Proc. Nat. Acad. Sci. USAVol. 71, No. 12, pp. 5004-5008, December 1974

A Quantal Analysis of the Synaptic Depression Underlying Habituationof the Gill-Withdrawal Reflex in Aplysia

(synaptic plasticity/behavior)

VINCENT F. CASTELLUCCI AND ERIC R. KANDELDivision of Neurobiology and Behavior, College of Physicians and Surgeons, Columbia University, 630 West 168th Street, New York,N.Y. 10032; and Department of Physiology, New York University School of Medicine, 550 First Avenue, New York, N.Y. 10016

Contributed by Eric R. Kandel, October 10, 1974

ABSTRACT Habituation, one of the simplest behav-ioral paradigms for studying memory, has recently beenexamined on the cellular level in the gill-withdrawal reflexin the mollusc Aplysia and in the escape response in cray-fish. In both cases short-term habituation involved a de-crease in excitatory synaptic transmission at the synapsesbetween the sensory neurons and their central target cells.To analyze the mechanisms of the synaptic depression inAplysia, we applied a quantal analysis to synaptic trans-mission between the sensory and motor neurons of the gill-withdrawal reflex. Our results indicate that short-termhabituation results from a presynaptic mechanism: a de-crease in the number of transmitter quanta released perimpulse. The sensitivity of the postsynaptic receptor re-mains unaltered.

Habituation, the decrease in a behavioral response to repeatedpresentation of a novel stimulus, is the most ubiquitous be-havioral modification found in animals and man and one of thesimplest paradigms for probing memory mechanisms (1-5).Habituation in different response systems and in differentanimals probably cannot be explained by a single neuronalmechanism. But in animals having a well-differentiatedcentral nervous system, certain types of habituation sharesimilar parametric features and time course, making it likelythat in these cases a restricted family of mechanisms is in-volved. Thus certain reflex responses of vertebrates and higherinvertebrates undergo short-term habituation lasting minutesand hours following a single training session of TO to 15stimuli, but repeated training sessions can produce long-termhabituation that lasts weeks. The acquisition of long-termhabituation is sensitive to the pattern of stimulation. Spacedtraining is more effective in producing the long-term effectsthan is massed training. Finally, immediate restoration of ahabituated response (dishabituation) can be produced bystimulating another pathway (2, 6).Because habituation can be studied in higher invertebrates

and simple vertebrate systems, it is the only behavioralmodification that has been successfully studied on the cellularlevel. In the gill-withdrawal reflex in the mollusc Aplysia (7)and in the escape response in crayfish (8), short-termhabituation has been found to involve a similar cellular locusand a common synaptic change. There is a decrease in excita-tory synaptic transmission (homosynaptic depression) at thecentral synapses made by the sensory neurons on their centraltarget cells. Synaptic depression, although at a different locus,may also underlie habituation of the flexion reflex in verte-brates (9).

The mechanism producing the synaptic depression is notknown. It could be either a presynaptic change in transmitterrelease or a postsynaptic change in receptor responsiveness. Todistinguish between these alternatives a quantal analysis isrequired. With rare exception (10, 11) this has been difficult toachieve in central neurons because they receive many synap-tic inputs, making the analysis of spontaneous release (12)difficult, and because the evoked quantal size is usually small(10-100 MV) in relation to the background noise and to thesynaptic bombardment produced by neural activity.Using a high divalent cation solution to reduce neuronal

activity, electrical filtering to reduce noise, and computeraveraging for signal recognition, we have been able to apply aquantal analysis to synaptic transmission between the sensoryand motor neurons of the gill-withdrawal reflex in Aplysiaand to analyze the synaptic depression underlying habitua-tion. Our results indicate that short-term habituation resultsfrom a presynaptic decrease in the number of transmitterquanta released per impulse. The sensitivity of the post-synaptic receptor remains unaltered.

METHODS

Abdominal ganglia were isolated from Aplysia californicaweighing 80-200 g. Analyses were carried out at 20 23° on theexcitatory postsynaptic potentials produced by stimulatingintracellularly single sensory neurons (7, 13). Single electrodesconnected to a bridge circuit were used to stimulate and recordfrom sensory cells; low resistance (less than 5 MO) doubleelectrodes were used for the follower cells (usually gill motorcell L7, with some additional data on siphon motor cell LBS-3and interneuron L16; see ref. 14 for nomenclature). The signalsfrom the follower cells were fed to a cathode follower and thento a dc and a high gain ac amplifier (Tektronix no. 2A61). Tominimize baseline noise, the bandwidth of the ac amplifierused was set between 0.6 Hz and 60 Hz. The filtering reducedpostsynaptic potential amplitude by about 15%. Data weretape recorded (Hewlett-Packard no. 3960) and displayed on aBrush Recorder (no. 440). Normal solutions of artificial seawater contained 55 mM Mg++, 11 mM Ca++. When cationswere substituted, osmolarity was maintained by deletion ofNaCl. Solutions were buffered (pH 7.6) with Trise HCl.

RESULTS

Synaptic Depression Underlying Habituation of the Gill-Withdrawal Reflex. A weak or moderate intensity stimulusapplied to the siphon of Aplysia produces a gill-withdrawalreflex that is controlled by the abdominal ganglion [Fig. 1A;(15) ]. With 10 to 15 repeated stimuli at 10- to 60-sec intervals

5004

Abbreviations: EPSP, excitatory postsynaptic potential; I.S.I.,interstimulus interval; S.N., sensory neuron.

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Synaptic Depression and Habituation in Aplysia 5005

c

SN. g o1 2 5 10 15

REST 15 MIN

e- e - I2mV

!,A.- - X.I. A - -> I 2OnV1 2 5 10 15 #FeCVl.SJ.==10eC

FIG. 1. Synaptic depression at the synapse between mechano-receptor neurons and motor neurons. (A) Diagram of the abdom-inal ganglion of Aplysia showing schematic innervation of gill andsiphon and illustrating simultaneous recording from gill motorneuron (L7) and a mechanoreceptor sensory neuron. (B) Criteriafor monosynapticity of the synaptic connection between motorcell (L7) and sensory neuron (S.N.). Independence of synapticlatency on level of transmitter release. (B.) Comparison of synap-tic latency with normal and low release. Superimposed recordsfrom the same pair of neurons obtained first (normal release) innormal artificial sea water (55 mM Mg++, 11 mM Ca++); andthen (low release) in a solution that depresses release (165 mMMg++, 8 mM Ca++). (B2) Comparison of synaptic latency withcontrol and increased release. Superimposed records from a pairof neurons before (control) and 10 min after (high release) theamplitude and duration of the presynaptic spike of the sensoryneuron were increased by intracellular injection of tetraethyl-ammonium chloride (TEA, 3 M) through the recording electrode.(C) Synaptic depression of the monosynaptic EPSP produced inmotor cell L7 by stimulation of a single sensory neuron in high di-valent cation solution (138 mM Mg++, 62 mM Ca++). Inter-stimulus interval (I.S.I.) 10 sec. Two series of 15 stimuli eachwere presented with a rest of 15 min in between. The rest led topartial recovery, but a slight build-up of the depression is evidentin the second run.

the reflex habituates (16). The neural circuit of this reflexcontains six identified motor neurons, a cluster of 24 sensoryneurons, and three identified interneurons (13, 15). Studies ofthe cellular correlates of habituation have shown that tactilestimulation of the siphon skin initiates a complex excitatorypostsynaptic potential (EPSP), produced largely by directsynaptic connections from the sensory cells (7, 13), that dis-charges the motor cells and causes gill contraction. Habitua-tion produces a progressive decrease in the complex EPSP(17). Analyses of the various components of the reflex duringshort-term habituation (including measurements of the thresh-old and input conductance of the motor cell) indicate that thecritical change occurs at the synapses between the sensory celland the motor neurons and interneurons (refs. 7 and 17, andmanuscript in preparation). With repeated stimulation, theelementary EPSP produced by the sensory neurons decreasesin amplitude (Fig. 1C), paralleling the time course of be-havioral habituation (16).

Monosynapticity of the Connections between Sensory Cells andMotor Neurons. To analyze the mechanism of the depressionin this synaptic potential, it was essential to determine thatthe connections between sensory and motor cells are mono-synaptic. To assess monosynapticity we have used the follow-

ing criteria (7, 18-20). (1) One-for-one following. At normallevels of transmitter release every action potential in the sen-sory neuron produces an elementary EPSP in the follower cell.(2) Short and constant latency. At 20-23' the latency betweenthe peak of the presynaptic spike in the sensory neuron andthe onset of EPSP in the follower cell is 5-10 msec (Fig. 1B).The latency did not change in solutions containing high (2.6to 3.0 X normal) concentrations of divalent cations that in-crease the threshold of neurons and reduce the likelihood of aninterneuron's being fired (21, 22). (3) Independence of latencyon amount of transmitter released. The latency of the EPSPremained unaltered when release was enhanced (Fig. 1B) byincreasing the duration of presynaptic spike (intracellularinjection of tetraethylammonium into the sensory cell) andreduced (20-fold) by increasing the Mg++ content (165 mM)of the artificial sea water. These criteria are consistent with amonosynaptic connection.

Quantal Fluctuations under Conditions of Low TransmitterRelease. We carried out a quantal analysis to examine themechanism of the synaptic depression. This analysis is basedon the finding that the transmitter at chemical synapses isreleased in multimolecular packets (quanta) and that anysynaptic potential is made up of an integral number of thesequanta (12).With reduced transmitter output (increasing Mg++ to 165

mM and reducing Ca++ to 8 mM),a repetitive stimulation ofthe sensory neuron produced marked fluctuations in theamplitude of the EPSP, in an apparently quantal fashion, andoccasional failures (Fig. 2). Amplitude histograms of 30 to 100consecutive responses (Fig. 3A-D) revealed a multimodaldistribution, with the mean of each subsequent peak being anintegral multiple of the unit peak. To improve the signal-to-noise ratiob and insure that we correctly identified failures,we averaged all putative failures, using a PDP8-E computer,so as to determine whether a smaller occult unit EPSP hadgone unrecognized (Fig. 3EI). Averaging did not reveal asmaller, occult unit EPSPc; the averaged failures were

a This solution contains 2.6 X normal divalent cation concentra-tion and, therefore, also serves to increase the threshold of cells,dramatically reducing the spontaneous impulse activity in theganglion. The high Mg++ concentration does not seem to producea significant effect on the receptor. The kinetics of decrement areroughly similar in normal sea water and in high Mg++ solution.b We did not examine spontaneous miniature potentials because ofthe background noise, much of it probably produced by the spon-taneous transmitter release from presynaptic cells that synapse onfollower cells. The noise level dropped by 50-70 % on with-drawal of the electrode from the cell. Moreover, doubling the os-molarity of the solution with sucrose produced showers of smalldepolarizations and hyperpolarizations, suggesting a massive re-cruitment of miniature potentials. In three instances we madeamplitude histograms of the depolarizations by collecting the ex-cursion of the baseline that had a similar rise time to the evokedEPSPs. The peak of a unit size correlated with the unit peak ofthe evoked EPSPs in one case, but in the two other cases manypeaks were observed and the results were ambiguous.¢ In two experiments we also have used a blind procedure. Thehistograms of the original EPSPs were independently graphed bya naive observer. In both cases the q and failure values obtainedwere similar to ours. We also did amplitude histograms of 200samples of the background fluctuation taken at a fixed intervalafter a spike and several seconds after the EPSP was over. Thesehistograms contained no trends and no peaks corresponding tothe peaks of the evoked EPSPs.

Proc. Nat. Acad. Sci. USA 71 (1974)

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5006 Physiology: Castellucci and Kandel

1N/z 45 50 155; 160SN

~REGION HK

5 46 51 156 161

6 47 52 157 162

7 W 48 53 158 163

I~8Kll 49zL 54 159 164

5Cmsec

FIG. 2. Quantal fluctuations accompanying synaptic depres-sion at a low level of release (165 mM Mg++, 8 mM Ca++).Samples of successive EPSPs evoked in L7 by a series of 200 con-secutive intracellular stimuli to the sensory neuron at 10-secintervals. Total membrane potential shift during the series wasless than 1 mV. Samples are taken from consecutive responses inthree different plateau regions defined in the text. The first region(see examples 5 to 8) shows marked amplitude fluctuations and nofailures (see Fig. 3A for details). In regions 2 and 3 there is a pro-gressive increase in the number of failures (arrows; see also Fig.3A). Number beside each pair of traces refers to the stimulusnumber in the series. The amplitude histograms of the EPSPs(Fig. 3A to D) were obtained by measuring the magnitude of theresponse in time intervals (window) between the peak of the pre-synaptic spike and the peak of the-first EPSP (1).

indistinguishable from the averaged background fluctuations.We also averaged the unit EPSP and found that it had asimilar configuration to the average of the first few largeevoked EPSPs (Fig. 3E2), indicating that one can also success-fully recognize by eye the unit quantal signal. These findings,and the further documentation below, demonstrate the feasi-bility of a quantal analysis at this synapse.

Quantal Analysis of Depression at Low Release. When themean probability (p) of a quantum's being released is low andthe available mean population of quanta (n) is large, trans-mitter release can be approximated by a Poisson distribution.An assumption of this distribution is that the number ofquanta released by an action potential (m = the meanquantum content) is m = n X p. The average amplitude (E)of an EPSP is then'E = m X 4 where 4 is the mean size of theunit EPSP. Measurements of m and q provide the most directmethod for distinguishing between pre- and postsynapticprocesses. The m value indicates the amount of transmitterreleased by the presynaptic terminal and a constant q valueindicates an unchanging sensitivity of the postsynapticreceptor.d

Assuming a Poisson distribution, we estimated quantalcontent (m) and quantal size (q) with reduced transmitter

d A reduction in q value is more difficult to evaluate. Given nor-

mal filling it indicates a decrease in postsynaptic responsiveness.But some pharmacological agents (e.g., hemicholinium) seem tointerfere with normal "vesicular filling" and also cause q to de-crease.

A - s. C 0.leOl - -* ,^ -

I R1Y 11mEY D I1311325rlNr3T

11° 1- Sl -1r

1 5~~~~~~~~

I' l1 N 7(0I 5N57550-90~~~~5-

25- t 5

20 20- 0-

I, -, 12

0 r-ZT- 0

40t 20o 20iDW 34 I02= 170 V 31 93 15pIN 44 132 MOW

Isojov

FIG. 3. Amplitude histograms and computer averages of uni-tary EPSPs and failures during synaptic depression. (Parts A-D)Four sets of amplitude histograms of successive EPSPs during thesynaptic depression accompanying 144 to 219 consecutive intra-cellular stimuli to the sensory neuron. The regions refer to periodsof stable responses as described in the text. The first response peakwas assumed to be the amplitude of a unitary EPSP (qs, as de-fined in the text). The successive peaks were roughly integral mul-tiples of the initial peak. From the value of mi obtained from theseamplitude histograms a predicted curve (broken line) was gen-erated assuming a Poisson distribution and a coefficient of varia-tion for qi of 30%. Roman numerals and vertical arrows refer tothe successive multiples of the estimated unit potential. Arrowson the ordinate refer to the predicted number of failures. Arrowson the abscissa refer to the mode. The mode shifts to the left (morefailures) with repeated stimulation. N = number of stimuli; thetwo values in parentheses refer to the first and last EPSP of theplateau region (A and C, cell #2 of Table 1, I.S.I. 10 and 60 see;B, cell #1, I.S.I. 10 sec; D, cell #3, I.S.I. 60 sec). (E) Computeraverages of the unitary EPSPs and failures during the synapticdepression accompanying 200 consecutive stimuli (cell #2, part A).(El) Data were averaged on a PDP8-E computer. Using tape de-lay loops, the peak of the presynaptic spike triggered the averag-ing procedure; the first five EPSPs of the series were averaged todetermine average EPSP and to establish the interval window(see Fig. 2). Failures (N = 21) and unit EPSPs were averagedseparately (N = 21). The background noise (N = 21) was aver-

aged 2 sec before the occurrence of a presynaptic spike. The fail-ure average resembled the background noise average; there was no

time-locked depolarization. (E2) Time course of average EPSPand averageunit. The averaged EPSP and the unit average ofpart El were matched for amplitude and superimposed photo-graphically to illustrate the similarity in their time course.

output in 11 series of experiments on various follower cells (insix different preparations). The evaluation of a Poisson distri-bution requires many responses. Because of the plasticity ofthese synapses it is difficult to obtain a large number of evokedEPSPs at the low frequencies (once every 10 min) that do not

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Synaptic Depression and Habituation in Aplysia 5007

TABLE 1. Quantal analysis of synaptic depression;

Cell andamplitude lq qa q31st EPSP Trials mI ma ms (CV) (.V) (UV)

I.S.I. = 10 sec1-9*10-3940-109110-144

1-9*5-3940-139140-219

5-3940-119120-221

5-100101-200201-218

2-3940-79

7.33.91.70.9

4.73.01.81.2

4.02.21.4

0.90.60.4

3.52.4

2.I1 .1

0.

2.C1.6

3.21.5

0.70.50.3

1.9

20-89 2.2 2.290-159 1.8 1.5160-229 1.7 1.5

L.S.IL =

5-100 1.2 1.1101-136 1.0 0.9

5-6970-139140-213

1-19*20-119120-204

5-2930-8990-205

5-100101-200201-258

2.2 3.12.0 2.22.0 2.1

I.S.I. =

2.9 _2.1 2.21.2 1.2

2.71.8 2.30.8 0.8

I.S.I. =

0.9 0.80.7 0.60.5 0.4

9.87 4.45 1.87 1.0

(18.4)(9.6)(3.1)(2.6)

(29.4)(16.1)(2.6)

0.80.80.5

10.53.4

2.71.61.8

30 see

1.71.8

8.03.24.2

60 see

14.72.91.5

8.32.51.1

1 see

1.50.80.8

3437.53434

40.640.640.640.6

46.946.946.9

272727

16.816.8

49.639.340.1

3732.7

30.241.6

34.131.935.7

21.1

25.430.233.531.5

(10.4)(12.8)(23.2)(19.4)(6.1)(6.0)

(23.6)29.921.022.7

5.611.5

13.9 14.0 11.213.9 16.9 15.313.9 15.3 13.4

27 27.6 18.627 27.3 14.7

19.6 13.6 5.219.6 18.3 12.219.6 18.5 9.2

31.331.331.3

43.743.743.7

272727

29.432.1

35.844.9

62226.3

14.13231.3

29 17.233.8 23.132.6 17.0

Corrected for noise.* Not included in histograms and calculation.

produce behavior habituation or synaptic depression. Wetherefore stimulated the sensory neurons intracellularly for200 consecutive stimuli, at frequencies that produced habitua-tion (10- to 60-sec intervals). We then divided each run intotwo to three consecutive stable "plateau regions" (in whichthe average EPSP did not decrease by more than 15%)containing 30 to 100 EPSPs, using the means of consecutivegroups of 10 successive EPSPs as the index of stability. Wethen calculated and compared m and q values in the consecu-tive plateau regions and used them as an index of the synapticdepression accompanying repeated stimulation. At this re-duced level of transmitter release the EPSPs were 1-5% of thecomparable EPSPs at normal release. Thus, the 200 consecu-

tive stimuli are roughly comparable to 15 stimuli of a behav-ioral habituation training session and each consecutive regionat low release is comparable to consecutive groups of fiveEPSPs at high release.We estimated m (and q) in three ways.(1) Frequency histograms. Amplitude histograms were made

of the different plateau regions. The first peak was the failuresand the next peak was assumed to represent the quantal unit(EPSP) size 4i. We then calculated ml from the equationml -E/ where E is the mean amplitude of all the EPSPsin that plateau region. In 11 experiments (Table 1), the esti-mated ml ranged from 4.0 to 0.4 and those of quantal sizeranged from 47 ,uV to 14 /.V. These values for q are similar tothose obtained by Miledi (11) at the squid giant synapse. Asmight be expected from quantal release, the amplitude histo-grams were multimodal and successive peaks roughly corre-sponded to integral multiples of the unit peak (Fig. 3A-D).With repetition (compare regions 1, 2, 3 in Fig. 3A-D) the po-sition of the peaks including the unit peak did not change buttheir amplitude was altered. The incidence of failures in-creased progressively and the mode of the evoked EPSPsshifted from the 3 to 4 quantal unit peaks (in region 1) tothe unit and failure peaks (in region 3). This finding suggeststhat the EPSP depression during continued stimulation isdue to a decrease in quantal content (m) while quantal size(41) remains relatively unaffected. Similar changes occurred atall intervals (1, 10, 30, 60 sec) examined (Table 1).

(2) Failure analysis. One can obtain a second estimate of m(M2) from the Poisson equation by examining the ratio offailures (no) to total number of trials (AT) where M2 = InN/n0. These estimates of M2 (range 3.2-0.3) and 42 (range 50to 14 /AV) based on failures were highly correlatede with thoseobtained from the amplitude histograms (Table 1). Again,with continued stimulation m decreased while q2 remainedconstant (Table 1).To determine the fit between the observed data and a

Poisson distribution, we used ml and the Poisson equation[assuming a 30% coefficient of variation (10) in the unitresponse] to predict the number of responses containing 0,1,2,etc. quantal units. We found good agreement between thepredicted and observed values (Figs. 3A-D)f. We also checkedwhether the failures tended to occur in groups, indicatingpossible branch block, using the Poisson equation e-2m; e-"m,etc. to predict the occurrence of doublets, triplets, etc. In nocase did we see an excess of successive failures beyond thefrequencies predicted by the Poisson equation.

(3) Coefficient of variation. In a Poisson distribution thecoefficient of variation is equal to i/A/m. To arrive at a thirdestimate ofm we used M3 = E.2/c2R where T2E is the variance ofthe EPSPs' amplitudes and E1 is their mean amplitude.Because of the low signal-to-noise ratio, a correction for thenoise was sometimes used: m3 = Ia2_- a2n where O2n is thevariance of the noise obtained by measuring the baseline at afixed interval (2 sec) before the occurrence of the spike. Thistechnique is the least reliable of the three and tends to under-

e Pearson correlation test (r), r = 0.8, p < 0.01 (N = 26).fA x2 test was performed between predicted and observed valuesof the successive peaks (degrees of freedom = 1 less than the to-tal of groups having a minimum predicted frequency of 5); in 17of 18 cases the expected and observed values were not statisticallydifferent (p > 0.05). The values occurring between the peaks weredistributed in proportion to adjacent peaks.

Cell #1(L7)406 1V

Cell #2(L7)225 MV

Cell #3(L7)437 1.V

Cell #4(L7)108 uV

Cell #5(LBS-3)126 4AV

Cell #6(L16)178 1AV

Cell #4(L7)68 .V

Cell #5(LBS-3)95 /AV

Cell #2(L7)94 /AV

Cell #3(L7)212 1AV

Cell #4(L7)40 ,V

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estimate 4 (23)g. The estimate of m was consistently higherand q3 consistently lower than with the other two estimates(Table 1). Nonetheless, the trend was the same: with repeatedstimulation m decreased while q3 remained the same.

Quantal Estimates of Depression at Normal Levels of Release.In two preliminary experiments (in two preparations) weestimated m3 and q at normal levels of release using the coeffi-cient of variation and assuming a Poisson process. The resultswere also consistent with an unvarying q value (average 17/AV) and an average decrease in m (from 85 to 55).

DISCUSSIONA quantal analysis suggests that the critical change accom-panying habituation of the gill-withdrawal reflex is a decreasein the number of transmitter quanta released by the pre-synaptic spike in the sensory neurons. With repeated stimula-tion the value of q remains constant while the value of mdecreases by more than 50%. Thus, in six cells examined at 10-sec intervals the mean normalized value for q2 (or q,) in thethree consecutive plateau regions was 100%, 104%, and 98%,respectively, while that for m2 (or imn) decreased from 100% to64% to 46%. Similar trends were found at 30- and 60-secintervals (Table 1). This suggests that with repeated stimula-tion the sensitivity of the postsyuaptic receptor is unaffectedwhile the number of quantal units being released is decreased.There is considerable uncertainty in the application of the

coefficient of variation method to small signals found incentral neurons. But the graphic method of quantal analysis(combined with averaging) and the analysis of failure based onthe Poisson equation gave internally consistent results, andsupport the finding of Miledi (11) that, despite small quantalsize, the quantal analysis can be applied successfully to thecentral neurons of invertebrates. Because invertebrates areproving highly advantageous for studying behavior, the use ofquantal analysis opens the possibility of studying the mecha-nism of synaptic changes that underlie other behavioralmodifications and of analyzing their interrelationships. Thus, acritical question in the study of memory is whether short andlong-term memory represent two distinct processes or differentstages in a single process. A determination of whether quantalsize also remains constant during the synaptic depressionaccompanying long-term habituation could provide oneanswer to this question. Similarly, a quantal analysis of thesynaptic facilitation accompanying dishabituation (7) couldprovide further insight into its relationship to habituation.

g The coefficient of variation estimates are less reliable than thefailure technique when the value of p is larger or n is lower thandemanded by the Poisson assumptions. Also, the estimates of mare unduly sensitive to variations in the synchronization of releaseand will be higher than the estimates derived from the failuremethod when release is slightly desynchronized (23).

The finding that the mechanism underlying short-termhabituation is presynaptic makes it interesting to analyzemorphologically the changes in the number and distributionof the synaptic vesicles (the likely storage sites of transmitterquanta) in the terminals of the sensory neurons. Techniquesfor marking the presynaptic terminals of identified neuronsfor electronmicroscopic examination have recently been de-veloped (24) and could permit.a subcellular analysis of thisand related instances of behaviorally relevant synaptic plas-ticity.

We have benefitted from discussions with Drs. G. Fishbach andJ. Koester and from their comments and those of T. J. Carewon an earlier draft of this manuscript. We also thank KathrinHilten for help with the illustrations. This research was supportedby Research Career Development Award 5K04 NS 70346-02 toV.C., Research Scientist Award MH 18,558 to E.R.K. anD NIHGrants MHS-19795 and NS-09361.

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ded

by g

uest

on

Mar

ch 8

, 202

1