Entorhinal Inputs to Dentate Gyrus Are ActivatedMainly by Conditioned Events With Long TimeIntervals

Arkadiy N. Talnov, Rodrigo Quian Quiroga,Martin Meier, Gen Matsumoto, and Jurij Brankack*

Brainway Group, Brain Science Institute,RIKEN, Saitama, Japan

ABSTRACT: Brain mechanisms of mammalian learning and memoryhave long been associated with the hippocampus. Although the role of thehippocampus in spatial behavior is well established, there is no generalconsensus on the function of the hippocampus in nonspatial tasks. Task-related changes in evoked potentials were first reported by Deadwylerand colleagues in the dentate gyrus, with a peak at 100 ms in rats trainedin an auditory discrimination task (Deadwyler et al., Brain Res 161:211–225, 1979). Surprisingly, these results have not been confirmed by otherinvestigators. In the present report, we set out to assess the task param-eters that induce, modulate, and suppress this potential. Using multielec-trode probes and current source density analysis, we monitored theentorhinal input to the dentate gyrus in behaving rats. Both differentialand simple auditory conditioning led to the appearance of a large negativepotential at 100 ms in the perforant path zone of the dentate gyrus. Thisnegativity was found in averaged and in single-trial, evoked potentials.Current source density analysis revealed sinks in the perforant path zoneof the molecular layer of the dentate gyrus with corresponding sources inthe hilus. Once trained for differential conditioning, decrements of targetprobability consistently increased the amplitude of this negativity. Whenusing a single-tone, stimulus-response task, the negative potential oc-curred with long (70-s), but not with short (10-s) intertrial intervals.Appearance of the potential coincided with a switch in response strategyand disappeared under stereotypical behavior. These data are best ex-plained under the assumption that long intertrial intervals lead to transi-tions in task-related reference frames enabling a switch to more appro-priate response strategies. Such transitions are not required during shortinterval stereotyped performance. © 2003 Wiley-Liss, Inc.

KEY WORDS: freely behaving rat; auditory differential conditioning;perforant pathway; current source density; intertrial time interval

INTRODUCTION

Eye-blink conditioning with nonoverlapping time de-lays between the conditioned stimulus (a tone) and theunconditioned stimulus (an air puff directed to the ani-mal’s eye), so-called trace conditioning, depends on anintact hippocampus (Moyer et al., 1990; Steinmetz,2000; Beylin et al., 2001). Classical fear conditioning(McEchron et al., 1998) and conditioned heart rate re-sponding (McEchron et al., 2000) also are hippocampal-dependent when a trace interval is introduced. The trialspacing effect (Walker et al., 1972; Barnet et al., 1995),according to which animals learn tasks with less trainingwhen long intertrial intervals (ITI) are used, is absent inhippocampal-lesioned rats (Walker et al., 1972; Chan etal., 2001). One explanation for this result is that distract-ing events cause interference with conditioning duringlong ITIs compared with shorter ITIs (Walker et al.,1972) and that the hippocampus is involved in “tuning-out” interfering contextual stimuli (Moore, 1979; Han etal., 1998).

It has been argued that hippocampal damage impairsthe ability to alter response strategies efficiently (Day etal., 1999). Switching to a different response strategy cer-tainly leads to resets and transitions of the animal’s inter-nal representations of its environment, so-called “refer-ence frames,” which are believed to occur in theentorhinal cortex or subiculum (Gothard et al., 2001),areas that could be considered to be afferent to the den-tate gyrus.

During the late 1970s, Deadwyler et al. (1979a,b) de-scribed an evoked potential with a peak latency of 100 msin the perforant path zone of the dentate gyrus, whichappeared with the acquisition of an auditory discrimina-tion task; this potential was absent in naive rats. At thetime, it was thought that this tone-evoked potentialemerged during task learning as a result of long-termpotentiation (LTP) of perforant path synapses (Dead-wyler et al., 1979a,b; West et al., 1982). In an earlier

Grant sponsor: RIKEN Brain Science Institute.Rodrigo Quian Quiroga is currently at the Sloan-Swartz Center for Theo-retical Neurobiology, Division of Biology, California Institute of Technol-ogy, Pasadena, CA.Martin Meier is currently at the Brain Research Institute, University ofBremen, Bremen, Germany.*Correspondence to: Jurij Brankack, Brainway Group, Brain Science Insti-tute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.E-mail: jurij@brainway.riken.go.jpAccepted for publication 25 October 2002DOI 10.1002/hipo.10126

HIPPOCAMPUS 13:755–765 (2003)

© 2003 WILEY-LISS, INC.

study in untrained rats, we demonstrated sensory evoked potentialsin the molecular layer of dentate gyrus, with a peak latency as earlyas 27 ms (Brankack and Buzsaki, 1986). Those sensory evokedpotentials varied with ongoing behavior, but they did not dependon task learning (Buzsaki et al., 1979; Brankack and Buzsaki,1986). We therefore assumed that the training-related potential at100 ms does not represent a potentiated sensory activation of den-tate gyrus, but rather corresponds to an “endogenous” or task-related potential signaling activation of the entorhinal input todentate gyrus.

The aim of the present experiment was to determine (1) whetherthe dentate gyrus potential at 100 ms would also be present insingle, raw trials; and (2) whether it would depend on temporaltask parameters, such as target probability, reward contingency,and ITI, dependence of which would be typical for endogenouspotentials. Based on the data presented in this report, we hypoth-esize that the training-related dentate potential at 100 ms repre-sents the activation of entorhinal input to the dentate gyrus, whichsignals a reference frame transition in relation to a nonstereotypedreward-related response. Moreover, we hypothesize that such ref-erence frame transitions occur only with long ITIs. In contrast,short ITIs induce stereotyped learned responses that require noreference frame transitions. However, both response strategieshave to be learned by the animal, and the animal may attempt bothstrategies. Some of these results have been presented in preliminaryform (Brankack et al., 2001; Brankatschk et al., 2001; Brankack,2002).

MATERIALS AND METHODS

Animal Care

This study was carried out in accordance with the U.S. NationalInstitutes of Health Guide for the Care and Use of LaboratoryAnimals (1996) and has been approved by RIKEN’s Animal Ex-periment Supervisory Panel. Long Evans rats were maintained on a12/12-h light/dark cycle with free access to food. Water was avail-able until behavioral training began, after which the rats receivedwater as reinforcement in the testing apparatus and for 15 min aftereach training session.

Animal Preparation and Histology

Twenty male Long Evans rats (300–400 g) were anesthetizedwith equithesin (3 ml/kg i.p.) and placed in a stereotactic appara-tus. In this study, 8- and 16-channel electrode probes (Barna et al.,1981; Jellema et al., 1991) were custom made from 25-�m, var-nish-insulated, stainless-steel wires. Either of these multichannelelectrode probes or conventional pairs of 80-�m varnish-insulated,stainless-steel wires were permanently implanted in the dentategyrus (4.2 mm posterior bregma, 2 mm lateral to the midline, 2.5and 3 mm ventral to bregma), perpendicular to the cell layers(Paxinos and Watson, 1998). Stimulation electrodes (pair of80-�m varnish-insulated nichrome wires, 0.5-mm tip exposure,

1.0-mm tip separation) were placed in the angular bundle (8 mmposterior to bregma, 5.7 mm lateral to the midline, 5 mm ventralto bregma), to stimulate fibers of the perforant path. Pairs of stain-less steel watch screws were fixed permanently into the skull overthe cerebellum and served as ground and reference electrodes. Toavoid the well-known phase and amplitude differences of thetarhythm recordings resulting from minor depth variations of intra-hippocampal electrodes, the theta rhythm was recorded from ascrew electrode placed over the anterior occipital cortex (areaV2ML: �5.0 mm posterior, 3 mm lateral). This cortical area over-laying the dorsal hippocampus in rat permits reliable recordings oftheta rhythm with theta amplitudes and phases comparable amongdifferent animals. After the conclusion of the experiments, theanimals were deeply anesthetized and perfused transcardially with0.9% physiological saline and 10% formalin. Brains were carefullydissected, and coronal frozen sections were cut (50 �m), mounted,and stained with cresyl violet. The position of the electrodes andmultichannel electrode probes was verified by light microscopy inthe Nissl-stained sections. Figure 7 displays Nissl-stained sectionsfor the animals with multielectrode probes shown in Figures 3, 4(rat 36), 5 (rat 37), and 6 (rat 41).

Behavioral Task and Electrophysiology

One week after surgery, animals were mildly water deprived (23h, �90% body weight), after which both behavioral training andelectroencephalographic (EEG) recording began simultaneously.After each training session, the rats were allowed free access towater for 15 min. The recording and training chamber consisted ofa box (40 cm � 40 cm � 40 cm) with a round opening in theceiling for the recording cables and a glass front door. The record-ing apparatus allowed the animals to freely move about the cham-ber. Anatomical landmarks were identified at the beginning andwere regularly verified throughout the task training (Brankack etal. 1993). Using perforant path stimulations and clicks (Brankackand Buzsaki, 1986), verification took place twice a week eitherbefore or after training sessions. For perforant path stimulation,100 single pulses (pulse duration, 0.1 ms, 150% of threshold;interstimulus interval, 3–9 s) were used. Several hundred clicks(0.1 ms, 65 dB, interstimulus interval: 3–9 s) were delivered sepa-rately. Trials with artifacts, high-voltage spindles, or sleep were notused for averaging perforant path evoked potentials. For averagingclicks, only trials during immobile wakefulness were used(Brankack and Buzsaki, 1986). Clicks were identical during theentire experiment and were never used in combination with waterreward. No difference was found among averages before training,during early training or at late training.

One group of rats (N � 16) was trained to perform a go/no-goauditory differential conditioning task. Tones of 50-ms durationwere presented, each having a 5-ms rise and 5-ms decay time andan intensity of 84 dB. Infrequent target (CS�: 8 kHz, 25%) andfrequent nontarget (CS�: 4 kHz, 75%) tones were presentedpseudo-randomly by a loudspeaker in the rear upper right corner ofthe chamber. The interstimulus interval varied pseudo-randomlyat 9–20 s, with an average across 16 animals of 12.58 � 1.83 s(range, 11.02–17.36 s). After presentation of a target tone, the

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animal’s task was to walk to the drinking tube that protruded fromthe left wall to receive a water reward. Only responses made within6 s after target presentation were rewarded with a few drops ofwater. A light beam just above the drinking tube was interruptedby the rat’s nose approaching the drinking tube. Beam interruptionwas detected by a computer, which triggered the release of thewater reward as well as suspending tone presentation. The rat’sresponse to nontarget tones was neither rewarded nor punished.On average, the training proceeded for 50 sessions, with each ses-sion containing an average of 265 trials (range, 215–317). One ortwo training sessions were run per day. All sessions were used. Forgroup statistics, the number of used trials and sessions was normal-ized among animals.

Another group of rats (N � 4) was trained to a simple condi-tioning task rewarding appropriate responses to a single tone (CS�:8 kHz). One-half of the animals began training with long ITIs(60–120 s); the remaining rats began training with short ITIs(9–12 s). After task acquisition, the ITI durations were reversed foreach group. All remaining task parameters were the same as in thego/no-go auditory differential conditioning task.

EEG epochs of 6-s duration, including a 1.5-s period beforetone onset, were recorded in the freely behaving animals. Record-ings were filtered (high-pass: 0.1 Hz and 1 Hz, low-pass: 1 kHz),digitized (2 kHz sampling rate), sorted according to the stimulustype and animal’s response, and stored on hard disk for offlineanalysis. Response time corresponds to the time difference betweenthe end of the tone and the interruption of the light beam by therat’s nose. Response rates corresponded to the number of targetsresponded to divided by the total number of targets presented.Ignorance rates were calculated as equaling the number of ignorednontarget (CS�) tones divided by total number of nontarget(CS�) tones presented.

Data Analysis

EEG epochs from all trials were visually inspected. Only arti-fact-free trials, those without spike and waves, mechanically in-duced noise transients, and trials void of any signs of sleep ordrowsiness, were accepted for further analysis. For averages acrossanimals, the first and final 100 trials of each behavioral responsetype were averaged for each animal. Single-trial analyses were per-formed with a denoising implementation (Quian Quiroga, 2000).This procedure diminished the contribution of the backgroundEEG significantly. Briefly, the method consisted of the followingsteps: (1) time-frequency components of average evoked potentialwere decomposed using multiresolution decomposition based onthe wavelet transform; (2) wavelet coefficients corresponding intime and frequency to components of the average evoked potentialwere identified, with some additional coefficients also considered,to allow for latency variations; and (3) single-trial potentials weredecomposed and then reconstructed from only those coefficientschosen in the previous step.

Continuous wavelet transformation using the Morlet motherwavelet was used to estimate in single trials the power within afrequency range of 4–12 Hz, for a period of 1 s before tone onset(�1.05 and �0.05 s).

Current source density (CSD) analysis was calculated fromnearest-neighbor electrodes (Nicholson and Freeman, 1975;Mitzdorf, 1985). (For further details of the CSD calculation, seeMitzdorf, 1985, and Brankack et al., 1993.) The actual electrodedistances were measured between adjacent electrode probe wires(center to center) with an accuracy of �5 �m, using precisionmicroscales from Fine Science Tools (Cat. No. 29025-02). Theresulting electrode distances and an impedance value of 300 �-cm(Ranck, 1963) were used to compute the CSD. The average elec-trode distance was 215 � 14 �m (range: 110–280 �m). Thefollowing electrode distances were used in Figures 3–6: (1) Figure3: 200, 250, 180, 240, 120, 220, 180 �m; (2) Figure 4: 250, 180�m; (3) Figure 5: 180, 110 �m; and (4) Figure 6: 200, 270 �m.

For statistical analysis, the medians, means, and standard errorswere calculated. Only animals with conventional electrodes lo-cated at the molecular layer of dentate gyrus were used for groupstatistics in Figures 1 and 2B. Statistical differences among groupswere examined with the Kruskal-Wallis one-way analysis of vari-ance (ANOVA) on ranks; pairwise multiple comparisons were per-formed using Dunn’s method. Comparisons between two groupswere performed using the Mann-Whitney rank-sum test. For cor-relation analysis, the nonparametric Spearman rank-order test wasused.

RESULTS

Differential Conditioning

Mildly water-deprived rats with chronically implanted elec-trodes in the dentate gyrus were trained to perform a go/no-goauditory differential (CS�, CS�) conditioning task. On average,the training proceeded for 50 sessions, with each session contain-ing an average of 265 trials (range, 215–317). One or two trainingsessions were run per day. Reference to trial number in the follow-ing discussion is to artifact-free trials; the corresponding total trialnumber is given in parentheses.

Dentate Gyrus Averaged Potentials

Figure 1 presents grand averages and standard errors of fieldpotential traces recorded in the molecular layer of the dentate gyrusof 7 rats. The left column of traces shows the averages for the first100 trials at the beginning of training (naive), and the right columnshows the averages for the final 100 trials at the end of training(trained). Large-amplitude dentate gyrus potentials with peak la-tencies of 90–100 ms occurred only in trained rats and only duringtrials with correct target responses (CS�, black curves). Ignoredtargets (CS�, gray curves) evoked long-latency (205 ms) potentialswith significantly smaller amplitudes (P 0.01). To examine ef-fect reliability, here, and in all other statistical comparisons, theamplitudes of the averaged potentials of the two groups were sub-jected to the Mann-Whitney rank-sum test. The potentials evokedby nontarget tones (CS�) were significantly smaller (P 0.01) andoccurred 10–30 ms later than those to target tones (CS�). The

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former followed the same trend between naive and trained animalsand between trials with runs to the water spout and trials withignored tones compared.

Dentate Gyrus Single-Trial Potentials

The question arose as to whether the averaged field potentialsemerged with task learning as a result of averaging the ongoingEEG or whether the evoked responses would also be apparent insingle trials. To examine this issue, we next used a recently devel-oped method for visualizing single-trial evoked responses (QuianQuiroga, 2000). Figure 2A shows a color-coded plot of the first500 target-evoked dentate potentials of one representative animalafter wavelet denoising (Quian Quiroga, 2000). Field potentialswith a peak at 90–100 ms after tone termination appeared in thedentate gyrus outer molecular layer during task learning only dur-ing trials in which the rat responded correctly to the target tone,never during trials in which the rat ignored targets or ignorednontarget tones. In 7 of 7 rats, amplitudes were significantly larger(P 0.01) in trials with correct target responses (Fig. 2B, red solidline) compared with either the baseline EEG amplitude (Fig. 2B,red dotted line) or amplitudes during trials with error runs afternontarget tones (Fig. 2B, blue solid line). In 5 of 7 rats, amplitudesduring trials with error runs after nontarget tones were significantlylarger (P 0.05) compared with the baseline EEG amplitude (Fig.2B, blue dotted line). Dentate gyrus field potentials larger than the

FIGURE 1. Grand averages (solid lines) and standard errors (dot-ted lines) of tone-evoked potentials recorded from the outer molecu-lar layer of the dentate gyrus in seven rats during differential condi-tioning. Top row shows field potentials evoked by target (CS�) tones;bottom row shows field potentials evoked by nontarget (CS�) tones.Plots on the left are averages of the first 100 trials in each of the sevenrats at the beginning of training (Naive). Plots on the right are aver-ages of the final 100 trials in the same seven animals at the conclusionof training (Trained). Only responses to targets (upper row, blackcurves) were rewarded. Black curves show grand averages of trialswith visits to the water spout; gray curves show grand averages of trialsfor ignored tones. Numbers indicate the time (in ms) of the peakamplitudes.

FIGURE 2. Emergence of dentate gyrus training-related poten-tials in single trials during task acquisition. A: Color-coded plot ofsingle potential amplitudes as a function of time shows the first 500denoised trials of one representative animal. Progressively more redrepresents more positive amplitudes, and progressively more bluerepresents more negative amplitudes. A denoising procedure (QuianQuiroga, 2000) was used to permit component detection and ampli-tude measurement in single trials. Only trials with correct responsesto targets were used. A negative potential occurring 95–100 ms aftertarget tone presentation (red rectangle) appears at trial 200, stabiliz-ing at trial 300. The dark blue plot on the right shows theta (4–12 Hz)power during the 1 s preceding target presentation. The vertical reddashed line corresponds to a theta power of 20 mV2; the red ticks onthe top of the plot are equal to 10 mV2 and 30 mV2. B: Grand averagesof evoked potential amplitudes and task performance among sevenrats. Amplitude: dentate gyrus potential amplitudes measured in sin-gle trials and averaged among seven animals. Solid lines show ampli-tudes at 100 ms after correctly responded targets (red) and after non-target (CS�) tones leading to response errors (blue) compared withbaseline EEG (corresponding red and blue dotted lines) recorded 100ms before tone onset. Task performance: grand averages of responserates (number of responded targets divided by total number of targetspresented) and ignorance rates (number of ignored nontarget [CS�]tones divided by total number of nontarget [CS�] tones presented).Note that response rate for targets (red line) and ignorance rate fornontarget (CS�) tones (blue line) both increased after trial 300. Re-sponse time: grand averages of response time to targets (red) and errorresponse time to nontarget (CS�) tones (blue) in seconds. Number ofruns: grand averages of number of runs per target (red) and number oferror runs per nontarget (CS�) tone (blue). Both response time andnumber of runs decreased between trial 200 and 300 to stable lowlevels, correlating strongly with the appearance of dentate potentials.All curves shown in B were smoothed using a moving average (n � 50,step: 1).

FIGURE 3. Current source density (CSD) analysis and identifi-cation of anatomical landmarks. The left part of Figure 7 and thedrawing on the right of Figure 3B show the anatomical position of theelectrode probe at 4.52–4.80 mm posterior and 1.8–1.9 mm lateral(Paxinos and Watson, 1998). The 3rd electrode position with elec-trode distances to the nearest neighbors of 250 and 180 �m wasmarked. The electrode probe protruded 480 �m of the dorsalblade’s molecular layer. The estimated distance between granule celllayers of dorsal and ventral was 360 �m, and the thickness of theventral blade’s molecular layer was 300 �m. With the electrode dis-tances (see also depth on the ordinate) from 120–250 �m the depthresolution of the electrode probe did not allow differentiation be-tween molecular sublayers and the medial/lateral perforant pathwayinputs. Color-coded contour maps of CSD as a function of time anddepth for one representative animal are shown. Depth equals thedistance to the center of the uppermost electrode wire of the probe. A:Submaximal electrical stimulation (0.1 ms) of the medial perforantpathway (at 0 ms) showed a pair of current sinks (blue-green colors) inthe middle molecular layer of the dorsal and ventral blades of thedentate gyrus; the corresponding current source(s) (red-brown colors)originated in the hilus. B: Auditory clicks (0.2 ms duration, 82 dBintensity) evoked a pair of sinks starting at 27 ms in the outer molec-ular layer of the dorsal and ventral blades, with sources in the hilus. C:Auditory tones (8 kHz, 50-ms duration, 84-dB intensity with slowlyrising and falling intensity flanks) in naive animals did not generateany prominent sinks or sources in the dentate gyrus. D: The same toneused as a target (25%) in an auditory differential conditioning taskduring training session 14 evoked a pair of strong sinks starting at 110ms in the outer molecular layer of the dentate gyrus. The positions ofdorsal and ventral granule cell layers are indicated by rows of fivesmall circles; mol: molecular layer, hil: hilus. The red asterisk in Dshows the channel used for the same animal in Figure 4 and based onthe anatomical landmarks corresponds to the recording in Figs. 5 and6 in two different animals.

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FIGURE 2

FIGURE 3

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baseline EEG appeared in single trials as early as the 100th targettrial (105th), and in one animal as late as the 1,500th target trial(1764th). In 7 rats, the median trial of first occurrence was 350target trials (404).

The negative potential in the molecular layer of the dentategyrus appeared with task learning as a well-defined negative peak inindividual raw trials, and not as a result of EEG averaging. Theemergence of these large dentate gyrus potentials was strongly cor-related with the animal’s response behavior. Specifically, with (1)the drop of the number of the animal’s visits to the water spout persecond to stable low levels (r � 0.967, P 0.001, N � 10), andwith (2) reaching 60% ignorance rate of nontargets (r � 0.833,P 0.01, N � 8). The emergence of those potentials, however,did not correlate with the rat’s performance to target tones indi-cated by reaching 80% of correct responses to targets (see also redlines in Fig. 2B).

In order to examine a possible behavioral effect on the amplitudeof the negative potentials at the molecular layer, the pre-stimulus(1-s) theta (4–12-Hz) power and the amplitudes of the last 500single trials of seven well trained animals were correlated. None ofthe seven animals demonstrated a correlation. In addition, the first500 single trials during early training were analyzed. The plot onthe right of Figure 2A shows the raw pre-stimulus theta power ofthe first 500 single trials. The vertical red dashed line correspondsto 20 mV2 and the red tick mark to 10 mV2 and 30 mV2. Therewas no correlation between pre-stimulus theta power and negativepotential at 100 ms in this and another five animals. Only one ofseven animals showed a correlation (inverse) between the ampli-tude of the negative potential and the pre-stimulus theta power forthe first 500 single trials. No correlation was found for the last 500single trials in the same animal. The inverse correlation for the first500 trials in this animal may result from correlations of opposingpolarity for the potential amplitude and the pre-stimulus thetapower both with the trial number. Theta power for the animalswith multielectrode probes and CSD analysis shown in the plots ofFigures 4A, 5A, and 6A was calculated from all single trials and incorrespondence with the CSD smoothed with a moving average.None of the rats revealed a correlation between CSD magnitudesand pre-stimulus theta power.

Current Source Density Analysis andIdentification of Anatomical Landmarks

Using multielectrode probes implanted perpendicular to thegranule cell layer, CSD was calculated from extracellular field po-tentials (see Materials and Methods; for details, see Brankack et al.,1993). Briefly, CSD estimation permits the identification of grosstransmembrane currents (sinks and sources) that generate extracel-lular potentials (Nicholson and Freeman, 1975). The anatomicalposition of the multielectrode probe within the dorsal hippocam-pus and dentate gyrus was verified histologically (see left part ofFig. 7; see also drawings in Figs. 3, 4, 5, and 6 based on Paxinos andWatson, 1998). The distances between electrodes (average dis-tance 215 � 14 �m, range 110–280 �m; see Materials and Meth-ods, for further details) did not allow further differentiations ofsublayers at the dentate molecular layer. Figure 3 shows color-

coded contour plots of current sinks (green-blue colors) andsources (brown-red colors), with yellow coding the low-magnitudeCSD activity. Plots for three conditions are shown for one repre-sentative animal after (1) electrical stimulation of the perforantpath (Fig. 3A), (2) auditory click stimulation (Fig. 3B), and (3)8-kHz tone stimulation, both, before training (Fig. 3C) and ontraining session 14 (Fig. 3D), respectively. Electrical stimulation ofthe perforant path (Fig. 3A) and auditory click stimulation (Fig.3B) were used to identify the anatomical landmarks around themultichannel electrode probe in the dentate gyrus. The contribu-tion of synaptic potentials to perforant path-evoked responses inthe dentate gyrus have been well described previously (Andersen etal., 1966; Lomo, 1971). The similarity between current sinks andsources evoked by perforant path stimulation and those activatedby auditory clicks was reported earlier (Brankack and Buzsaki,1986). Both types of stimulation were repeated periodically to testthe electrode probe stability and the preservation of the surround-ing tissue. Typically, the position and magnitude of the anatomicallandmarks were stable for more than 60 days. Tone stimulation (8kHz) caused no changes in CSD activity (Fig. 3C) before training.With advancing task training, the same tone used as the targetstimulus induced prominent sinks at 100 ms at the perforant pathzones of the dorsal and ventral blades of the dentate gyrus andcorresponding sources at the hilus (Fig. 3D). In the following, weinvestigated in trained animals correlations between various taskparameters and the magnitude of the current sink in the molecularlayer of the dorsal blade of dentate gyrus (asterisk in Fig. 3D).

Target Probability

Next, we investigated the effect of target probability on themagnitude of the dentate gyrus sink (see asterisk, Fig. 3D), after itbecame established with task acquisition. Figure 4A shows a color-coded plot of target-evoked dentate gyrus CSD in one animalduring acquisition of a go/no-go auditory differential (CS�, CS�)conditioning task, the same task used for detecting the dentatepotential in single trials described above. A prominent dentategyrus sink (shades of green and blue colors in Fig. 4A) appearedwithin the first 500 trials signaling the activation of the entorhinalinput. Subsequent manipulations of the target probability (aftertrial 1200) led to corresponding significant changes of the CSD(P 0.01), without an effect on target response rates (Fig. 4C, redcurve). Extreme increases of target probability (Fig. 4A,B, meantarget percentage: 87 � 5%) also led to a decrease in the animal’signorance rate of nontargets (Fig. 4C, top of blue curve). Lowtarget probabilities caused an activation of the entorhinal input,whereas high target probabilities led to a weaker entorhinal input.No correlation was found between magnitudes of the CSD andpre-stimulus theta power.

Simple (Single-Tone) Conditioning and theEffect of Intertrial Interval

We then examined whether changing intertrial intervals in asingle-tone stimulus-response task would affect the magnitude ofthe training-related dentate gyrus current sink. In the previousexperiment, the mean ITI between targets with probability of 0.25

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was 70 s. Using a mean of 70 s for the pseudo-random stimulusinterval, we trained naive rats to respond to a single rewarded tone.A dentate gyrus current sink emerged within the first 100 trials(Fig. 5A) in the dentate gyrus molecular layer (see asterisk, Fig. 3D)signaling activation of the entorhinal input. For the electrodeprobe position of this animal, see the middle of Figure 7 and thedrawing at the top of Figure 5A. Decreasing the mean intertrialinterval from 70 s to 10 s (trials 414–1000) gradually, but signif-icantly, diminished (P 0.01) the magnitude of the current sinkbelow original levels. This manipulation also increased the numberof runs to the water spout per second (Fig. 5C, blue curve), withouteffecting the target response rate (Fig. 5C, red curve). Increasingthe mean ITI to 80 s (after trial 1000) led to the recovery of thecurrent sink. Target probability did not change in this experiment.

Only the decrease of the intertrial time interval diminished themagnitude of the entorhinal input to the dentate gyrus. At thesame time, the number of visits to the water spout per secondincreased significantly (P 0.01). The data for the second animalwere consistent with those for the rat shown in Figure 5. Nocorrelation was found between magnitudes of the CSD and pre-stimulus theta power.

That dentate gyrus potential amplitudes depended on ITI wasstatistically evaluated in seven animals performing a go/no-go au-ditory differential (CS�, CS�) conditioning task. We consideredonly those target trials that immediately followed a target, andthese were sorted according to ITI. ITIs were normalized with 10 sset to 100% and were grouped in steps of 5% increments/decre-ments. Dentate gyrus potential amplitudes were also normalized

FIGURE 4. Manipulations of target probability change the mag-nitude of the dentate gyrus potential. A: The left part of Figure 7 andthe drawing on the top of Figure 4A show the anatomical position ofthe electrode probe at 4.52–4.80 mm posterior and 1.8–1.9 mmlateral (Paxinos and Watson, 1998). The 3rd electrode position withelectrode distances to the nearest neighbors of 250 and 180 �m wasmarked. Color-coded contour plot of current source density (CSD)from one location in the outer molecular layer of the dentate gyrus ofa representative animal (Fig. 3D, asterisk) is displayed. Note the emer-gence of the current sink at 100 ms with task acquisition during thefirst 500 trials. Mean target percentages (�SE) are shown in red at leftof plot. Only trials in which the animal responded correctly to targetswere used. For clarity, low-magnitude CSD from �0.3–0.3 mA/cm3

is shown homogeneously in yellow. Increasing target percentage to

57% (trials 1420–1700) diminished the dentate sink significantly(P < 0.001). Lowering target percentage to 24% (trials 1701–2194)increased the dentate sink significantly (P < 0.001). The dark blueplot on the right shows theta (4–12 Hz) power during the 1 s preced-ing target presentation. The vertical red dashed line corresponds to atheta power of 15 mV2, the red ticks on the top of the plot equal to 10mV2 and 20 mV2. B. Target percentage is shown. C: Correspondingresponse rates for targets (red curve) and ignorance rates for nontarget(CS�) tones (blue curve) are shown. CSD and all curves weresmoothed with a moving average (n � 100; n � 30 in B). Please notethat an increase in target percentage to 87% in trained rats (top of Aand B) led to an decrease of ignorance rate below 0.5 (C, top of bluecurve).

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such that values corresponding to 10 s ITI were set to100% andmeans of larger and shorter ITIs were calculated accordingly. Av-eraged across seven animals dentate potential amplitudes corre-lated significantly with ITI (r � 0.879, P 0.001). There was amain effect of ITI on dentate gyrus pontential amplitudes (H �18.191, P 0.05) with ITI differences �20% inducing reliablechanges in dentate gyrus potential amplitudes (P 0.05).

Initial Training With Short Intertrial Intervals

In the previous experiment, rats were initially trained with long(e.g., 70 s) ITIs; later, the ITI was modified. We trained animalsfirst with short (e.g., 9–12-s) ITIs. Figure 6A illustrates the dentateCSD at 100 ms in one representative animal. For the electrodeprobe position of this animal, see the right part of Figure 7 and thedrawing at the top of Figure 6D. After completing the experimentin this animal the electrode positions at the pyramidal cell layer and

at the granule cell layer of the ventral blade were marked by electriccoagulations.

Both rats acquired the task (80% corrected responses) within thefirst 100 (400) trials (see the representative data for one animal in Fig.6D). For this animal, the short ITI forced the animal to emit a stereo-typed response with a correspondingly high number of visits to thewater spout per second (Fig. 6C) and a somewhat less stable responserate (Fig. 6D). Even after more than 480 (1727) trials, no current sinkemerged in the dentate gyrus molecular layer (Fig. 6A). Increasing theITI to 60–110 s (Fig. 6B) at trial 484 (1747) led to an improvementin the stability of the response rate, and at trial 633 (2048) the numberof visits to the water spout dropped significantly to stable, low levels.Almost instantaneously at trial 635 (2056) a strong sink emerged inthe dentate gyrus molecular layer signaling an activation of the ento-rhinal input. No correlation was found between magnitudes of theCSD and pre-stimulus theta power.

FIGURE 5. Altering magnitudes of the dentate gyrus sink bychanging stimulus intertrial intervals in a single tone task. A: Thedrawing at the top (and middle of Fig. 7) show the anatomical posi-tion of the electrode probe at 4.16–4.30 mm posterior and 1.8 mmlateral (Paxinos and Watson, 1998). The 4th electrode position withelectrode distances to the nearest neighbors of 180 and 110 �m wasmarked. Color-coded map of target-evoked current source density(CSD) calculated from three recordings in the outer molecular layerof the dentate gyrus. Note a current sink at 100 ms after targetappears within the first 50–100 trials. Decreasing the intertrial inter-

vals (ITI) (trials 414–1000) from 60–10 s slowly diminished the sink.Returning stimulus intervals back to 60–70 s slowly restored the sink.The dark blue plot on the left shows theta (4–12 Hz) power duringthe 1 s preceding target presentation. The vertical red dashed linecorresponds to a theta power of 10 mV2, the red ticks on the top of theplot equal to 5 mV2 and 15 mV2. B: Intertrial interval. C: Correspond-ing response rate to targets (red curve) and number of visits to thewater spout (blue curve). CSD and all curves were smoothed with amoving average (n � 100).

762 TALNOV ET AL.

DISCUSSION

The present results demonstrate that (1) strong and early (90–100-ms) activation of the dentate gyrus via entorhinal inputsemerged during task acquisition with correct responses to targets,both in averaged and in single trial responses; (2) ignoring targetsled to much later (210-ms) and weaker dentate gyrus activation intrained but not in naive rats; (3) no correlation was found betweendentate gyrus activation at 100 ms and pre-stimulus theta power;(4) in trained animals, strong and early (90–100-ms) dentate gyrusactivation depended on low target probability or long intertrialintervals, both resulting in less visits to the reward location; and (5)initial training with short ITIs did not cause activation of theentorhinal input to the dentate gyrus, even after more than 2,500tones had been presented. This is the case even though the animalsdid acquire the task.

In an earlier report by Deadwyler et al. (1979b), tone-evokedpotentials in the perforant path zone of the outer molecular layer ofthe dentate gyrus were shown to appear with the acquisition of

operant tone discrimination. At that time, it was considered, how-ever, that those potentials with peak latencies at 100 ms repre-sented sensory evoked potentials “potentiated” via an LTP mech-anism (Deadwyler et al., 1979a,b; West et al., 1982). In a previousreport, we demonstrated click and tooth pulp evoked potentialsoccurring much earlier at 27 ms in the same perforant path zone ofthe molecular layer of the dentate gyrus. Those evoked potentialsvaried with ongoing behavior, but they did not depend on tasklearning (Buzsaki et al., 1979; Brankack and Buzsaki, 1986). Bothclicks and tooth pulp stimulations evoked short-latency (27-ms)responses at the hippocampus, but both also caused startle re-sponses and head jerks (Brankack and Buzsaki, 1986), which didnot occur in our experiments with slowly rising beeps. Further-more, we have no data that show that early responses appeared withtask training. One explanation would be that the short-latency (27ms) response and its motor part may be linked to innate circuitriesrelated to possibly dangerous signals that may require immediateescape responses. Clicks and slowly rising beeps appear to activatedistinctive neuronal circuitries at the auditory cortex (Y. Yanovsky,unpublished observations). The slow rising beep may not activate

FIGURE 6. Effect of manipulating the intertrial intervals (ITI) ina single-tone task, initially training with short ITIs (9–12 s). A: Thedrawing at top of D (and right of Fig. 7) show the anatomical positionof the electrode probe at 4.5 mm posterior and 2.2 mm lateral (Paxi-nos and Watson, 1998), protruding the dentate gyrus slightly underoblique angle to lateral. The 4th electrode position with electrodedistances to the nearest neighbors of 200 and 270 �m was marked.Current source density (CSD) at 100 ms post-target from one locationat the outer molecular layer of the dentate gyrus (equivalent to theasterisk for the animal in Fig. 3). The gray plot on the left shows theta

(4–12 Hz) power during the 1 s preceding target presentation. Thevertical gray dashed line corresponds to a theta power of 10 mV2, thegray ticks on the top of the plot equal to 5 mV2 and 15 mV2. Note thecomplete absence of a dentate sink with short ITIs (9–12 s), lastingfor as long as 483 trials (1744) with correct responses or 2545 totaltone presentations; in contrast, a dentate sink slowly emerges aftertransition to long ITIs (60–110 s) at trial 484 (1747). B: Intertrialinterval. C: Number of runs per second based on the total number ofruns per session divided by total session time. D: Response rate.Curves in B and D were smoothed with a moving average (n � 30).

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those innate circuitries and therefore may not evoke early responsesor motor jerks. Currently, we are working to further elucidate thisquestion.

The present data clearly demonstrate that the learning-relateddentate potential, with a peak latency at 100 ms, depends on taskparameters in a manner similar to other endogenous event-relatedpotentials, such as the P300, a positive wave recorded at the surfaceof the brain or scalp occurring 300 ms after an infrequent, or“oddball” target (for review, see Pritchard, 1981; Picton, 1992). Itis also similar to the mismatch negativity potential (Sato et al.,2000). Moreover, the appearance of the dentate potential coin-cided with both the drop in the number of visits to the water spout,a feature that was also demonstrated in the early report by Dead-wyler et al. (1979b), and the emerging (60%) ignorance of non-target tones. In other terms, the appearance of entorhinal-to-den-tate potentials coincided with the animal’s adjustment of its motorresponses from excessive runs after every tone to more appropriateand “economic” responses restricted to just one visit to the waterspout after reward-related targets. Furthermore, we clearly demon-strated a strong correlation between short ITIs, frequent visits tothe water spout and the absence of dentate potentials and con-versely with long ITIs, infrequent runs and large amplitude dentatepotentials in well trained or even overtrained rats. We also dem-onstrated the permanent absence of dentate potentials in ratstrained with short ITIs, even though the animals acquired the task.

Animals respond in a stereotyped and somewhat excessive mannerduring periods with short intertrial intervals without strong ento-rhinal inputs to the dentate gyrus. In contrast, during periods withlong intertrial intervals, conditioned responses are adequate andpunctuate, correlating with strong entorhinal inputs to the dentategyrus.

One possible explanation, therefore, is a functional relationshipbetween entorhinal inputs to the dentate gyrus and adequate re-sponses or perhaps shifts in response strategies. The ability tochange response strategies depends on the integrity of the hip-pocampus (Day et al., 1999). In terms of recent ideas about hip-pocampal network function, shifts in response strategies woulddefinitely cause transitions in the animal’s reference frames. It wasrecently suggested that reference frame transitions occur in struc-tures “before CA3, possibly in the entorhinal cortex or subiculum”(Gothard et al., 2001). Activation of the entorhinal input to den-tate gyrus may be related to a reference frame transition, triggeredby a target tone only after a long ITI. During short ITI, there maybe no need for reference frame transitions because the animal doesnot change its stereotyped response behavior, focusing its attentioninstead on the task. Long ITIs may exceed the attention span of theanimal, allowing it to get distracted by other activities. The targettone will cause a shift back to the learned task context (or referenceframe). Therefore, an alternate interpretation of our results wouldbe a switch of attention toward task context on long ITIs, which

FIGURE 7. Nissl-stained brain sections showing the multielec-trode probe position for the three animals with current source density(CSD) estimations shown in Figs. 3, 4 (rat 36), 5 (rat 37), and 6 (rat41). The anatomical position of the electrode probe in rat 36 (left) wasat 4.52–4.80 mm posterior bregma and 1.8–1.9 mm lateral to themidline. The electrode probe in rat 37 (middle) was located at 4.16–4.30 mm posterior and 1.8 mm lateral. In rat 41, the electrode probe

protruded the dorsal hippocampus at 4.5 mm posterior and 2.2 mmlateral (Paxinos and Watson, 1998). The brain section in the rightpart was taken from positions 4.3 mm posterior bregma. After com-pleting the experiment in rat 41, the electrode positions correspond-ing to the pyramidal cell layer and to the granule cell layer of theventral blade were marked by electric coagulations.

764 TALNOV ET AL.

may not be needed with short ITIs. Understanding the functionalsignificance, informational meaning and cellular consequences ofstrong entorhinal inputs to the dentate gyrus at long time intervalswill require future well-designed experiments.

Acknowledgments

The authors thank K. Shionoya and Dr. A.I. Pilyavskii for ex-cellent technical assistance; V.I. Kukushka for programming; andDr. R. Tamura and Dr. Y. Yanovsky for valuable comments on anearlier version of the manuscript.

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