, 5-ms rise/fll, 80 ± 5 db, open field) delivered t 1 Hz, emitted from speker mounted ner the ceiling of the opernt ox. The use of multiple uditory presenttions s single CS llowed greter smpling of neuronl dt within ech tril 8. The US ws mild electric footshock (0.4 ma, 0.5 s) delivered through the grid floor of the test ox. A constnt ckground pseudo-white noise (55 db) produced y ventiltor fn ws present throughout the sessions. The experiment consisted of three phses. During hitution, the CS ws presented lone for 8 trils. These were immeditely followed y 16 conditioning trils in which the CS nd US co-terminted. The rt ws then plced in its home cge for 1 h, fter which it ws returned to the test ox for 20 extinction trils, during which the CS ws gin presented lone. Trils were seprted y vrile men intervl of 4 min (rnge, 3 5 min). The entire trining session lsted out 5 h. To test for effects of trining on spontneous neurl ctivity, 10 min of spontneous ctivity were recorded t four times during the experiment: efore hitution, immeditely following conditioning, just efore extinction nd following extinction. The spontneous ctivity ws recorded while the suject rested in the conditioning ox, with wll preventing ccess to the rpressing lever. In ddition to the pired group descried ove, seprte group (unpired) received explicitly unpired CS nd US presenttions during the conditioning phse of the experiment, to control for non-ssocitive influences of conditioning. Dt nlysis. Dt were nlyzed using comintion of NEX spike trin nlysis softwre (Plexon, Dlls, Texs), Mtl nd Excel, s descried elow. Br-press suppression 15 ws mesured using the suppression rtio (r pre r cs )/(r pre + r cs ), in which r pre nd r cs indicte the men press rtes during the 60 s efore the CS nd during the CS, respectively. This yields vlue of 1 for complete suppression, 0 for no suppression, nd negtive vlues down to 1 for fcilittion elicited y the CS. Freezing ws defined s cesstion of ll movement other thn respirtory ctivity or slight er twitches to the onset of uditory stimuli, nd ws mesured y lind oserver using stopwtch, from videotped recording of the experiment. Testing for chnges from hitution levels, for oth ehviorl nd neurl nlyses, used the finl six hitution trils s seline. PETHs of uditory responses were constructed for ech cell, using 10-ms ins. As ech CS consisted of 21 uditory pip presenttions 8, the lst of which overlpped with the US during conditioning, shock rtifct mde recording during the twenty-first pip of conditioning trils impossile. Therefore, nlyses of uditory responses were lwys sed on the first 20 stimuli per CS. To investigte the effects of trining on the entire popultion of LAd cells, PETHs for ech cell were summed over ll stimuli for the trils eing nlyzed, nd normlized to the 500-ms pre-stimulus seline using stndrd Z-score trnsformtion. Tht is, ech in of the normlized PETH expressed the numer of stndrd devitions ove or elow the men seline firing rte. The CS response of given cell in one phse of the experiment, expressed s n verge Z-score, ws then sutrcted from the cell s CS response during nother phse. The distriution of the resulting Z difference scores cross the popultion of cells yielded n index of plsticity for the region. In ddition, neurons were nlyzed on cell-y-cell sis to determine whether they showed sttisticl evidence of trining-induced enhnced responding. First, the envelope of the CS-evoked response ws determined for ech cell y finding the erliest nd ltest ins tht showed elevted CS-evoked ctivity. Specificlly, PETH ws constructed using ll trils from ll phses of the experiment. All PETH ins following stimulus onset tht exceeded the verge firing rte during the 500-ms prestimulus period y 1.65 stndrd devitions or more were included, until 2 consecutive ins filed to rech this criteri, or until 25 post-cs ins (250 ms) hd een considered. In ddition, t lest one of the ins ws required to e three stndrd devitions ove pre-stimulus levels, or the cell ws clssified s not CS-responsive. The resulting response time window, clculted seprtely for ech cell, ws then nlyzed further for increses in CS-elicited firing (the firing rte during the cell s response 730 nture neuroscience volume 4 no 7 july 2001

8 rticles time window minus the rte during the 500-ms pre-stimulus period) over the course of the experiment. Histology. At the end of the experiment, smll lesions were mde y pssing current (4 µa, 8 s) through recording wires from most of the wire undles from which cells were recorded. Animls were trnscrdilly perfused with uffered formlin. The rins were removed nd stored in formlin-sucrose solution, with 2% nitroferrocynide dded to visulize iron deposits left y the lesioned wires (Prussin lue rection). Frozen sections (40 µm thick) were cut on sliding microtome or cryostt, nd sections were stined for Nissl odies. Lesion sites were used to locte the regions of the recorded cells. The known configurtion of the electrode wire undles llowed the reconstruction of ll recording sites 47. ACKNOWLEDGEMENTS This reserch ws supported in prt y NIH grnts RO1 MH46516, KO2 MH00956, R37 MH38774 nd F31 MH The work ws lso supported y grnt from the W.M. Keck Foundtion to N.Y.U. 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L., O Keefe, J. & Brnes, C. A. The stereotrode: new technique for simultneous isoltion of severl single units in the centrl nervous system from multiple unit records. J. Neurosci. Methods 8, (1983). nture neuroscience volume 4 no 7 july