Proc. Nati. Acad. Sci. USAVol. 83, pp. 2733-2737, April 1986Neurobiology

Conditioning-specific membrane changes of rabbit hippocampalneurons measured in vitro

(ionic mechanisms/afterhyperpolarizatlon/associative learning/brain slice/nictitating membrane conditioning)

JOHN F. DISTERHOFT*, DOUGLAS A. COULTER, AND DANIEL L. ALKONSection on Neural Systems, Laboratory of Biophysics, IRP, National Institute of Neurological and Communicative Diseases and Stroke, National Institutes ofHealth at the Marine Biological Laboratory, Woods Hole, MA 02543

Communicated by Richard F. Thompson, December 6, 1985

ABSTRACT Intracellular recordings were made from hip-pocampal CAl pyramidal neurons within brain slices ofnictitating membrane conditioned, pseudoconditioned, andnaive adult male albino rabbits. All neurons included (26conditioned, 26 pseudoconditioned, and 28 naive) had stablepenetration and at least 60 mV action potential amplitudes.Mean input resistances were --60 Mfl for the three groups. Amarked reduction in the afterhyperpolarization (AHP) follow-ing an impulse was apparent for conditioned (x = -0.98 mV)as compared to the pseudoconditioned (x = -1.7 mV) andnaive (x = -2.0 mV) neurons. The AHP has been attributedpreviously to activation of a Ca2+-dependent outward K+current. The distribution of AHP amplitudes for the condi-tioned group included a new lower range of values for whichthere was little overlap with the other groups. The condition-ing-specific reduction of AHP may be due to reduction ofICa2+-K+ as shown previously for conditioned Hermissendaneurons. This conditioning-induced biophysical alteration ofthe CAl pyramidal cell must be stored by mechanisms intrinsicto the hippocampal slice and cannot be explained as a conse-quence of changes of presynaptic input arising elsewhere in thebrain. Our experiments demonstrate the feasibility of analyz-ing cellular mechanisms of associative learning in mammalianbrain with the in vitro brain slice technique.

It is well known that bilateral hippocampal lesions in humanscause permanent inability to store most new memories (1, 2).Hippocampal units recorded from freely moving rats duringclassical conditioning of an appetitive response to foodshowed marked involvement of the hippocampal subfieldsduring and after learning (3-6). Berger and Thomson andco-workers (7-9) have shown that hippocampal neurons fromrabbit show large changes in firing rate that precede acqui-sition of the nictitating membrane (NM) conditioned re-sponse and form a "model" of the NM extension as the rabbitbecomes well trained. In a very elegant paper, they haveshown that it is the CA1 and CA3 pyramidal neurons thatbecome so engaged (10).

In addition to an excellent in vivo base of recording data onhippocampus during learning, there is extensive hippocampalslice literature that provides baseline biophysical parameters(11-16). Most biophysical data are from CA1 and CA3pyramidal cells, precisely the cell group whose conditionedstimulus (CS)-elicited responses form the neural model (men-tioned above) of the rabbit NM response in vivo.Our first question was, Can we demonstrate learned

alterations in biophysical parameters in the slice?-that is,can we train an animal, remove a part of the brain in whichCS-elicited impulse activity is known to be altered byassociative learning (the hippocampus), and show that effectsof the learning are retained in vitro (even though neurons in

the slice are denied their normal afferent and efferent con-nections)? Learning-induced biophysical alterations within aslice would have to be stored by intrinsic mechanisms andwould not be explainable as a consequence of changes ofpresynaptic input arising in other brain regions. If learnedeffects are present in the slice, then we could exploit thetechnical advantages of the slice technique to analyze them.To date, studies ofplasticity in the brain slice have dealt withlong-term potentiation (17) and kindling (18). No in vitrostudies of associative learning in mammalian brain have beenreported.Our second goal was to determine if common mechanisms

might underlie learning in mammalian and invertebrate neu-rons. A biophysical sequence of alterations that occur in thetype B photoreceptor of Hermissenda and that underlieassociative learning in this nudibranch mollusc has beendescribed (19). In brief, an excitability increase in the type Bcell is caused by a reduction of an early, rapidly inactivatingoutward K+ current, IA, and of a Ca2+-dependent outwardK+ current, IC (20, 21). Prolonged elevation of intracellularCa2+ may be an important initial step for producing thelearning-induced reduction of IA and ICa2+_K+ (22-24). Pro-longed Ca2' elevation can be electrically induced in hip-pocampal pyramidal cells (25). IA and ICa2+-K+ have also beenshown to occur in these cells (26, 27). We wished to examinethe possibility, therefore, that ionic changes occur in thesehippocampal neurons after classical conditioning similar tothose changes measured previously after classical condition-ing in Hermissenda neurons. We have published preliminaryreports of our findings (28, 29).

METHODSYoung adult male albino rabbits (Oryctolagus cuniculus)(1.0-1.5 kg) were used as subjects. Behavioral trainingprocedures similar to those described (30, 31) were used.Conditioned animals (n = 11) were trained on 3 successivedays in 80 trial sessions. In each trial, a 400-ms white noise(80 decibels) CS was delivered by way of a closed soundsystem (32) to the right ear. The 150-ms periorbital shockunconditioned stimulus (US) (0.1 ms, 200 Hz) was justsufficient to give a reliable unconditioned response (1-2 mA).The average intertrial interval was 60 s. In every 10th trial,the CS was presented alone. We measured eyeball retraction,the response which causes NM extension, directly with aphotodiode technique (33). The conditioned rabbits weretrained to a behavioral criterion of 80-100% conditionedresponses (three sessions for all but two animals). Pseudo-

Abbreviations: AHP, afterhyperpolarization; NM, nictitating mem-brane; EPSP, excitatory postsynaptic potential; CS, conditionedstimulus; US, unconditioned stimulus.*To whom reprint requests should be sent at: Department of CellBiology and Anatomy, Northwestern University Medical School,Chicago, IL 60611.

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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"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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conditioned animals (n = 13) received 80 CS and 70 US trialsin a pseudorandom nonoverlapping sequence with an average

intertrial interval of 30 s for three sessions. Every 10th UStrial was blank. Five of the 26 pseudoconditioned cells weretaken from four animals who received 40 CS and 35 US trials.Parameters for these five cells were not distinguishable fromparameters obtained for the other (n = 21) pseudocondi-tioned cells. Statistical significance levels were not alteredby inclusion of these cells for the purpose of testing betweengroup differences. All 26 pseudoconditioned cells weretherefore included in the data presentation and analysis.Naive rabbits (n = 16) received no surgery or behavioraltraining.On the day after the final training session, slices of the left

hippocampus were prepared using standard procedures (34).The incubation medium contained 3.5 mM KCl, 124 mMNaCl, 2 mM NaH2PO4, 2 mM MgSO4, 2 mM CaCl2, 25 mMNaHCO3, and 10 mM dextrose and was oxygenated with95% 02/5% CO2. Slices (350 jm) were cut at an angle of 700to the fimbria. This is the approximate position that Andersonet al. (35) described for the hippocampal lamellae of therabbit.Four to 6 slices were transferred to a surface recording

chamber (36) and maintained at 30-32°C. The volume of thechamber was 0.6 ml; the flow rate ofoxygenated medium was1.9 ml/min. The atmosphere above the slices was humidifiedand oxygenated. Another 6-12 slices were placed in a holdingchamber containing oxygenated incubation medium at roomtemperature and used if needed. The slices were allowed toequilibrate for 1 hr before recording was begun.

Intracellular recordings were made with microelectrodesfilled with 3 M KCl, with impedances that measured 70-100MQi. A high-input impedance bridge amplifier was used. Datawere measured from a storage oscilloscope or stored on aHewlett-Packard FM tape recorder (bandpass DC-2500 Hz)for later analysis.The recording microelectrode was placed into the CA1 cell

body layer under visual control. In addition, bipolar stimu-lating electrodes were often placed on the Schaffer collateralsfor orthodromic stimulation and on the alveus for antidromicstimulation. Stable recordings could be held for 1-2 hr withrelative ease. The electrode was generally withdrawn toconfirm the membrane resting potential. The values for

membrane resting potential in Table 1 were gained in thismanner. Action potential height was measured from baseline.

RESULTS

Action Potential Amplitude, Resting Potential, and InputResistance. Most cells for which stable recordings could bemade had action potential amplitudes of 60 mV or more,resting membrane potentials of 50 mV or greater, and inputresistances of 20 MCI higher. Cells with characteristics thatdid not meet these criteria were not included in the presentstudy. A total of 26 neurons from 11 conditioned rabbits, 26neurons from 13 pseudoconditioned rabbits, and 28 neuronsfrom 16 naive rabbits did meet these criteria. Recordingscould usually be maintained for 1-2 hr but were terminated(after 30-45 min) by withdrawing the electrode to check theresting membrane potential. Membrane resting potential andaction potential amplitudes (elicited by a positive currentpulse) were similar among groups (Table 1). In general, stablerecordings showed little or no spontaneous action potentialactivity but were characterized by marked baseline activity.

Input resistance measurements showed delayed and sub-threshold anomalous rectification when estimated with aseries ofhyperpolarizing and depolarizing pulses, as reportedfor CA1 cells (37). We estimated input resistance in the linearregion of the current-voltage relation with hyperpolarizingpulses from 0.1 to 0.3 nA. Input resistance measured in thisway was similar among groups (Table 1).

Afterhyperpolarization (AHP). CA1 impulses are typicallyfollowed by an AHP. The AHP has been attributed to aCa2+-dependent potassium current, ICa2+-K+ (38-40). Evi-dence that the AHP is due to ICa2+-K+ includes its reductionby EGTA iontophoretic injection (41-43), by Ca2+ channelblockers as Mn2' and Co2+ (38, 40, 42, 44), and by substi-tution of Ba2+ for Ca2' in the external perfusion medium (40,44). Perfusion of our own slices with the Ca2+ channelblocker Co2+ (2.3 mM) or Cd2+ (100-200 uM) reliablyreduced or eliminated the AHP (-1.67 mV vs. -0.67 mVafter Co2+, Cd2+; P < 0.02). For our measurements here theAHP was defined as the largest negative shift of membranepotential from baseline during the 350-ms interval followingthe 100-ms current pulse used to elicit impulse(s) (Figs. 1 and2). In most cases these values were the averages of fiveseparate measurements (Fig. 1). The AHP following one

Table 1. Measured parameters and statistics

Group StatisticsParameter C PC N Comparison P*

AHP, mVOne spike -0.98 ± 0.80 -1.7 ± 0.83 -2.0 ± 0.79 C vs. P <0.004

(21) (23) (26) C vs. N <0.0001Two spikes -1.89 ± 1.47 -2.26 ± 1.27 -2.82 ± 0.92 C vs. N <0.02

(19) (18) (24)Sag, mV 2.28 ± 2.0 3.36 ± 1.8 5.4 ± 1.8 C vs. P <0.04t

(25) (22) (25) C vs N <0.0001P vs. N <0.001

Spike height, mV 80.3 ± 11.8 80.8 ± 12.7 77.5 ± 11.5 -(23) (24) (26)

Resting potential, mV -65.0 ± 6.1 -67.2 ± 7.5 66.0 ± 6.5(15) (17) (21)

Input resistance, MU 59.3 ± 22.6 61.2 ± 16.2 60.6 ± 17.6(26) (23) (27)

EPSP, mV 9.2 + 0.6 9.9 ± 1.7 12.3 ± 1.3 C vs. N <0.02(11) (8) (13) P vs. N <0.05t

All values are expressed as mean ± SD; n is given in parentheses for each value. EPSP, excitatorypostsynaptic potential; C, conditioned; PC, pseudoconditioned; N, naive.*Two-tailed significance level unless otherwise indicated.tOne-tailed significance level.

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/

J5 mV

11.0 nA/---

FIG. 1. Voltage responses of CA1 cell to positive intracellularcurrent pulses. The AHP of a CA1 cell from a conditioned animal isconsistently minimal following repeated injections (at 30-s intervals)of positive current sufficient to elicit one (0.1 nA) or two (0.15 nA)impulses. Note the small variability ofresponses elicited by the samecurrent injections.

action potential for the conditioned group (-0.98 ± 0.8) wassignificantly lower than for the pseudoconditioned (-1.7 ±0.8 mV; P < 0.004) and the naive (-2.0 ± 0.8 mV; P <0.0001)groups (Table 1, Fig. 2). The AHP following two actionpotentials was also lower for the conditioned (-1.9 ± 1.4 mV)as compared to the pseudoconditioned (-2.3 ± 1.3 mV; notstatistically significant) and naive (-2.8 ± 0.9 mV; P < 0.02)groups.The observed AHP between group differences cannot be

explained as secondary to conditioning-induced alteration ofinput resistance or resting membrane potential since theseparameters were similar among the groups. That the AHPdifferences could not be attributed to differences of inputresistance was further indicated by the fact that there was nosignificant correlation in any group between input resistanceand AHP (calculated correlation coefficients of 0.07 forconditioned cells, 0.014 for pseudoconditioned cells, and 0.12for naive cells were not statistically significant). Furtherindication that the between-group differences ofAHP couldnot be attributed to differences of resting membrane potentialwas provided by closely matching resting potentials for sevenconditioned (x = -62.1 mV), seven pseudoconditioned (x =-62.6 mV), and seven naive (x = -61.4 mV) neurons. AHPmagnitude remained significantly smaller for the conditioned(x = -0.68 mV) as compared to the pseudoconditioned (x =1.79 mV; P < 0.01) and naive (x = -1.76 mV; P < 0.01)groups.

Naive

15 mVS^,50 ms

_1_*..... -

Pseudoconditioned

/ \_- - \- ------

A distribution ofthe actualAHP values for the three groupswas also constructed (Fig. 3). This distribution showed aclear overlap of higher AHP values for all three groups.However, 48% of the conditioned neurons had AHP magni-tudes between 0 and -0.5 mV, values which appeared for onepseudoconditioned but no naive neurons.

Blind Samples. Two additional data analyses were con-ducted to control for possible experimenter bias. First, arepresentative sample (7 cells each) was chosen from each ofthe three groups. Photographed records of taped measure-ments were then coded, mixed, and analyzed blindly by anindividual not familiar with the experimental protocol orobjectives. This "blind" analysis of recorded data yieldedAHP values that were still significantly lower for the condi-tioned (x = 1.09 mV) as compared to the pseudoconditioned(x = 2.17 mV; P < 0.01) and naive (x = -1.61 mV; P < 0.05)neurons.

In addition, a subpopulation of conditioned, pseudocondi-tioned, and naive animals was presented for electrophysi-ologic protocols by using a double-blind procedure. The smallnumber of cells (n = 5 for each group) obtained in this waydid not prevent observation of a statistically significantdifference between AHP amplitude of the conditioned (x =-0.9 ± 0.5 mV) as compared to that of the naive group (x =-2.3 ± 0.9 mV; P < 0.02). The pseudoconditioned groupAHP response (x = -1.6 ± 0.8 mV) was larger than that ofthe conditioned group though not significantly different withthe small sample.Sag Response. When large hyperpolarizing current pulses

were injected, the voltage responses showed an initial rapiddecrease and then a subsequent slower small increase beforereaching a steady-state level (Fig. 4). This type of responsehas been termed "sag" or droop and has been seen in avariety of neurons (45-47). Sag in hippocampal pyramidalneurons may represent the voltage-dependent activation ofan active inward K+ current (i.e., at potentials more negativethan the K+ equilibrium potential there is a reversed outwardK+ current) or an inward Na' current by the large hyperpo-larizing pulses (45). We measured the amount of sag presentas the difference (mV) between the maximal voltage deflec-tion and the steady-state response to 100-ms, 1-nA negativepulses (Fig. 4). The response to five current pulses was

.,o 6

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oW)

0

c 640

Conditioned

FIG. 2. AHP after one spike in a naive, pseudoconditioned, andconditioned neuron. A 100-ms depolarizing current pulse sufficient toelicit one action potential was injected into the cell. The AHP can beseen by comparing the voltage response following the depolarizingpulse to the baseline (indicated by the dashed line to facilitatecomparison). The point where the AHP was measured in these tracesis indicated by the arrow. Note that the AHP is considerably reducedin the conditioned as compared to the naive and pseudoconditionedneuron. One trace is illustrated for each neuron.

6~

n = 21

n = 23

n = 26

.f-0.5 -1.5 -2.5 -3.5AHP magnitude, mV

FIG. 3. Histogram of the AHP response to one spike for theconditioned, pseudoconditioned and naive cell groups. The distri-bution of AHPs for the conditioned neurons overlaps the other twogroups except for the responses of -0.5 mV and less.

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2736 Neurobiology: Disterhoft et al.

Naive Pseudoconditioned Conditioned

110 mV20 ms

FIG. 4. Sag response in a naive, pseudoconditioned, and condi-tioned neuron: voltage response to 100-ms hyperpolarizing squarecurrent pulses injected through the recording electrode. Five suc-cessive pulses at six values (-1.0 nA, -0.8 nA, -0.6 nA, -0.4 nA,-0.2 nA, 0 nA) were overlaid on a storage oscilloscope screen anda picture was taken. The sag is particularly noticeable in the responseto the 1-nA pulses in the naive and pseudoconditioned neurons. Thevoltage response loses the characteristic of a passive charging curveat about 40 ms, changes direction, and becomes less hyperpolarizedfor the remainder of the pulse. The change in trajectory of the voltageresponse reflects activation of Iq (45) by the hyperpolarizing pulse.

averaged for each neuron. The magnitude of sag was signif-icantly less for the conditioned (x = 2.3 ± 2.0 mV) ascompared to the pseudoconditioned (x = 3.4 ± 1.8 mV; P <0.04) and naive (x = 5.4 ± 1.8 mV; P < 0.0001) neurons. Forthis measurement the pseudoconditioned neurons had de-creased values compared to those for the naive neurons (P <0.001). Thus, the differences of sag observed were notconditioning-specific.Orthodromic EPSP. The size of an orthodromic EPSP that

elicited one action potential was also somewhat less for theconditioned (x = 9.2 0.6 mV; P < 0.02) andpseudoconditioned (x = 9.9 1.7 mV; P < 0.05) as comparedto naive (x = 12.3 + 1.3 mV) cells. Thus, by this measure theconditioned and pseudoconditioned cells were not differentfrom each other.

DISCUSSIONOur major finding is that the AHP response that follows oneor two action potentials is reduced in CA1 pyramidal cells ofhippocampal slices taken from rabbits who have been con-ditioned in the NM paradigm but not in slices taken frompseudoconditioned or naive rabbits. The AHP response inCA1 and CA3 pyramidal cells has been well studied by othersin the hippocampal slice. The AHP response measured afteran action potential caused by the injection of positive currentis primarily caused by a Ca2l-mediated K+ conductance (38,40, 43). It is not presently known in which cellular compart-ment(s) the AHP arises. Thus, the conditioning-specificdifference in AHP reported here cannot be assigned at thistime to a specific locus within the CA1 pyramidal cell.Madison and Nicoll (42) have presented evidence that avoltage-dependent K+ current, the M current, may contrib-ute slightly to the AHP.The AHP's presumed function in vivo is to slow or stop

firing of hippocampal pyramidal neurons that tend to fire inbursts. In vivo recordings in rabbits and rats demonstrate thathippocampal pyramidal cells become more responsive to a

CS after conditioning (3-10). A reduction in the AHP couldcause, or at least contribute to, this increased excitability. Itis of interest also that the AHP response in hippocampalpyramidal cells has been reported to decrease after long-termadministration of ethanol (48) and to be prolonged in timeduring aging (49).A decrease in sag was also observed for the conditioned

compared to the pseudoconditioned cell groups. It wasreduced in conditioned and pseudoconditioned groups incomparison to the naive cell group as well. Single-electrodevoltage-clamp studies in CAl pyramidal neurons have indi-

cated that this sag in the voltage response to large hyperpolar-izing pulses is carried by an inward current termed Iq (45).This current is apparently carried by Na' and K+ ions and isactivated at hyperpolarized levels less than -80 mV. Thefunction of a current activated at such extreme hyperpolar-izing levels is not clear.The basic finding of a conditioning-specific reduction of

AHP has been confirmed and extended in an entirely separatesubsequent study (50). In the second study conducted with adifferent recording protocol-i.e., from submerged vs. sur-face slices (see Methods)-between-group differences ofAHP were significant following depolarizing pulses thatelicited one, two, three, or four impulses. In addition, theAHP duration was measured and showed a conditioning-specific reduction. Statistically significant, conditioning-specific AHP amplitude and duration reductions were seen ina large subset of the second study carried out with blindprocedures.Learned Alteration Is Intrinsic and Postsynaptic. The alter-

ation in AHP that we measured was elicited by intracellularinjection of depolarizing current. We usually recorded verylittle or no spontaneous firing in impaled neurons. A reducedAHP could have functional expression in vivo as a result ofsynaptic input but was manifest in our population of condi-tioned neurons in the absence of such input. It is alsoimportant to note that we recorded no conditioning-specificalteration in the size of EPSPs evoked by Schaffer collateralstimulation due to conditioning. Thus, our data at this pointdo not support the well-known hypothesis (51) that associa-tive learning causes alterations at synapses, which, in turn,cause increased postsynaptic responsivity to a constantafferent input.Our tentative conclusion is that the reduced AHP in the

CA1 pyramidal cells is a postsynaptic phenomenon, one thatis intrinsic to a portion of the pyramidal cell population. Invivo studies of cat pericruciate cortex after eyeblink condi-tioning have suggested that the alterations recorded there arepostsynaptic as well (52). Further studies of other cell groupswithin the hippocampal slice, especially the CA3 pyramidalcells and interneurons, will be necessary to determine whatbiophysical effects of conditioning they manifest and whatpossible relation such effects might have to the measure-ments made here.Learned Changes Are Retained in Vitro. One of the major

issues we addressed in these experiments was whether it waspossible to demonstrate learned alterations in biophysicalparameters in the slice-that is, Could we train an animal,remove a part of the brain known to be altered by associativelearning, and show that effects of the conditioning wereretained in vitro? Our findings confirm that the effects ofassociative learning are retained in the hippocampus in vitro.A final piece of evidence regarding the utility of the in vitro

brain slice preparation for analysis of learning is the strikingconvergence between our in vitro data and that recorded byBerger et al. (10) in vivo. They found that 62% of identifiedsingle pyramidal neurons increased their firing rate in re-sponse to the tone CS and before US onset after conditioning.We found that 48% (10/21) of CA1 pyramidal cells showedAHP responses less than -0.5 mV after conditioning (Fig. 2).This size ofAHP response was lower than almost all of thoseobserved in the pseudoconditioned or naive groups. If weassume that a cell with a markedly reduced AHP is a"learned" cell, the convergence of the two types of data isquite striking.

Role of Hippocampus in NM Conditioning. It is wellestablished that hippocampal lesions do not eliminate NMconditioning in rabbits when the simple delay paradigm(where CS and US overlap) is used (53). But scopolamine, acholinergic blocker that disrupts hippocampal activity, re-tards NM acquisition only when hippocampus is intact (54).

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And, as we have reviewed above, a large percentage ofhippocampal neurons becomes markedly involved duringNM conditioning (7-9).Although humans who have sustained bilateral hip-

pocampectomy are unable to permanently store new mem-ories (1, 2), they can acquire some motor tasks, includingblink conditioning. Weiskrantz and Warrington (55) havereported successful blink conditioning of such patients whohad no recall of the task following their training. This wouldsuggest that the hippocampus is involved in storing some typeof higher-order, cognitive information concerning NM orblink conditioning rather than its motor program. Such dataare consistent with the quite reasonable proposal that learn-ing is a distributed process in mammalian brain.We are not suggesting that we have demonstrated the ionic

mechanism for NM conditioning. Rather, we have beguninvestigating the cellular substrate of information storage orplasticity intrinsic to hippocampus that occurs in this asso-ciative paradigm.

Is There a Comparable Mechanism of Learning in Mammaland Invertebrates? A second major question that we ad-dressed in this series of experiments was whether commonmechanisms may underlie learning in mammalian and inver-tebrate systems. Alkon and his associates have studied themechanisms of an avoidance conditioning task in thenudibranch mollusc Hermissenda (19). In a variety of exper-iments, they have shown a causal sequence of biophysicalchanges in the type B photoreceptors that occur duringconditioning and that are sufficient to cause the learnedresponse. The excitability increase in the type B photorecep-tor was caused in part by a reduction in Ca2l-dependentoutward K+ current and also by a reduction of an earlyoutward K+ current, IA (20, 21).

Biophysical analyses have demonstrated that the AHP re-sponse in CA1 pyramidal neurons is primarily caused byCa2+-dependent K+ current (38, 42, 44). Our data clearly showareduction inAHP after conditioning. However, we cannot ruleout the possibility that a reduced voltage-dependent Ca2+current, ICa2+, is indirectly responsible for the conditioning-specific reduction of jc. A conditioning-specific reduction ofeither ICa2+-K+ or ICa2+ would provide a biophysical memorytrace remarkably similar to such a trace demonstrated to encodeHermissenda associative learning (19).

CONCLUSIONWe have demonstrated with an in vitro preparation an ionicmechanism that accompanies associative learning in mam-malian brain. The indication is very strong that the ionicmechanism is postsynaptic and intrinsic to the CA1 pyrami-dal cell. The reduction in the AHP response is clearly presenteven when the pyramidal cells are separated from the rest ofthe conditioned reflex arc. It is not dependent upon afferentneuronal drive for its expression. Our data lend support to theproposition that the in vitro brain slice preparation, used inconjunction with in vivo single-neuron recording and behav-ioral techniques, will be a useful tool for determining thecellular basis of associative learning in mammals.

This research was partially supported by a research grant from theNational Science Foundation (BNS-8302488) and by a NationalInstitute of Neurological and Communicative Disorders and StrokeIPA Fellowship to J.F.D.

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