Proc. Natl. Acad. Sci. USAVol. 93, pp. 9931-9936, September 1996Neurobiology

Enhancement of sensorimotor connections by conditioning-related stimulation in Aplysia depends upon postsynaptic Ca2+

(long-term potentiation/synaptic plasticity/classical conditioning/learning and memory)

GEOFFREY G. MURPHY* AND DAVID L. GLANZMANtt*Interdepartmental Graduate Program in Neuroscience, School of Medicine, University of California, Los Angeles, CA 90095; and tDepartment of PhysiologicalScience and Brain Research Institute, University of California, 2859 Slichter Hall, Los Angeles, CA 90095-1568

Communicated by Mark R. Rosenzweig, University of California, Berkeley, CA, June 17, 1996 (received for review April 11, 1996)

ABSTRACT Classical conditioning of Aplysia's siphon-withdrawal reflex is thought to be due to a presynapticmechanism-activity-dependent presynaptic facilitation ofsensorimotor connections. Recent experiments with sensori-motor synapses in dissociated cell culture, however, provide analternative cellular mechanism for classical conditioning-Hebbian long-term potentiation (LTP) of sensorimotor con-nections. Induction of Hebbian LTP of these connections ismediated by activation ofN-methyl-D-aspartate-related recep-tors and requires the postsynaptic elevation of intracellularCa2+. To determine whether the enhancement of sensorimotorsynapses during classical conditioning inAplysia-like LTP ofsensorimotor synapses in culture-also depends upon theelevation of postsynaptic Ca2 , we carried out experimentsinvolving the cellular analog of classical conditioning ofsiphon withdrawal. We examined changes in the strength ofmonosynaptic siphon sensorimotor connections in the abdom-inal ganglion of Aplysia following paired presentations ofsensory neuron activation and tail nerve shock This trainingregimen resulted in significant enhancement of the monosyn-aptic sensorimotor excitatory postsynaptic potential, as com-pared with the sensorimotor excitatory postsynaptic potentialin preparations that received only test stimulation. Infusingthe motor neuron with 1,2-bis(2-aminophenoxy)ethane-N,N-N',N'-tetraacetic acid, a specific chelator of intracellularCa2+, prior to paired stimulation training blocked this syn-aptic enhancement. Our results implicate a postsynaptic,possibly Hebbian, mechanism in classical conditioning inAplysia.

Classical conditioning of the siphon-withdrawal reflex in themarine molluskAplysia (1, 2) is thought to be mediated, in part,by changes in the strength of the monosynaptic connectionsbetween sensory and motor neurons in the abdominal gan-glion. According to a prominent model (3, 4), the pairing ofaction potentials in sensory neurons induced by the condi-tioned stimulus (CS)-weak tactile stimulation of the animal'ssiphon or mantle-with activation of facilitatory interneuronsinduced by the unconditioned stimulus (US)-electricalshocks delivered to the animal's tail-leads to increasedneurotransmitter release by the sensory neurons with subse-quent siphon stimulation. This enhancement of sensorimotorsynaptic transmission is associative because greater enhance-ment is produced by paired CS-US stimulation than byunpaired (CS-) stimulation, or by presentation of the US alone(3, 4). The cellular mechanism that underlies this associativesynaptic change-known as "activity-dependent (enhance-ment of) presynaptic facilitation" (3) or "activity-dependentneuromodulation" (4)-is thought to be due exclusively topresynaptic processes (3-6).

Recent experiments (7, 8) indicate that the sensorimotorsynapses of Aplysia in dissociated cell culture possess thecapacity for homosynaptic long-term potentiation (LTP). In-duction of LTP of Aplysia sensorimotor synapses in vitroresembles LTP of synapses in the CAl region of the mamma-lian hippocampus in its dependence upon: (i) strong postsyn-aptic depolarization (9-11), (ii) a significant rise in postsyn-aptic intracellular Ca2+ (12, 13), and (iii) activation of N-methyl-D-aspartate receptors (14) or N-methyl-D-aspartate-related receptors (15). Aplysia sensorimotor synapses in vitrofurther resemble CAl hippocampal synapses in their capacityfor Hebbian (16) LTP (9, 10). Thus, LTP of cultured Aplysiasynapses can be induced by pairing brief presynaptic stimula-tion, which by itself is insufficient to induce LTP, with strongpostsynaptic depolarization (8). This finding suggests thepossibility that classical conditioning of Aplysia's siphon with-drawal reflex (1, 2) is mediated, in part, by Hebbian enhance-ment of sensorimotor connections (17, 18), because tail shock(the US) produces strong depolarization of the siphon motorneurons (19). Thus, paired CS-US stimulation during classicalconditioning training would be expected to result in a patternof neuronal activity within the central nervous system ofAplysia similar to that used to induce LTP of sensorimotorsynapses in cell culture (8). Accordingly, we have begun to testthe potential role of Hebbian potentiation of sensorimotorsynapses in classical conditioning of Aplysia's siphon with-drawal reflex. Our experiments make use of the cellular analogof classical conditioning of the withdrawal reflex (3, 4) andemploy semi-intact preparations of the nervous system ofAplysia. We now report that the enhancement of sensorimotorsynapses that results from paired CS-US stimulation in thecellular analog of classical conditioning is blocked when thepostsynaptic motor neuron is infused with the Ca2+ chelator1,2-bis(2-aminophenoxy)ethane-N,N-N',N'-tetraacetic acid(BAPTA) (20).

METHODSAplysia california (100-200 g, obtained from local suppliers)were anesthetized by injection of MgCl2 and the centralnervous system (except for the buccal ganglia), together withthe two posterior pedal nerves (P9) that normally innervatethe animal's tail, were removed. After lightly fixing the neuralsheath by soaking the abdominal ganglion with glutaraldehyde[0.5% in 50%- MgCl2 and 50% normal artificial seawater(ASW)], each preparation was pinned to the Sylgard-linedbottom of a plastic recording chamber. The abdominal gan-glion was then partially desheathed in a solution of50% MgCl2and 50% normal ASW. After desheathing the abdominal

Abbreviations: BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N-N',N'-tetraacetic acid; CS, conditioned stimulus; US, unconditioned stimu-lus; ASW, artificial sea water; EPSP, excitatory postsynaptic potential;5-HT, serotonin; LTP, long-term potentiation.tTo whom reprint requests should be addressed. e-mail: dglanzman@physci.ucla.edu.

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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ganglion, the two tail nerves were drawn into suction elec-trodes, and the MgCl2-containing solution was washed outwith normal ASW. The preparation was perfused with normalASW for at least 30 min before each experiment. Intracellularrecordings were made from a single siphon sensory (LE)neuron (21) and a single small siphon (LFS) motor neuron(19). The neurons were impaled with micropipettes (20-30Mfl) filled with a solution of 2.0 M potassium acetate, 0.5 Mpotassium chloride, and 10 mM Hepes (pH 7.2). Recordingswere made using an Axoclamp 2A amplifier (Axon Instru-ments, Foster City, CA) and the data were stored on amodified VHS recorder (Model 400, A. R. Vetter, Rebersburg,PA). In each experiment a single LE neuron and a single LFSneuron were impaled. The motor neuron was hyperpolarizedto 50 mV negative to the resting potential in the soma toprevent it from firing spontaneously during the experiment;this was accomplished by passing negative current through thebridge circuit of the amplifier. The hyperpolarizing current wasmaintained throughout the experiment. Action potentials inLE neurons were elicited by 20-ms-long pulses of depolarizingcurrent. We used two basic types of perfusion medium in ourexperiments: (i) normal ASW (460 mM NaCl/11 mMCaCl2/10 mM KCl/55 mM MgCl2/10 mM Hepes buffer, pH7.6); and (ii) "2:1" ASW (368mM NaCl/13.8mM CaCl2/8mMKCl/101 mM MgCl2/20mM MgSO4/10 mM Hepes buffer, pH7.6). The 2:1 solution, which comprised 2.2x the normalconcentration of extracellular Mg2+ and 1.25x the normalconcentration of Ca2+, significantly raises spike thresholds,thereby reducing the contribution of interneurons to thesensorimotor excitatory postsynaptic potential (EPSP) (22).There were four experimental groups. Two of the experi-

mental groups received training with paired stimulation,whereas the other two groups received only the test stimuli. Inone of the groups that received the paired stimulation ("CS+"group) we did not attempt to buffer intracellular Ca2+ in thepostsynaptic motor neuron. In the second group that receivedpaired stimulation ("CS+/BAPTA" group) the motor neuronwas loaded with the Ca2+ chelator BAPTA (20) before thestart of the experiment. This was done by dissolving 200 mMBAPTA (tetrapotassium salt; Sigma) in the recording solutionof the intracellular micropipette, and allowing 30-60 min forthe BAPTA to diffuse from the micropipette into the motorneuron before the start of training. We believe this wassufficient time for the BAPTA to diffuse into dendritic regionsof the postsynaptic cell because in pilot experiments we foundthat similar loading with BAPTA of sensory neurons, whichare approximately the same size as the LFS motor neurons,blocks synaptic transmission within 15 min, as indicated by thedisappearance of the sensorimotor EPSP. To assess the effi-cacy of the paired stimulation, as well as to control for possiblenonspecific effects of BAPTA, we included preparations thatreceived only the test stimuli, both with and without BAPTAin the postsynaptic recording electrode ("Test Alone/BAPTA" aand "Test Alone" groups).The paired stimuli were presented using a forward-pairing

regimen, similar to that of Hawkins et al. (3), in which the onsetof the CS preceded the onset of US by 500 ms (Fig. 1). Therewere five bouts of paired CS-US stimulation in each condi-tioning experiment; these occurred at a rate of 1/5 min. TheCS consisted of firing 12 action potentials in the sensoryneuron (at a rate of 25 Hz), using a current 2x the spikethreshold level. The US consisted of 1 s of tail nerve shock(3-ms-long pulses at 25 Hz) delivered via suction electrodes;the extracellular current was 3 X the threshold level for evokingan EPSP in the motor neuron. To enable us to assess the statusof the sensorimotor synapse during training, we included seventest trials (T1-T7) at 1/5 min. Each test trial consisted of firinga single action potential in the sensory neuron and recordingthe resulting EPSP in the motor neuron. In the experimentsinvolving paired stimulation there was a test trial 1 min before

2:1

PRE 1 PRE 2 PRE 3 Ti T2 f T3 I

2:1

T4+ T5A T6J T7 PT15 PT 60

~~~ 20 MV

SN20 mV160m

Cs llllllll

us lrTFIG. 1. Experimental protocol for the cellular analog of classical

conditioning of siphon withdrawal. The first two pretests were carriedout in normal ASW. After the second pretest 2:1 ASW was perfusedinto the experimental chamber. The third pretest was performed in the2:1 ASW, which was then washed out. Training proceeded in normalASW. The training included seven test trials at 1/5 min. There werefive presentations of the paired CS-US stimuli (indicated by arrows).Following training 2:1 ASW was again perfused into the experimentalchamber for the two posttests. Note that some preparations receivedonly the test stimuli (Test Alone experiments; see Materials andMethods. This figure also presents sample recordings from a sensoryneuron and a motor neuron during a bout ofpaired CS-US stimulation(records are from a CS+/BAPTA experiment). The US producedstrong depolarization and firing of the motor neuron, as well as slowdepolarization of the sensory neuron. (The deflections on the sensoryneuron record following the CS are stimulus artifacts due to the US.)

each CS-US bout. [Paired stimulation commenced 1 min afterthe second test trail (T2).] In addition to the seven test trialsduring training, each experiment had three pretests (Prel-Pre3) and two posttests. The pretests were separated by 15 min;there were also 15 min between the last pretest and T1. Theposttests (PT15 and PT6o) were conducted at 15 and 60 minafter the last CS-US bout. One of the pretests (Pre3) and bothof the posttests were carried out in the presence of a modified(2:1) ASW that contained elevated levels of Mg2+ and Ca21(see above). The 2:1 ASW was perfused into the recordingchamber after the second pretest and washed out immediatelyafter the third pretest. The 2:1 ASW was then reintroducedafter T7, and remained in the recording chamber during bothPT15 and PT60. Note that all seven test trials, T1-T7, as well asthe paired stimulation, were performed in normal ASW. Theprotocol for the Test Alone experiments was identical to thatused for the CS+ experiments, with the exception that thepaired stimuli were omitted.The summary statistics are presented as mean ± SEM.

Statistical significance for multiple group comparisons wasassessed with either parametric or nonparametric ANOVAs.In the case of the nonparametric ANOVAs (Kruskal-Wallistests), subsequent between-group comparisons were madewith Dunn's tests, which correct for multiple comparisons.

Proc. Natl. Acad. Sci. USA 93 (1996)

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Planned comparisons involving only two experimental groupswere made with nonparametric Mann-Whitney U tests. Intra-group comparisons (e.g., between pre- and posttests) weremade with paired Student's t tests or nonparametric Wilcoxonsigned-rank tests. The choice of parametric or nonparametrictests was determined by the outcome of Bartlett's tests forhomogeneity of variances performed beforehand on the groupdata. All reported levels of statistical significance representtwo-tailed values.

RESULTS

Fig. 2 presents representative EPSPs for Pre3 (the pretestperformed in 2:1 ASW) and the 15-min posttest for eachexperimental group. The group data for the 15-min and 60-minposttests are summarized in Fig. 3. For the histograms in Fig.3 the amplitude of the EPSP on each of the posttests wasnormalized to the amplitude of the EPSP on Pre3. The pairedstimulation produced significant enhancement of the EPSP inthe CS+ group (n = 7), as indicated by comparing the meanEPSPs for the 15-min and 60-min posttests (182 + 32% and174 ± 29%) with the Pre3 EPSP (P < 0.05 for each compar-ison). By contrast, the mean EPSP of the Test Alone group(n = 6) exhibited significant homosynaptic depression (23) onboth posttests (60 ± 9% for the 15-min posttest and 72 + 3%for the 60-min posttest), as indicated by comparisons with thePre3 EPSP (P < 0.05 for each comparison). To determine theeffect of postsynaptic BAPTA we compared the posttest data

Pretest Posttest (15')

4mV

CS+

5mV

CS+/BAPTA

4mV

Test Alone

Test Alone/BAPTA

20 mV

40 ms

FIG. 2. Representative EPSPs from the third pretest and the15-min posttest for each of the four experimental groups. Note thatthese tests were carried out in 2:1 ASW.

-250.(

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- CS+EJ Test AloneE CS+/BAPTA!Z Test Alone/BAPTA

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Posttests

FIG. 3. Effect of postsynaptic BAPTA on the cellular analog ofclassical conditioning. The amplitude of the EPSP on each of theposttests was normalized to the amplitude of the EPSP on the thirdpretest (Pre3 in Fig. 1). The data are presented as means ± SEM. Thepaired CS-US stimulation produced significant enhancement of themonosynaptic sensorimotor EPSP on both the 15- and 60-min post-tests, as indicated by the data for the CS+ group. Infusing BAPTA intothe motor neuron before training blocked this synaptic enhancement,as indicated by the data for the CS+/BAPTA group. The data for theTest Alone and Test Alone/BAPTA groups indicates that postsyn-aptic BAPTA, by itself, did not disrupt normal synaptic transmissionnor homosynaptic depression.summarized in Fig. 3 for all four groups. Kruskal-Wallis testsindicated that the differences among the four group meanswere highly significant (P < 0.005 for the 15-min posttest andP < 0.002, for the 60-min posttest). Subsequent individualcomparisons indicated that the presence of BAPTA in themotor neuron blocked the conditioning-related enhancementof the sensorimotor EPSP. Thus, the mean CS+/BAPTAEPSP (n = 7) was significantly smaller than the CS+ EPSP onthe posttests (CS+/BAPTA EPSP = 69 ± 9% for the 15-minposttest and 66 ± 11% for the 60-min posttest; P < 0.05 foreach of the two comparisons). By contrast, the size of theCS+/BAPTA EPSP did not differ significantly from that ofthe Test Alone EPSP on either of the two posttests (P > 0.05for each comparison). The disruptive effect of postsynapticBAPTA cannot be attributed to nonspecific effects of the drugon the sensorimotor synapses, or to interference with presyn-aptic transmitter release, because (i) there were no significantdifferences among the mean raw EPSPs evoked on Pre3 in thefour experimental groups (CS+ EPSP = 6.3 ± 2.1 mV;CS+/BAPTA EPSP = 6.8 + 1.2 mV; Test Alone EPSP =

8.9 ± 1.6 mV; Test Alone/BAPTA EPSP = 6.5 ± 0.5 mV; P >0.4), and (ii) the mean amplitude of the Test Alone/BAPTAEPSP (n = 6) was not significantly different from that of theTest Alone EPSP on either posttest (Test Alone/BAPTAEPSP = 79 ± 10% on the 15-min posttest, and 69 ± 7% on the60-min posttest; P > 0.05 for each comparison).The effect of postsynaptic BAPTA on the conditioned

enhancement of the sensorimotor EPSPs was also apparentduring the training phase of the experiments. Fig. 4 presentsthe mean EPSPs for the seven test trials performed duringtraining. The data in this figure have been normalized to theamplitude of the EPSP on the second test trial (T2) for eachexperiment. Note that the test trials were carried out in normalASW; consequently, the test stimulus frequently evoked apolysynaptic EPSP. We did not attempt to determine themonosynaptic component of the EPSPs evoked on the testtrials during training, and will therefore refer to these EPSPsas "compound" to distinguish them from the monosynaptic

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FIG. 4. EPSPs evoked on the test trials during training. Theamplitude of the EPSP on each test trial was normalized to theamplitude of the EPSP on the second test trial. Data are presented as

mean ± SEM. These tests were carried out in normal ASW, and no

attempt was made to isolate the monosynaptic component of theEPSPs. The CS-US pairings in the CS+ experiments are indicated byarrows. The training produced clear enhancement of the EPSP in theCS+ group. The amplitudes of the EPSPs declined in the other threeexperimental groups.

EPSPs evoked in 2:1 ASW. As Fig. 4 shows, the pairedstimulation produced clear enhancement of the compoundsensorimotor EPSP in the CS+ group during training. Forstatistical comparisons we summed the compound EPSPsacross T3-T7 (the tests that occurred after the onset of pairedstimulation in the CS+ groups) for each group. A Kruskal-Wallis test performed on the differences among the groupmeans of the summed compound EPSPs indicated that thedifferences were highly significant (P < 0.0001). Individualcomparisons indicated that the mean CS+ compound EPSPwas significantly greater than that of each of the other threegroups (P < 0.001 for each comparison). There was nosignificant difference between the Test Alone compoundEPSPs and Test Alone/BAPTA compound EPSPs, or be-tween the Test Alone compound EPSPs and the CS+/BAPTAcompound EPSPs (P > 0.05 for each comparison). Thus,during training the paired stimulation appeared to have littleeffect on EPSPs in CS+/BAPTA preparations, EPSPs thatwere, at least in some cases, clearly compound. This factsuggests that postsynaptic BAPTA not only disrupted en-hancement of the monosynaptic connections between sensory

neurons and motor neurons, but also disrupted conditioning-related enhancement of synapses between interneurons andthe motor neurons (see refs. 19 and 22).The lack of significant differences between the results for

the Test Alone and Test Alone/BAPTA groups suggests thatthe BAPTA did not diffuse from the motor neuron into theterminals of the presynaptic sensory neurons (or interneu-rons). Furthermore, at the end of each experiment involvingBAPTA, we strongly depolarized and repeatedly fired themotor neuron while recording from the sensory neuron, andnever observed any evidence of retrograde electrical couplingbetween the sensory and motor neurons.

Infusion of BAPTA into the motor neurons blocked allenhancement of the sensorimotor EPSP in response to pairedstimulation (Figs. 3 and 4). This result was unexpected: we

originally anticipated that the paired stimulation would yieldsome residual enhancement of sensorimotor EPSP due toactivation of facilitatory interneurons (24-26) by the US. Wewere therefore concerned that our experimental conditionsmight somehow have interfered with activation of these inter-

neurons. Trudeau and Castellucci (22) found that the 2:1 ASWinterferes with excitatory interneuronal pathways within theabdominal ganglion for up to 30 min after reintroduction ofnormal ASW, as indicated by the amplitude of the compoundsensorimotor EPSP. In our experiments there was a 15-minperiod between the pretest carried out in 2:1 ASW (Pre3) andthe first test trial (Ti) (Fig. 1). During this period -10 volumesof normal ASW were perfused through the preparation cham-ber. Furthermore, there was another 6 min between T1 and thefirst bout of paired stimulation. To determine whether exci-tatory interneuronal input might, nonetheless, have remaineddepressed during training, we compared the unnormalizedamplitudes of the compound sensorimotor EPSPs on Pre2 (thetest that immediately preceded the introduction of the 2:1ASW) and T1. One-wayANOVAs performed on the data fromall the experimental groups indicated that the differences inthe amplitudes of the compound EPSPs among the four groupswere not statistically.significant for either Pre2 (P > 0.3) or T1(P > 0.2). We therefore combined the data from the fourgroups and compared the mean overall amplitude of thecompound EPSP on Pre2 to that on T1. The mean overallamplitude of the compound EPSP on T1 (27.0 ± 2.4 mV),which followed washout of the 2:1 ASW, was actually greaterthan the mean overall amplitude of the compound EPSP onPre2 (25.4 ± 2.5 mV), although this difference was notstatistically significant (P > 0.08, paired Student's t test). Wetherefore conclude that activation of excitatory interneuronswas not suppressed by the start of paired stimulation followingwashout of the 2:1 solution.

DISCUSSIONBased on data from experiments that, like the present ones,utilized the cellular analog of classical conditioning of siphonwithdrawal, Carew et al. (5) concluded that "impulse activityin the postsynaptic cell is neither necessary nor sufficient toproduce the... change in synaptic efficacy that underliesclassical conditioning inAplysia" (p. 1218), and that, therefore,a Hebbian mechanism does not play a role in this form oflearning. The actual generation of sodium spikes by thepostsynaptic cell does not, however, play an essential role in themodern reformulation of the concept of Hebbian synapticmodulation (9, 27). What is instead critical to this concept isthe requirement for coincident (or correlated) presynapticactivity and strong postsynaptic depolarization. According tothis contemporary reformulation of Hebb's (16) "neurophys-iological postulate," it is now apparent, from work on synapsesin cell culture and from the results presented here, thatHebbian modulation of sensorimotor synapses might well playa role in classical conditioning in Aplysia. With respect towhether postsynaptic depolarization is sufficient for enhance-ment of the sensorimotor synapses, Lin and Glanzman (8) haveshown that pairing presynaptic activity with postsynaptic de-polarization suffices to induce LTP of sensorimotor synapsesin the complete absence of any heterosynaptic modulatoryinput. With respect to whether postsynaptic changes are nec-essary for enhancement of sensorimotor synapses observedafter conditioning-related stimulation (3, 4, 28): (i) Lin andGlanzman (7) found that postsynaptic hyperpolarization canblock LTP of these synapses, and (ii) the present resultsdemonstrate that chelating postsynaptic Ca2+ can block thecellular analog of classical conditioning. These results there-fore provide further support for the hypothesis (8, 17, 18) thatHebbian LTP of sensorimotor synapses mediates, at leastpartly, classical conditioning of Aplysia's siphon withdrawalreflex. Previous work has implicated a presynaptic mechanism(activity-dependent presynaptic facilitation) in classical con-ditioning inAplysia (3, 4, 6, 29). This form of synaptic plasticityis thought to be due to firing of sensory neurons, activated bythe CS, paired with release of the transmitter serotonin (5-HT)

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from the terminals of facilitatory interneurons (see refs. 26, 30,and 31), activated by the US. Our data suggest that thispresynaptic mechanism is not the sole form of associativeplasticity responsible for classical conditioning in Aplysia; amechanism involving a postsynaptic rise in Ca2+ appears toplay a role as well.We did not include preparations that received specifically

unpaired presentations of the CS and US (a CS- group) in ourstudy. However, we believe that the paired stimulation resultedin associative, rather than nonassociative, enhancement of thesensorimotor EPSP. A comparison between our results andthose of Hawkins et al. (3) reinforces this belief. The amountof synaptic enhancement we observed in the CS+ group on the15-min posttest is similar to that reported by Hawkins et al. fortheir CS+ group on a comparable posttest. Thus, the meannormalized CS+ EPSP was 182 ± 32% in our experiments and161 ± 27% in the Hawkins et al. study. Moreover, the EPSPsin Hawkins et al.'s unpaired (CS-) group, unlike the EPSPs inour CS+ group, did not exhibit a significant increase inamplitude above their pretest values (see Fig. 3).Because we did not include either a CS- or US alone group

in the present study, we were unable to assess the extent towhich heterosynaptic modulatory pathways were engaged inour experiments. It is possible that under our experimentalconditions the paired stimulation selectively engaged a homo-synaptic associative mechanism and that facilitatory interneu-rons (24-26) were not activated or were minimally activated.The original study by Hawkins et al. (3) did not employ themodified (2:1) ASW we used to isolate the monosynapticcomponent of the sensorimotor EPSP. Although we saw noevidence of persistent suppression of interneuronal pathwaysafter washout of the 2:1 ASW (see Results), we cannot excludethe possibility that neuromodulatory neurons within the cen-tral nervous system of Aplysia are particularly susceptible toresidual effects of elevated divalent cations. We are currentlycarrying out experiments utilizing unpaired stimulation to testwhether the US activates heterosynaptic modulatory inputunder our experimental conditions.The present results indicate that the postsynaptic motor

neuron represents a crucial cellular site for convergence of theCS and US pathways during classical conditioning in Aplysia,at least insofar as changes in the sensorimotor connections areconcerned. Nevertheless, these results do not exclude a pre-synaptic site for convergence of CS and US pathways (3-6),nor do they exclude a role for 5-HT in classical conditioningof the withdrawal reflex. Indeed, the evidence is compellingthat the US tail shock activates serotonergic interneuronscapable of facilitating sensorimotor connections in the abdom-inal ganglion of Aplysia (26, 31). It is therefore attractive tothink that US-stimulated release of 5-HT by these interneu-rons plays an important role in classical conditioning, perhapsin addition to Hebbian plasticity (32, 33). An interestingpossibility is that 5-HT might interact with Hebbian plasticity,perhaps prolonging it (see below).

Based on the finding that paired training with sensoryneuron activation and tail (or tail nerve) shock (3), or trainingwith sensory neuron activation and application of 5-HT (29,34), yields an associative increase in the duration of thepresynaptic action potential, it has been argued that the locusof 5-HT's action during classical conditioning is presynaptic.However, the present results suggest the intriguing alternativepossibility that 5-HT may participate in a postsynaptic asso-ciative effect during classical conditioning, in addition towhatever presynaptic actions it might have. For example,US-stimulated interneuronal release of 5-HT might enhanceLTP of sensorimotor synapses (7, 8) during conditioning, orincrease the probability that LTP of these synapses will beinduced by conditioning-related stimuli. Such a modulatoryrole for 5-HT with respect to LTP of Aplysia synapses wouldbe analogous to that of norepinephrine with respect to LTP of

central vertebrate synapses (35-38). Additional support for theidea of a postsynaptic locus for 5-HT's role in classical condi-tioning is provided by data suggesting that 5-HT can directlymodulate membrane currents in LFS siphon motor neurons (19).Although our results indicate that at least some of the

cellular processes leading to the enhancement of sensorimotorsynaptic transmission during* classical conditioning of thesiphon-withdrawal reflex are initiated postsynaptically, it isunclear whether the locus of expression of this enhancementis pre- or postsynaptic. The synaptic enhancement may involvean increase in presynaptic release, an increase in postsynapticresponsiveness to presynaptic transmitter, or both. Eliot et al.(29) have recently found that the associative increase intransmission of sensorimotor synapses in cell culture due topaired presynaptic tetanus and 5-HT does not result in anincrease in the quantal size. This activity-dependent, pairing-specific enhancement of in vitro sensorimotor synapses there-fore appears to be mediated by increased probability oftransmitter release. Perhaps the previously reported associa-tive increase in spike broadening following various regimens ofpaired stimulation tracks some ongoing presynaptic processrelated to the expression of the associative synaptic change, forexample, persistent activation of a protein kinase (see ref. 39).

Because preparations in the CS+/BAPTA group receivedrepeated presentations of tail nerve shock, a regimen that hasbeen previously shown to induce significant heterosynapticfacilitation of Aplysia sensorimotor synapses (6, 40), we wereinitially surprised by the complete absence of enhancement ofthe CS+/BAPTA EPSPs, relative to the Test Alone EPSPs(Figs. 3 and 4). This result may indicate, as discussed above,that heterosynaptic pathways were not significantly activatedby the US under our experimental conditions. Alternatively,the delivery of the CS and US in presence of postsynapticBAPTA might have constituted an anti-Hebbian, or "pre-not-post," stimulus regimen, a regimen that has been proposed toinduce long-term depression (41, 42). This long-term depres-sion of the sensorimotor synapses might have been sufficientto override the heterosynaptic facilitation that would be ex-pected to result from repeated presentations of the US. Insupport of this idea, our laboratory has recently found thatAplysia sensorimotor synapses possess the capacity for long-term depression (43).

Previous studies have concluded that the cellular mechanismby which sensorimotor synapses are enhanced during classicalconditioning in Aplysia does not involve postsynaptic changesbut, rather, represents merely an elaboration of the presynapticmechanism of heterosynaptic facilitation (3-6). By contrast,the present study indicates that a postsynaptic rise in Ca2+plays an important role in conditioning-related modulation ofsensorimotor synapses. This finding, in turn, supports thehypothesis that N-methyl-D-aspartate-receptor-related poten-tiation is significantly involved in classical conditioning inAplysia (8, 17, 18).

We thank M. B. Barish, M. S. Fanselow, F. B. Krasne, and T. J.O'Dell for their helpful comments on the manuscript. This work wassupported by grants from the National Institutes of Health (NS29563),the National Science Foundation (IBN-9410579), the Alzheimer'sDisease Program, State of California/Department of Health Services(95-23335), and the Academic Senate, University of California, LosAngeles, to D.L.G., as well as by grants from the National Institute ofMental Health (F31-MH11136) and the Los Angeles Chapter of theAchievement Rewards for College Scientists Foundation to G.G.M.

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