Enhanced Synaptic Transmission in CA1 Hippocampus After Eyeblink Conditioning

RAPID COMMUNICATION

JOHN M. POWER, LUCIEN T. THOMPSON, JAMES R. MOYER, JR., AND JOHN F. DISTERHOFT Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611

Power, John M., Lucien T. Thompson, James R. Moyer, Jr., stimulation evoked larger summating excitatory postsynaptic and John F. Disterhoft. Enhanced synaptic transmission in CA1 potentials (EPSPs) in CA1 pyramidal neurons from condi- hippocampus after eyeblink conditioning. J. Neurophysiol. 78: tioned rabbits (LoTurco et al. 1988). [ 3H] a-amino-3-hy- 1184±1187, 1997. CA1 ®eld potentials evoked by Schaffer collat-

droxy-5-methyl-4-isoxazolepropionic acid (AMPA) binding Downloaded from eral stimulation of hippocampal slices from trace-conditioned rab- in CA1, CA3, and dentate gyrus sub®elds is increased after bits were compared with those from naive and pseudoconditioned controls. Conditioned rabbits received 80 trace conditioning trials conditioning (Tocco et al. 1992). In the present study, daily until reaching a criterion of 80% conditioned responses in a Schaffer collateral evoked synaptic responses in slices from session. Hippocampal slices were prepared 1 or 24 h after reaching trace-conditioned rabbits are compared with those in naive criterion (for trace-conditioned animals) or after a ®nal unpaired and pseudoconditioned controls at intervals where maximal stimulus session (for pseudoconditioned animals); naive animals postsynaptic changes have been observed. were untrained. Both somatic and dendritic ®eld potentials were http://jn.physiology.org/ recorded in response to various stimulus durations. Recording and METHODS data reduction were performed blind to the conditioning state of the rabbit. The excitatory postsynaptic potential slope was greater Subjects and surgeries in slices prepared from trace-conditioned animals killed 1 h after conditioning than in naive and pseudoconditioned controls (re- Animal care was approved by the US Department of Agriculture peated-measures analysis of variance, F Å 4.250, P õ 0.05). Asso- and managed by Northwestern University's Animal Care and Use ciative learning speci®cally enhanced synaptic transmission be- Committee. Behavioral treatment and slice preparation methods tween CA3 and CA1 immediately after training. This effect was were identical to those previously described (Moyer et al. 1996; Thompson et al. 1996). Forty-six young (1.5 mo) female rabbits

not evident in the population ®eld potential measured 24 h later. by 10.220.33.3 on November 3, 2016 (New Zealand White) received either trace conditioning or pseudoconditioning or were naive. Trace-conditioned rabbits received 80 INTRODUCTION pairings daily of a 100-ms tone CS, followed by a 500-ms trace interval, then a 150-ms air puff, until reaching a criterion of 80% The hippocampus plays an essential role in the learning CRs in a given session. Pseudoconditioned subjects received an equal number of unpaired stimuli for a matched number of training of certain spatial and temporal associative tasks, with hippo- sessions. campal lesions producing severe de®cits (Squire 1987). One model of associative learning is eyeblink conditioning, in Slice preparation which a conditioned stimulus (CS) is followed by an uncon- ditioned stimulus (US) that induces an eyeblink. After re- Transverse hippocampal slices (400 mm) were prepared from peated pairings, the subject exhibits conditioned responses rabbits 1 or 24 h after training and held in arti®cial cerebrospinal (CRs), blinks after CS onset before US onset. Although ¯uid (aCSF, composition, in mM: 124 NaCl, 26 NaHCO3 ,10 the hippocampus is not required for all forms of eyeblink D-glucose, 3 KCl, 2.4 CaCl2 , 1.3 MgSO4 , and 1.24 NaH2PO4 ,pH conditioning (Thompson 1986), when the CS and US are 7.4, 23ЊC). All recordings and analyses were performed blind to separated by a 500-ms trace (no stimulus) interval, hippo- the animal's conditioning state. campectomized rabbits fail to acquire the eyeblink conditioning task (Moyer et al. 1990; Solomon et al. 1986). Electrophysiology Hippocampal synaptic enhancement may underlie the ac- Extracellular recordings were made 1±6 h after slice preparation, quisition of conditioned eyeblink responses. Multiunit stud- during the period of optimal slice health (Green and Greenough ies have demonstrated a learning-dependent increase in the 1986). Slices with crisp, well-de®ned cell layers were perfused ®ring of CA1 and CA3 neurons that models the time course with 31ЊC oxygenated aCSF in a submersion slice chamber. Field and amplitude of the CR and is not seen in unpaired controls potentials were evoked by Schaffer collateral stimulation with the (Berger et al. 1976; Solomon et al. 1986). Additionally, use of 75-mm twisted stainless steel bipolar electrodes (250 kV) perforant-path-evoked ®eld potentials in dentate gyrus are aligned with the end of the dentate gyrus (see Fig. 1). Field poten- enhanced during delay conditioning (Weisz et al. 1984). In tials were recorded in both stratum radiatum and stratum pyrami- dale with a 1- to 5-MV glass electrode ®lled with 2 M NaCl, vivo experiments, however, cannot eliminate extrahippo- positioned 750 mm from the stimulating electrode. Slices were campal factors and localize changes to the hippocampus. stimulated (100 ms, 100 mA) every 30 s until evoked potentials In vitro experiments, which can localize changes to the stabilized (usually 15±20 min). Slices were discarded if a 200- hippocampus, also suggest that synaptic alterations underlie ms, 100-mA stimulation evoked a somatic ®eld potential with popu- eyeblink conditioning. High-frequency Schaffer collateral lation spike amplitude õ1.5 mV or exhibited multiple low-ampli-

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FIG. 1. Diagram of a rabbit hippocampal slice depicting locations of stimulating electrode (SE) and recording electrodes (R1, R2) and representative somatic (R1) and dendritic (R2) ®eld potentials 1 h after conditioning. Schaffer collaterals (sc), perforant path (pp), dentate gyrus (DG), mossy ®bers (mf), CA1, and CA3 are labeled for clarity. http://jn.physiology.org/ tude epileptic form responses. A stimulus-response function was largest responses were chosen to represent the animal, the generated by evoking four potentials with 100-mA current at differ- results were unchanged [F(4,41,7) Å 2.705, P õ 0.05; Fish- ent stimulus durations (ranging from 20 to 600 ms). To control er's PLSD: trace 1 h, naive, P õ 0.025; trace 1 h, pseudo for drift, ®eld potentials were evoked with the use of a ®xed 100-ms, 1h,Põ0.015; trace 1 h, pseudo 24 h, P õ 0.02; trace 1 100-mA stimulus both before and after determination of stimulus- h: trace 24 h, P ú 0.15). No signi®cant differences in the response function. Slices were excluded if the pre- versus posttest ®eld potentials differed by ¢15%. population spike amplitude were seen between groups [F(4,41,7) Å 3.119, P ¢ 0.25, see Fig. 2, C and D), al-

though a trend was seen toward a conditioning-associated by 10.220.33.3 on November 3, 2016 Data analysis enhancement of the population spike amplitude 1 h after Evoked potentials were digitized (10±20 kHz) and analyzed conditioning. There were no signi®cant differences in spike with the use of custom software. Somatic ®eld potentials measured latency, nor was there any correlation between ®eld measure- population spike amplitude and latency. The EPSP slope of den- ments and time of recording after slice preparation. dritic ®eld potentials measured synaptic current. Recordings were made from three to ®ve slices per animal. Measurements at each stimulus duration were averaged for a given animal and used in DISCUSSION subsequent statistical analysis. Repeated-measures analyses of variance (Abacus Concepts, Statview) were performed with the use These results demonstrate an enhancement of CA1 Schaf- of behavioral treatment as a factor. Signi®cant effects (a Å 0.05) fer collateral synaptic transmission, intrinsic to the hippo- were additionally analyzed with the use of Fisher's Protected Least campus, as a result of learning a hippocampally dependent Signi®cant Difference (PLSD) post hoc tests. task. The lack of pseudoconditioning effects indicates that the synaptic enhancement was due to a learned association RESULTS between the CS and US, not simply stimulus presentations. Similar behaviorally induced synaptic potentiation in hippo- Conditioning speci®cally enhanced synaptic potentials at campus has been reported in vivo in rats during and after intervals shortly after rabbits reached behavioral criterion. exploratory learning (Moser et al. 1994), tone-shock condi- The initial EPSP slopes of dendritic potentials from conditioning (Laroche et al. 1987), operant conditioning (Skelton tioned animals killed 1 h after the ®nal conditioning session et al. 1987), and environmental enrichment (Sharp et al. were greater [F(4,41,7) Å 3.119, P Å 0.0250, see Fig. 2, 1985), and in vitro after environmental enrichment (Green A and B] than the slopes from naive (Fisher's PLSD, P õ and Greenough 1986). 0.02) and pseudoconditioned controls (1 h, P õ 0.005; 24 Possible mechanisms for the observed synaptic enhance- h, P õ 0.01). There were no signi®cant differences between ment include changes in the following. naive and pseudoconditioned control animals (either 1 or 24 1) Synaptic structure. Synaptic remodeling (e.g., increas- h) or between conditioned and control animals killed 24 h ing perforated synapses with multiple transmission zones) after conditioning. (Geinisman 1993) could contribute to an enhanced EPSP. It has been suggested that slices exhibiting the largest 2) Transmitter release or binding. Increased glutamate responses were likely to be the healthiest, and the most release from hippocampal slices has been reported after representative of the in vivo condition (Green and paired tone shock conditioning (Laroche et al. 1987). Addi- Greenough 1986). When data from the slice exhibiting the tionally, enhanced AMPA (Tocco et al. 1992) and N-methyl-

9K17/ 9k16$$jy02 J965-6RC 08-05-97 15:14:07 neupa LP-Neurophys 1186 J. M. POWER, L. T. THOMPSON, J. R. MOYER, AND J. F. DISTERHOFT Downloaded from http://jn.physiology.org/ by 10.220.33.3 on November 3, 2016

FIG. 2. Effects of conditioning on Schaffer collateral evoked ®eld potentials recorded in CA1. Means { SE are given for trace-condititioned (1 h, n Å 9; 24 h, n Å 13), pseudoconditioned (1 h, n Å 9; 24 h, n Å 8), and naive (n Å 7) animals for each stimulus intensity value. A: excitatory postsynaptic potential (EPSP) slope recorded in dendrites was greater in slices prepared from conditioned animals 1 h after conditioning. B: no signi®cant differences in initial EPSP slope were seen between conditioning groups 24 h after conditioning. No conditioning effect was seen in population spike amplitude input- output function1h(C)or24h(D) after conditioning.

D-aspartate (Stewart et al. 1992) receptor binding has been for several days. The time course of the current generating demonstrated after learning. It is unlikely that the synaptic the slow AHP in CA1 pyramidal neurons suggests that AHP potentiation observed here is analogous to long-term potenti- changes do not directly contribute to short-latency changes ation (LTP) at the Schaffer collateral CA1 synapses. Unless in the EPSP slope. However, the AHP reduction may have depotentiated, LTP can last for days (Bliss and Lomo 1973). facilitated the synaptic enhancement. Pharmacological The synaptic potentiation observed here diminished within blockade of the AHP can convert the potentiation resulting 24 h. In addition, we observed no potentiation of the spike from a weak tetanus from a short-term to a more sustained beyond that predicted from the enhancement of the EPSP potentiation (Sah and Bekkers 1996). (E-S potentiation) after learning, often present in LTP (An- None of the proposed mechanisms are mutually exclusive, dersen et al. 1980). and further work is needed to fully examine these and other 3) Postsynaptic conductances. Postsynaptic conductance possibilities. The transient learning-speci®c enhancement of changes contributing to the observed synaptic enhancement dendritic ®eld potentials that we observed in CA1 appears would likely be preferentially localized to the apical den- to be different both from the previously reported AHP and drites, because similar changes in somatic conductance accommodation changes and from LTP. We hypothesized would further enhance the population spike, and E-S potenti- that the AHP reductions could act as an adjustable gain ation was not observed. We have previously reported post- control, amplifying speci®c synaptic inputs (Moyer et al. synaptic reductions in postburst afterhyperpolarization 1996; Thompson et al. 1996). Thus enhancement of the CA1 (AHP) and in spike-frequency accommodation after trace Schaffer collateral synapses may amplify inputs from CA3 conditioning (e.g., Moyer et al. 1996; Thompson et al. neurons. Our data would be consistent with a process in 1996). Unlike the synaptic enhancement seen here, AHP which generalized synaptic alterations (observable in popu- and spike-frequency accommodation changes were maximal lation ®eld potentials) occurred early during learning, and up to 24 h after the ®nal conditioning session and persisted later became localized to a much more speci®c pattern or

9K17/ 9k16$$jy02 J965-6RC 08-05-97 15:14:07 neupa LP-Neurophys ENHANCED SYNAPTIC TRANSMISSION AFTER EYEBLINK CONDITIONING 1187 set of synapses (not observable in population ®eld poten- tic potentials in rabbit CA1 pyramidal neurons following classical condi- tials). The speci®c pattern of synaptic alterations may in- tioning. Proc. Natl. Acad. Sci. USA 85: 1672±1676, 1988. MOSER, E. I., MOSER, M., AND ANDERSEN, P. Potentiation of dentate syn- clude both potentiation and depression that obscure each apses initiated by exploratory learning in rats: dissociation from brain other in population measures but may play important roles temperature, motor activity, and arousal. Learn. Memory 1: 55±73, 1994. in the learning process. MOYER, J. R., JR., DEYO, R. A., AND DISTERHOFT, J. F. Hippocampectomy disrupts trace eyeblink conditioning in rabbits. Behav. Neurosci. 104: 243±252, 1990. This work was supported by National Institutes of Health Grants 1F31 MOYER, J. R., JR., THOMPSON, L. F., AND DISTERHOFT, J. F. Trace eyeblink MH-10826, R01 DA-07633, R01 MH-5734, R01 MH-52409, and R01 AG- conditioning increases CA1 excitability in a transient and learning speci®c 08796. manner. J. Neurosci. 16: 5536±5546, 1996. Present address of J. R. Moyer: Dept. of Psychology, Yale University, SAH,P.AND BEKKERS, J. M. Apical dendritic location of slow afterhyperpo- New Haven, CT 06520. larization current in hippocampal pyramidal neurons: implications for the Address for reprint requests: J. M. Power: Dept. of Cell and Molecular integration of long-term potentiation. J. Neurosci. 16: 4537±4542, 1996. Biology, Northwestern University Medical School, 303 East Chicago Ave., SHARP, P. E., MCNAUGHTON, B. L., AND BARNES, C. A. Enhancement of Chicago, IL 60611-3008. hippocampal ®eld potentials in rats exposed to a novel, complex environ- ment. Brain Res. 339: 361±365, 1985. Received 13 December 1996; accepted in ®nal form 13 March 1997. SKELTON, R. W., SCARTH, A. S., WILKIE, D. M., MILLER, J. J., AND PHILLIPS, A. G. Long-term increases in dentate cell responsivity accompany operant

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