Proc. Ntl. Acd. Sci. USA Vol.76, No. 12, pp. 6675-6679, December 1979 Neurobiology Clssicl conditioning in Aplysi clifornic (ssocitive lerning/locomotion). T. WALTRS*, T. J. CARW*t, AND. R. KANDL*t *Division of Neurobiology nd ehvior, Deprtments of Physiology nd Psychitry, College of Physicins nd Surgeons, Columbi University, New York, New York 132; nd tnew York Stte Psychitric Institute, New York, New York 132 Contributed by ric R. Kndel, September 24, 1979 ASTRACT A form of versive clssicl conditioning is described in which chemosensory conditioned stimulus rpidly cquires the bility to modulte defensive response (escpe locomotion). ecuse Aplysi show both sensitiztion nd clssicl conditioning, it is now possible to begin to exmine the reltionship between nonssocitive nd ssocitive lerning on behviorl nd cellulr levels. A centrl problem in the study of behvior is the nlysis of the mechnisms underlying the vrious forms of lerning. Wht re the cellulr mechnisms of ssocitive lerning nd how do they relte to those of nonssocitive lerning? Are different forms of lerning governed by seprte mechnisms or by vritions on common mechnism? Answers to these questions re beginning to emerge. They hve come primrily from the use of simple experimentl systems in which the nimls re cpble of vrious forms of lerning nd re ccessible to nlysis on the cellulr level (1-7). ecuse of the reltive simplicity of its nervous system nd the detiled knowledge vilble on the biophysicl, biochemicl, nd morphologicl properties of its neurons (8), the mrine snil Aplysi hs been useful for nlyzing the mechnisms of two forms of nonssocitive lerning: hbitution nd sensitiztion. ch of these forms hs been shown to be due to chnge in strength t specific set of synptic connections (2, 9), nd in ech cse the chnge in synptic strength results from n ltertion in the clcium current controlling trnsmitter relese t the presynptic terminls (1, 11). A level of complexity beyond hbitution nd sensitiztion is ssocitive conditioning. A mjor limittion to the further study of the mechnisms of lerning in Aplysi hs been the filure to demonstrte ssocitive lerning in this niml (12-14). We here describe powerful form of clssicl conditioning in Aplysi. The prdigm we hve used ws bsed upon clssicl defensive or versive conditioning (15). In this prdigm one stimulus, the conditioned stimulus (CS), is repetedly pired with n versive unconditioned stimulus (US). This trining commonly gives rise to set of conditioned responses, which cn be studied in two different wys. First, overt motor responses.to the CS sometimes develop nd they cn be exmined directly (5, 15). A second pproch-which we hve used here-is to exmine the bility of the conditioned stimulus to modulte other (test) behviors not involved in the originl conditioning procedures (16, 17). The test behvior we hve used is escpe locomotion. Despite its technicl dvntges nd theoreticl interest, this second pproch, commonly used in vertebrtes, hs not been directly explored in invertebrtes. This pproch is useful for two resons. (i) xmintion of conditioned modultory effects llows investigtion of dditionl dimensions of lerning not previously explored in invertebrtes, dimensions tht my illustrte new prllels between more complex spects of lerning in vertebrtes nd invertebrtes. Such prllels could encourge the use of simple systems not only for mechnistic nlysis but lso for the behviorl study of fundmentl psychologicl issues, such s the distinction between lerning nd performnce (18). (ii) This protocol permits the use of test system tht survives restrint of the niml nd surgicl exposure of its centrl nervous system, prerequisite for cellulr nlysis. Our behviorl index of versive clssicl conditioning ws the modultion by the CS of escpe locomotion (Fig. 1). This response offers severl dvntges for the study of lerning in Aplysw on both behviorl nd cellulr levels: (i) Locomotion is esy to quntify becuse the individul steps re discrete nd redily identifible. (ii). Locomotion displys short-term nd long-term sensitiztion (ref. 19 nd unpublished dt). (iii) The centrl progrm for locomotion is locted within the circumesophgel gngli nd cn be mesured relibly in the ptterned ctivity of both peripherl nerves nd identified motor neurons in simplified preprtions (2-22). RSULTS Conditioning Procedures. The experimentl procedures we used re summrized in Fig. 2. All nimls underwent the pretest on dy 1 to ssess bseline locomotor responsiveness before trining. The test stimulus ws trin of wek pulsed electric shock pplied cross the til with spnning Ag/AgCl electrodes. The ltency of the first step nd the number of steps tken within 5-min period fter the shock were recorded by using criteri described in the legend of Fig. 1. Animls were then mtched on the bsis of their pretest scores nd ssigned to one of three trining groups. The "untrined" group received no further trektment during the 2-dy trining period. The "unpired" group received trining with the shrimp CS nd the hed shock -US explicitly unpired. The "pired" group ws trined with specific temporl piring of the CS nd US (see Fig. 2). Three trining trils per dy (intertril intervl, 3 hr) were given for 2 dys. The trining procedure in ech tril ws similr to tht of Mpitsos nd Collins (5). Animls in the pired group received the CS pplied over the nterior hed region; 6 sec fter onset of the CS the US ws pplied vi spnning Ag/AgCl electrodes cross the front of the hed. The unpired group ws trined with the sme CS nd US s the pired nimls, but received them specificlly unpired; on ech tril the CS ws delivered 9 min fter the US. ighteen hours fter the lst trining tril ll nimls were tested, using blind procedure (by n observer who did not know the nimls' individul trining histories). In the test The publiction costs of this rticle were defryed in prt by pge session ech niml received the CS for 6 sec nd then, with chrge pyment. This rticle must therefore be hereby mrked "dvertisement" in ccordnce with 18 U. S. C. 1734 solely to indicte Abbrevitions: CS, conditioned stimulus; US, unconditioned stimulus. this fct. 6675
6676 Neurobiology: Wlters et l. A Pretest (Dy 1) Test JL-period---4 5 min Trining (Dys 2 nd 3) 3 hr Pired _FL L -U- Proc. Ntl. Acd. Sci. USA 76 (1979) 1.5 hr 1.5 hr AW 3JL Til shock 15sec Hed shock (US) 3 sec -FJI-Shrimp (CS) 9 sec JL - -,-m JAL C Test (Dy 4) Test period -l D F 'i;_- r :- - 7. Ar- lw.*. FIG. 2. ehviorl protocol. Animls (15-35 g) were housed individully in perforted circulr pns (28-cm dimeter, 7.6-cm depth) tht were suspended in 75-liter tnk of erted rtificil se wter (15'C). The test stimulus ws 15-sec trin of 5-mA c pulses (1.5 sec ech),.33 Hz, to the til. The US ws hed shock, 3 sec of 4-mA c pulses (1.5 sec),.33 Hz. Lrge currents were necessry for effective stimultion becuse of the sewter shunt between the electrodes nd skin. The CS ws 1.5 ml of crude shrimp extrct. Immeditely prior to CS onset plstic bg ws plced round ech pn to seprte its contents from the wter in the tnk. CS offset ws ccomplished by siphoning ll the wter out of the pn nd then removing the bg. These procedures were identicl for pired nd unpired groups. presenttions or no trining t ll (Fig. 3). efore trining there were no mrked differences mong the three groups in the mount of locomotion elicited by til shock (the test stimulus). After trining, one-wy nlysis of vrince indicted there ws n overll significnt difference (F2, 45 = 27.4; P <.1) mong the groups in their escpe locomotor responses to the test stimulus in the presence of the shrimp CS. Pired comprisons showed tht the number of steps tken by the untrined group ws not significntly different from tht of the unpired group, but the nimls in the pired group wlked more thn those in either control group (P <.5 in ech cse; Fig. 3A). A direct FIG. 1. One complete step in the locomotor sequence of Aplysi. (1/4 life size.) (A nd ) levtion nd extension of the hed; (C nd D) rching of the midbody; ( nd F) retrction of the til. Steps were counted s the number of til retrctions becuse these re discrete nd esily observed. the CS still present, the sme test stimulus used in the pretest (wek til shock) ws delivered. The ltency nd number of steps tken within 5 min were monitored for ech niml during the test session. These trining procedures resulted in form of conditioning chrcterized by (i) temporl specificity, (ii) requirement of CS presence for the lerning to be expressed, nd (iii) rpid cquisition. ch of these chrcteristics will be described in turn. Temporl Specificity. A criticl feture of ssocitive lerning is temporl specificity. In both clssicl nd instrumentl lerning the subject Displys chnge in behvior due to specific temporl reltionships between events. A common interprettion (23) is tht the subject chnges its behvior s it lerns tht one event (the CS in clssicl conditioning nd the opernt response in instrv-mentl conditioning) comes to predict the occurrence of nother (the US or reinforcement). The first question we exmined ws whether trining with pttern of temporlly pired CS nd US presenttions produces different outcome thn trining with pttern of explicitly unpired 12 A 4.~~~~~~~~~~~~ 2 C 6~ ~ ~ ~ ~ - FIG. 3. (A ) Responses in the test session. ch score is the men (± SM) number of steps tken within 5 min of the test stimulus. Pired nimls (n = 18) wlked significntly more thn untrined nimls (n = 12) or unpired nimls (n = 18), P <.5 in ech cse. All pired comprisons were by t tests for independent groups; ll probbility vlues re two-tiled. () Differences between test nd pretest scores (from sme experiment s A). Pired nimls wlked significntly more in the test thn in the pretest (P <.5, t for correlted mens). The decrese in locomotion of the untrined group is not sttisticlly significnt, wheres the decrese of the unpired group is (P <1.25). Such decreses re often, but not invribly, observed in control groups.
18 A 16,, 14 1 O~-12- (41 1 Neurobiology: U1 6 r c 4 Wlters et l. -2 - i-4-4..4 W[ c -2-4 Untrined US lone CS lone yl.j FIG. 4. (A) Stimulus site control. Differences (men + SM) between test nd pretest scores. US delivery during trining ws with sewter-filled cpillry electrodes (.5-cm dimeter,.2-cm seprtion) to obtin repetble, restricted stimultion of the nterior hed. Pired nimls (n = 8) showed significntly greter locomotion (P <.5) thn unpired nimls (n = 8). () Differences between test nd pretest scores of nimls trined with CS nd US unpired (n = 8) or with US lone (n = 8). There ws no significnt difference between these groups. (C) Differences between test nd pretest scores of untrined nimls (n = 8) nd nimls trined with CS lone (n = 8). There ws lso no significnt difference between these groups. Proc. Ntl. Acd. Sci. USA 76 (1979) 6677, comprison of the difference in locomotion exhibited by the different groups before, nd fter trining is shown in Fig. 3. The dt presented in this wy clerly show tht the pired group is different not only in the mount of escpe locomotion exhibited in the test session but lso in the direction of the effect: oth control groups showed decrese in locomotion in the test session wheres the pired group showed n increse. No significnt differences were found in the men ltencies to the first step mong these groups in the test or pretest. Although the testing in this experiment ws conducted blind, the trining ws not. Thus the possibility existed tht triner bis could hve contributed to the differences observed. To control for this nd relted rtifcts in the trining procedure, we crried out severl control experiments. We first replicted the experiment but used blind trining procedure s well s blind testing procedure. Trining ws done by person- who knew neither the design nor the purpose of the study. As in the previous experiment, nimls in the pired group (n = 8) exhibited significntly more escpe locomotion during the test session (P <.5) thn did the unpired control group (n = 8). ven when the trining ws crried out blind, the difference in trining procedures for pired nd unpired groups my hve produced n unintentionl bis on the prt of the triner (for exmple, he my hve delivered stronger US to one group or nother by direct contct with the spnning electrodes). Thus, functionlly stronger or weker US delivered to one group might hve contributed to the differentil effect we observed. To test this possibility we exmined the two conceivble US intensity differences by giving unpired trining with (i) functionlly stronger US nd (ii) functionlly weker US thn tht previously used. The wek US ws shock with hlf the current (2 ma) normlly used. The strong US consisted of the sme current s norml (4 ma), but the electrodes, insted of being held ner the skin, were brought into direct contct with the skin. Seprte pilot experiments hd indicted tht such contct cuses considerbly more sensitiztion of vriety of responses thn does shock from noncontcting electrodes. After trining these two groups (n = 8 in ech group) were not significntly different from ech other (men steps ± SM for strong US group = 2.25 + 2.25, nd for wek US group = 3. + 2.27) nd ppered similr to the unpired groups receiving the stndrd US. Finlly, we investigted the possibility tht, even if US intensity differences did not ccount for the effect, differences in the site of US stimultion might contribute to the differences between pired nd unpired groups. We were specificlly concerned tht, when nimls in the pired group withdrew their heds in response to the CS (s most nimls do during trining), the mount of hed nd neck surfce they exposed to the US might be different, nd therefore the pired group might hve received qulittively different US thn the unpired group (which received the CS nd US 9 min prt). We controlled for this possibility by replicting the bsic experiment shown in Fig. 2 with the US delivered through sewter-filled glss cpillry electrodes, which repetedly provided precise contct to the middle of the nterior hed (centered between the orl tentcles nd the rhinophores) even when the niml's hed becme withdrwn. With this different method of VS delivery the pired group still showed significntly more escpe locomotion in the presence of the CS thn the unpired group (P <.5; Fig. 4A). These experiments demonstrte tht trining with the CS nd US specificlly pired endows the CS with properties not observed with unpired presenttion of the sme stimuli. We lso found tht trining with either the US lone (Fig. 4) or with the CS lone (Fig. 4C) filed to produce the con- lo1- el 2 (A -6-4 "-6 I 6 A4 t MI Pired _8 Test Test without CS with CS -1 FIG. 5. Differences between test nd pretest scores in the bsence nd (3 hr lter) in the presence of the CS. Pired nimls (n = 8) wlked significntly more thn unpired nimls (n = 8) in the presence of the CS (P <.25). The pired nimls lso wlked significntly more thn they hd in the bsence of the CS (P <.1, t for correlted mens).
6678 Neurobiology: Wlters et l. 8-6- 14-12- 1- Q4-8 2-6- 4-2- A A - - I I I I I IU-- I 2 4 6 9 Trils FIG. 6. Acquisition. Different groups were given from zero to nine trining trils nd then tested 18 hr fter the lst tril. ch point is the men (+ SM) number of steps tken in the test session. Three conditions were exmined: pired trining (A, ), unpired trining (, ), nd no trining (e& in A nd, ech representing the sme dt). Numbers of nimls per group were: untrined, n = 27; pired nd unpired (respectively), one tril, n = 16, n = 16; two trils n = 24, n = 24; three trils, n = 24, n = 24; four trils, n = 16, n = 16; five trils, n = 16, n = 15; six trils, n = 26, n = 26; nine trils, n = 16, n = 16. The horizontl broken lines indicte bseline (untrined) performnce. Significnt differences between pired nd unpired scores were found on trils 5, 6, nd 9 (indicted by rrows, P <.25, P <.5, nd P <.5, respectively). lt Proc. Ntl. Acd. Sci. USA 76 (1979) ditioned fcilittory effect of the CS on escpe. These findings further support the conclusion tht the bility of the CS to enhnce escpe locomotion is dependent upon the specific temporl reltionship between CS nd US during conditioning. Requirement of CS Presence. Associtive lerning is usully chrcterized by mrked stimulus specificity (24). The presence of the CS is necessry for expression of the conditioned response: it is not elicited by other stimuli, except by those tht re quite similr. We hve begun to exmine these fetures by testing for the requirement of the CS during the test session. If pired trining hd merely produced generl increse in responsiveness, one could predict tht the test stimulus lone might elicit the conditioned effect in the bsenee of the CS. To test this possibility we first trined nimls with the stndrd pired nd unpired protocols (Fig. 2). These groups were then tested twice, first in the bsence of the CS, nd then, 3 hr lter, in the presence of the CS. After trining neither group exhibited much locomotion in response to til shock in the bsence of the CS (Fig. 5). However, in the presence of the CS, the pired group exhibited significntly more escpe locomotion in response to the test stimulus thn did the unpired group (P <.25), nd, in ddition, significntly more locomotion thn it hd shown in response to the sme test stimulus in the bsence of the CS (P <.1, t for correlted mens). In the bsence of the CS both groups showed decrese in locomotion reltive to the pretest scores. In the presence of the CS only the pired group showed n increse in locomotion reltive to the pretest. These results indicte tht the CS is required for the conditioned effect to be exhibited nd suggest tht the conditioning does not tke the form of nonspecific increse in responsiveness. Acquisition. We next exmined one spect of the cquisition of the lerned response by trining different groups of nimls with different numbers of trils. ch group ws tested the morning fter its lst trining tril. Thus nimls receiving one to three trils trils were tested on dy 3, while nimls receiving four to six trils, s well s nimls given zero trils (no trining), were tested on dy 4 (Fig. 2). Animls receiving nine trils were tested on dy 5. In ech cse pired nd unpired groups were run nd ech point on the composite cquisition curve (Fig. 6) consisted of t lest two experiments for ech group. The composite curve for the pired groups (Fig. 6A) ws sigmoid. In trils one through four there were no significnt differences between the groups. However, fter five trining trils the pired group exhibited significntly more locomotion in response to the test stimulus thn did the unpired group (P <.25), in prt due to n unexplined decrese in the control group. y trils six nd nine there were consistently lrge nd significnt differences between pired nd unpired groups (P <.5 in ech cse). Two spects of the cquisition curve (Fig. 6) deserve mention. First, lthough the dt were vrible, unpired groups tended to wlk more thn-pired groups fter receiving one to three trils. We re not sure how relible this smll trend is. Second, cquisition ppers to be quite rpid in the pired groups fter the fourth tril. Rpid cquisition is common chrcteristic of versive clssicl conditioning in vertebrtes (see below). DISCUSSION These experiments provide direct evidence tht Aplysi cn be clssiclly conditioned, forming powerful temporlly specific ssocition between chemicl CS nd noxious electricl US. The ssocition is rpidly cquired nd is dependent for its expression upon the presence of the CS. The fetures of temporl specificity, the requirement of the CS during testing, nd rpid cquisition re chrcteristic of versive clssicl conditioning in vertebrtes (18). Moreover, in Aplysi s in vertebrtes, versive conditioning hs the interesting feture tht conditioning often occurs without overt chnges in the externl behviorl response to the CS. Rther, conditioning leds to chnge in internl stte tht is mnifest in the cquired bility of the CS to modulte vriety of behviors (25, 26). For exmple, versive clssicl conditioning cn endow CS with the cpbility of suppressing ppetitively reinforced br pressing (16), enhncing instrumentl voidnce responding (27), fcilitting n unconditioned strtle response (17), nd ccelerting hert rte (28). These conditioned properties of the CS re specific to the temporl reltionships of the CS nd US during trining s well s to the prticulr CS used. The cquisition of conditioned versive responses s reveled by such tests is typiclly quite rpid in vertebrtes, often peking within 5-1 trining trils (17, 29). The similrity of the effects described for Aplysi to those of versive clssicl conditioning in vertebrtes encourges the serch for further similrities nd suggests tht mechnisms found in the study
Neurobiology: Wlters et l. of this form of ssocitive lerning in Aplysi my hve generl significnce. Wht is the lerned response? The ssocitive effect in our experiments is not simply the direct conditioning of locomotor response becuse no locomotion is elicited when the shrimp extrct is presented lone fter conditioning. Two other possibilities for the conditioned effect re: (i) conditioning of nother motor response (e.g., hed withdrwl or posturl response) tht itself fcilittes locomotor responses to til shock, nd (ii) conditioning of motivtionl or rousl stte (perhps nlogous to wht is clled "fer" in vertebrtes) tht fcilittes escpe responses. At present we cnnot distinguish between these possibilities. However, neither the withdrwls nor the postures of pired nd unpired nimls re noticebly different during the test session. Thus, in contrst to versive conditioning in Pleurobrnche, in which conditioning leds to the development of n overt withdrwl response to the CS (5), in Aplysi the conditioning ppers to tke the form of powerful chnge in internl stte tht becomes mnifest in the modultion of response not directly elicited by the CS. Tht specific motor response is not conditioned is consistent with theoreticl interprettions of versive clssicl conditioning s conditioned motivtionl stte rther thn conditioned motor response (18, 25, 26). Tht Aplysi disply well-defined ppetitive nd defensive motivtionl sttes (14, 3) further supports this possibility. The hypothesis tht Aplysi cn lern conditioned motivtionl sttes hs severl interesting implictions. One prediction is tht rnge of behviors will be ffected by the CS fter conditioning nd tht these effects will be motivtionlly consistent; defensive responses should be enhnced nd ppetitive responses suppressed by the CS (25). A second impliction is relevnt to hypotheses bout the reltionship between sensitiztion nd clssicl conditioning (8, 31, 32). ecuse sensitiztion is thought to be component of motivtionl sttes (3), the conditioning of motivtionl stte would suggest tht this form of ssocitive lerning is likely to involve, in prt, spects of sensitiztion. This notion is ttrctive becuse it suggests tht different forms of lerning my utilize vrious combintions of restricted set of fundmentl plstic mechnisms. Lerning in Aplysi persists fter the niml hs been restrined nd the hed gngli hve been surgiclly exposed. Moreover, we hve recently observed neurl correltes of versive conditioning in identified pedl motor neurons (unpublished dt). Three other forms of ssocitive lerning hve lso been demonstrted in relted gstropod mollusks: voidnce conditioning in Pleurobrnche (4, 5), bit-shyness in Limx (6), nd intersensory ssocitions in Hermissend (7). In ech of these nimls neuronl correltes of ssocitive lerning hve lso been reported (33-35). Thus, in ddition to its being possible to exmine the reltionship between nonssocitive nd ssocitive lerning within the sme species, it my be possible to exmine on the cellulr level the reltionships mong different forms of ssocitive lerning cross relted species. We thnk F. Adeshin nd J. Sliney for technicl ssistnce; R. Hwkins, I. Kupfermnn, V. Cstellucci, nd J. Koester for helpful comments; K. Hilton nd L. Ktz for rtwork; nd P. Kndel for photogrphic ssistnce. This work ws supported by Ntionl Institutes of Helth Fellowships to.t.w. (5T32MH1574), T.J.C. Proc. Ntl. Acd. Sci. USA 76 (1979) 6679 (5K2MH81), nd.r.k. (5K5MH18558) nd by grnts from the Ntionl Institutes of Helth (NS12744 nd GM2354) nd the McKnight Foundtion. 1. Spencer, W. A., Thompson, R. F. & Neilson, D. R. (1966) J. Neurophysiol. 29, 253-274. 2. Cstellucci, V., Pinsker, H., Kupfermnn, I. & Kndel,. R. (197) Science 167, 1745-1748. 3. 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