HZPPOCAMPUS, VOL. 1, NO. 2, PAGES 181-192, APRIL 1991

Learning-related Patterns of CAl Spike Trains Parallel Stimulation Parameters Optimal for Inducing Hippocampal

Long- term Potentiation Tim Otto,* Howard Eichenbaum,” Sidney I. Wiener,?

and Cynthia G. Wible* *Department of Biological Sciences, Wellesley College, Wellesley, MA 021 81 U.S.A. and tCNRS Laboratorie d e Physiologie Neurosensorielle, 15 Rue d e

L’Ecole de Medicine, 75270, Paris, France

ABSTRACT

Recent studies have revealed 3 stimulation parameters that together comprise the temporal pattern of neuronal activation optimal for the induction of hippocampal LTP: high-frequency bursts, activity 100-200 ms prior to a burst, and burst delivery in phase with the ongoing hip- pocampal theta rhythm. The present paper reports that these 3 aspects of patterned neural ac- tivity, collectively referred to as “theta-bursting,” are characteristic of the spike trains of CAI pyramidal cells in rats during the sampling and analysis of learning cues in an odor discrimination task and during performances of a spatial memory task. In contrast, theta-bursting occurs rela- tively infrequently during behavioral events less directly related to task-relevant mnemonic pro- cessing. These findings suggest that the optimal conditions for the induction of LTP occur nat- urally in behaving animals, time-locked to behavioral events critical to learning.

Key words: theta rhythm, theta-bursting, complex-spike, LTP, memory

Long-term potentiation (LTP), a lasting enhancement of synaptic efficacy induced by tetanic stimulation of afferent fibers, has been demonstrated in many monosynaptic path- ways (Racine et al., 1983). In discussing the possible func- tional significance of LTP, several investigators have cited the many similarities between the induction and maintenance characteristics of LTP and those of behaviorally defined memory (Teyler and DiScenna, 1984; Lynch and Baudry, 1984). These parallels have been pursued most vigorously with regard to LTP in hippocampal afferent and intrahippo- campal pathways for many reasons, most notably the im- portance of this structure to certain types of memory (Squire et al., 1989; Otto and Eichenbaum, 1991). Like memory, LTP in hippocampus can be long-lasting (Staubli and Lynch, 1987), and induced rapidly by brief bouts of activity, and is strengthened by repetition (Barnes, 1979; Teyler and Dis- cenna, 1987). Further, decay of LTP is correlated with the time course of forgetting, as assessed in hippocampus-de- pendent spatial learning tasks (Barnes, 1979). Finally, several recent studies suggest that hippocampal LTP induction and certain forms of hippocampal-dependent memory share a common dependence on activation of the NMDA receptor (Staubli et al., 1988; Morris, 1989; Oliver et al., 1989; Rob-

Correspondence and reprint requests to Tim Otto, Department of Biological Sciences, Wellesley College, Wellesley, MA 02181 U.S.A.

inson et al., 1989; Gilbert and Mack, 1990). Thus accumu- lating evidence implicating LTP in mnemonic processes, par- ticularly those dependent upon hippocampal circuitry, suggests that hippocampal LTP may indeed be a physiological substrate of some forms of memory.

Interest in LTP as a mnemonic device has spawned a con- siderable body of research attempting to characterized its op- timal induction parameters. In the hippocampal slice prepa- ration, LTP in CAI is preferentially induced by brief, high- frequency bursts of afferent stimulation (4 pulses at 100 Hz) repeated at 5-10 Hz (Larson et al., 1986). Moreover, LTP in CA 1 can be induced by a single burst if another burst (Larson and Lynch, 1986 or single pulse (Rose and Dunwiddie, 1986) precedes that burst by 130-200 ms (i.e., at latencies that par- allel frequencies of 5-7 Hz). Similar results are obtained in vivo in CA 1 following patterned stimulation of commissural afferents (Diamond et al., 1988) and in dentate gyrus follow- ing patterned stimulation of the perforant path (Greenstein et al., 1988). This “priming” effect (Larson and Lynch, 1986) suggests that very brief bouts of high-frequency activity can induce LTP given a suitable temporal relationship between successive episodes of activity. Finally, patterned stimula- tion is most effective in inducing LTP in dentate gyrus when delivered at the peak of the dentate theta rhythm (Pavlides et al., 1988). Together, these convergent data suggest that brief episodes of high-frequency stimulation, when applied in the appropriate temporal relationship to prior activity and to

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182 HZPPOCAMPUS VOL. 1, NO. 2, APRIL 1991

the ongoing theta rhythm, can reliably enhance synaptic ef- ficacy in a brain area critical to the formation of certain types of memory.

These characteristics of patterned stimulation are similar to naturally occurring patterns of hippocampal neuronal ac- tivity. First, the high-frequency burst mimics the repetitive discharges characteristic of hippocampal pyramidal cell firing commonly observed in behaving rats. These “complex- spike” discharges consist of 2- 1 1 spikes of progressively de- creasing amplitude with interspike intervals typically be- tween 2 and 6 ms (Ranck, 1973). Second, the optimal delay of 100-200 ms between successive bouts of activation cor- responds to the 5-10 Hz theta rhythm prevalent in the rat hippocampus during exploratory activity (Vandenvolf et al., 1975). Third, the optimal phase of burst stimulation within the theta cycle corresponds to the observation that complex- spike cell activity occurs preferentially near the positive peak of dentate theta in both walking and urethane-anesthetized rats (Fox et al., 1986). Although each of these aspects of temporal patterning has been observed in the cellular activity of waking rats, there is as yet no evidence that any of them, either individually or combined, are characteristic of hippo- campal neuronal activity during mnemonic processing. The present investigation sought to determine the extent to which this combination of patterns, which we will call “theta-burst- ing” (Arai et al., 1990), occurs naturally in the spike trains of hippocampal complex-spike cells during behavioral events associated with learning and memory. To that end, we as- sessed the bursting characteristics of CA I pyramidal neurons in rats during performance of 2 tasks: an odor discrimination task that has previously been demonstrated to engage hip- pocampal slow wave (Macrides et al., 1982) and unit (Wiener et al., 1989) activity, and for which performance is dependent on hippocampal function (Eichenbaum et al., 1988), and a spatial task conceptually similar in memory demands to the radial arm maze (Olton et al., 1979), in which hippocampal cells demonstrate place-related firing (Olton et al., 1978). We now report that theta-bursting is common in complex-spike cells during performance of these tasks and is time-locked to behavioral events associated with mnemonic processing.

MATERIALS A N D METHODS

The present study involves new analyses of data accu- mulated for a previous set of reports on the behavioral cor- relates of hippocampal unit activity (Wiener et al., 1989; Ei- chenbaum et al., 1989). Recording methods, data acquisition, and behavioral training procedures have been described in detail in those reports and will be summarized here only briefly.

Electrodes, surgery, and recording apparatus

All subjects receiving preoperative behavioral training (see below) were given water ad libitum for 48 hours before sur- gery. Rats were tranquilized with acepromazine, anesthetized with pentobarbital, and given atropine to reduce excess sa- livation. Body temperature was maintained using a 39” heat- ing pad. A bipolar electrode suitable for recording slow wave activity was implanted across the CAI stratum oriens and the granule cell layer of dentate gyrus in one hemisphere (dentate

electrode positive), a multiwire electrode for recording single unit activity was mounted in a microdrive assembly and im- planted just above hippocampal area CAI in the contralateral hemisphere, and a cannula, through which a thermocouple could be introduced to monitor sniffing, was implanted into the nasal cavity. The microdrive was advanced slowly over several days until CA1 pyramidal cells could be identified by their waveform and firing characteristics. Amplified and fil- tered (0.3- 10 KHz) cellular activity was routed in parallel to an oscilloscope, tape recorder, and multichannel window dis- criminator, the latter of which enabled the simultaneous iso- lation and discrimination of the action potentials of one or more units. In addition, the rats’ head movements were mon- itored by video-tracking a light bulb affixed to the recording headstage. A DEC microVax computer with custom-designed interface and interrupt software was used to simultaneously control all aspects of the behavioral task, sample the window discriminator at 1 kHz, and digitize the hippocampal EEG and sniffing at 100 Hz and movement at 10 Hz.

All behavioral training and unit recording took place in an electrically isolated behavioral arena (40 x 44 cm) with 50 cm high walls slanted 15” outward. A cul-de-sac located on one wall contained 2 conical sniff ports located 8 cm above the floor. Entry into the cul-de-sac and the sniff ports was monitored by separate photoelectric cells. Water cups were located in each of the 4 corners of the chamber and at the entry to the cul-de-sac. Delivery of all stimuli and reinforcers was controlled by computer. The behavioral arena was en- closed in a sound-attenuating chamber and was illuminated with a 12 V incandescent light.

Following behavioral training and unit recording, subjects were anesthetized deeply with sodium pentobarbital and per- fused intracardially with 10% buffered formalin. The brain was then removed and sectioned coronally at 30 pm, and the relevant sections (through the dorsal hippocampus) were mounted and stained with cresyl violet. lnspection of the sec- tions confirmed the appropriate location of the EEG and mi- croelectrodes.

Behavioral training procedures

Most subjects were trained concurrently on 2 tasks: a si- multaneous odor discrimination task and a spatial memory task. Each will be described in turn.

Olfactory fask

Odor discrimination training was conducted in the record- ing chamber described above. Entry into the cul-de-sac re- sulted in the simultaneous ejection of 2 different odors from adjacent conical sniff ports. A nosepoke into the sniff port emitting the arbitrarily designed S’ odor resulted in the de- livery of a 0.05 ml water reinforcer into a water port located on the floor of the chamber at the entry into the cul-de-sac; nosepokes into the sniff port emitting the S-odor went un- reinforced. The location of S+ and S- odors was varied randomly across trials to render irrelevant the spatial com- ponents of stimulus presentation. Animals were trained until they reached a criterion of 18 correct responses in 20 con- secutive trials on 3-5 successive days.

After training on a single discrimination problem, subjects were implanted with electrodes suitable for unit and EEG

LEARNING-RELATED PATTERNS OF HIPPOCAMPAL SINGLE CELL ACTIVITY Otto et a[ . 183

recording (see above). Following a 7-day postoperative re- covery period, animals performed a concurrent discrimina- tion task involving a random series of trials in which 1 of 2 distinct odor pairs was presented. On sessions in which at least 1 unit with a signal-to-noise ratio of at least 2 : 1 could be isolated, data were collected as the rat was trained for 100-250 trials.

Spatial task

During shaping, water was made available at irregular in- tervals at the 4 water cups located in the corners of the arena. Rats were considered ready for implantation when they ap- proached each of the water cups upon presentation of re- wards. Postsurgical training used positional information from the TV tracker to control trial onset and reward delivery. To initiate a trial, the rat was required to position the light bulb on headstage within a 5 cm radius of the arena center, thereby activating a “beeping” tone. Subsequent approach to within 2.5 cm of any of the 4 water cups was reinforced with the delivery of 0.05 ml water and simultaneously terminated the tone. The rat was then required to return to the center of the arena to restart the tone, and then approach a water cup not yet visited on that trial. Repeat visits to a corner within a trial and visits not preceded by initiating the tone were not rewarded. Completion of a trial (visiting all 4 corners) was followed by a 30-second intertrial interval.

Characterization of behavioral and spatial correlates of unit activity

Olfactory task

Preliminary characterization of the behavioral correlates of units in this task has been described in detail elsewhere (Wie- ner et al., 1989). Briefly, raster displays of unit firing time- locked to specific behavioral events were constructed and analyzed for statistically reliable increases or decreases in firing during relevant trial periods. Many cells exhibited in- creases in firing associated with 1 of 2 events: “goal-ap- proach” cells fired just prior to entry into the cul-de-sac or following the nospoke response as the subject approached the water cup; “cue-sampling’’ cells fired maximally just after odor onset while the subject evaluated the discriminative stimuli and prepared its response. The present study focused on cells of each type for which the peak firing rate was at least twice the average firing rate of that cell.

Spatial task

“Place” correlates of cell activity were determined as de- scribed previously (Wiener et al., 1989). Briefly, the area of the behavioral chamber was divided into an array of 18 x 16 2.5 cm square pixels. The mean firing rate for each pixel was calculated as the average over the recording session for all visits to a 9 pixel area centered on that pixel. A place field was defined as a set of at least 3 adjacent pixels with average firing rates at least 2.33 SEM above the average overall firing rate (1-tailed P < .01). The present study focused on cells with a place field for which the in-field firing rate was at least twice the out-of-field firing rate.

Data analysis

The firing patterns of 24 cue-sampling cells, 18 goal-ap- proach cells, and 23 place cells, recorded from 8 rats and chosen according to the criteria described above, were as- sessed in the present study. Detailed analyses of these cells were conducted in order to assess the extent to which their firing patterns corresponded to the features of patterned stim- ulation optimal for hippocampal LTP induction. Thus the analyses address 3 primary questions: first, whether these neurons do indeed “burst” time-locked to specific behavioral events; second, whether bursts are phase-locked to the on- going hippocampal theta rhythm; and third, whether bursts are preceded by neural activity preferentially at about 1 theta cycle, that is, whether they are “primed.”

Bursts were defined as a series of 2 or more spikes in which each interspike interval was not more than 10 ms. Histograms of interspike intervals were generated to characterize the pre- dominant firing rate within identified bursts. For cue-sam- pling and goal approach cells, raster displays and histograms indicating the occurrence of spike and burst activity time- locked to either the onset of odor sampling or the discrimi- native response were generated to permit comparison of the behavioral correlates of bursts with that of overall spike ac- tivity. In some cases, spike and burst activity was also time- locked to the peak of the ongoing theta cycle to visualize phase-related firing. For cells recorded in the spatial task, the rate of bursting for each pixel was plotted and maps of burst- ing were compared to the place field map.

Subsequent analyses focused on the period of maximal burst activity (odor task) or bursting within the place field (place task). The phase of dentate theta at which bursts oc- curred preferentially was determined using the method of Ku- perstein et al. (1986). Briefly, the theta phase of each burst was determined by computing the relative position of the first spike of the burst between 2 peaks of hippocampal theta (den- tate electrode positive). Phases were accumulated in 22.5” bins and the cumulative histogram fit to a series of sine waves using the least squares method to determine the preferred theta phase. The statistical significance of the Pearson cor- relation between the best-fitting sine wave and the histogram was used to evaluate how well bursting was predicted by that sine wave approximation. The phase of the bin corresponding to the peak of the best-fitting sine wave was used to represent the preferred phase of theta at which bursting occurred.

To examine firing patterns prior to each burst, either every spike or every burst occurring 50-300 ms prior to the iden- tified burst was accumulated in 10 ms bins of a histogram. The cumulative histogram was inspected for a prevalence of activity 80-140 ms (corresponding to 7-12.4 Hz) prior to the burst. The incidence of latencies within the 12 bins defining the theta interval was compared to that in the 6 bins on each side of that interval and evaluated using a chi-square analysis.

RESULTS

Average baseline firing rate for all 65 cells, regardless of their behavioral or spatial correlate, was 2.3 spike&, with an average modal interspike interval within identified bursts of 5.5 ms. The modal number of spikes within bursts was 2 for all cells. Mean peak-to-average firing ratio was 5.6 for

184 HZPPOCAMPUS VOL. 1, NO. 2, APRIL 1991

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cue-sampling cells (SEM, 0.6; range, 2.0-13.0) and 3.6 for goal-approach cells (SEM, 0.5; range, 2.0-9.0). Average in- field to out-of-field firing rate for place cells was 11.25 (SEM, 2.25; range, 2.1-26.25). Cue-sampling, goal-approach, and place cells were analyzed separately, and the results of each analysis will be discussed in turn.

Cue-sampling cells

Inspection of cue-sampling cell firing patterns across in- dividual trials revealed many examples of all 3 aspects of “theta-bursting.” When activated during the odor sampling period, these cells tended to fire in high-frequency bursts near

111 111 I1 I

the positive peak of the dentate theta rhythm, and often ex- hibited multiple bursts on consecutive theta cycles (Fig. 1A). When data from a series of trials were summarized by time- locking recordings to the first positive theta peak after odor onset, a consistent theta pattern emerged in the EEG over the period of cue analysis, and the occurrence of bursts was aligned in this period across trials (Fig. 1B). Quantitative analyses on all recorded cue-sampling cells confirmed these preliminary observations.

Of 24 cue-sampling cells analyzed, 22 cells burst on at least 10% of the trials (mean, 38.3%; SEM, 4.3). The remaining 2 cells burst on fewer than 2.5% of the trials, and bursting ap-

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Fig. 1 . (A) Hippocampal EEG (upward deflections indicate positivity) and unit activity on individual discrimination trials. (B) Hippocampal EEG and unit activity from a series of trials time-locked to the first peak of the dentate theta rhythm after odor onset. Rasters show single spikes (small dots) and bursts (large dots) for a subset of trials. Note that the 100 ms time scale applies to both (A) and (B).

LEARNING-RELATED PATTERNS OF HIPPOCAMPAL SINGLE CELL ACTIVITY i Otto et a1 . 1 85

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peared uncorrelated with trial events. The average modal within-burst interspike interval for bursting cells was 5.0 ms (SEM, 0.36; range, 2-9 ms). the modal interspike interval for 17 of these cells fell between 4 and 6 ms.

In each of the 22 cases bursting occurred coincident with the behavior-related increase in overall firing rate of the cell. The results from typical cue-sampling cells are shown in Fig- ures 2-4. The cell in Figure 2 burst time-locked to the onset

of odor sampling beginning approximately the third theta cycle into that period, with maximal activity at approximately the fifth theta cycle. Note that the occurrence of bursts mir- rored the increase of nonburst firing quite well. In this ex- ample the rat was relatively immobile during bursting, but this was not the case for all cells (see Figs. 3 and 4). For 9 cells, peak bursting occurred while the animal was relatively motionless, while for the remaining 13 cells, peak bursting

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Fig. 2. (A) EEG, movement, and unit activity across trials time-locked to the first dentate theta peak after odor onset. EEG was first normalized then averaged across trials; movement was averaged across trials as well. EEG (upward deflections reflect positivity) was first normalized within the 4-second sampling period, then averaged across trials. Separate X and Y movement traces, respectively, indicate averaged head movements toward the sampling ports and to the left (up) or right (down). Rasters show single spike (small dots) and burst (large dots) for a subset of analyzed trials. Note that spike and burst activity are aligned to the cycles of averaged EEG across trials. Histograms indicate the incidence of single spikes (open bars) and bursts (shaded bars). P(spike) refers to the probability of burst-related spike activity within each 100 ms bin. (B) Distribution of within-burst interspike intervals. (C) Distribution of the incidence of burst onsets across phases of the theta cycle. Zero and 360" indicate successive positive peaks of dentate theta rhythm. (D) Distribution of latencies of unity activity prior to identified bursts. Darkened bars indicate latencies corresponding to 7- 12.5 Hz. P(spike/burst) refers to the probability of a spike occurring during each 10 ms bin prior to an identified burst.

186 HIPPOCAMPUS VOL. 1, NO. 2, APRIL 1991

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occurred during movements within the cul-de-sac, indicating, therefore, that the occurrence of bursting was independent of movements during the odor sampling period.

In most cases there was a high degree of convergence be- tween peak burst and overall activity, suggesting that bursting occurred whenever a cell fired maximally. However, there were at least 2 instances in which bursting was not tightly correlated with increased nonburst firing. In the example shown in Figure 3, the cell increased nonburst activity throughout the period of odor sampling, but maximal bursting occurred as the rat moved to the left just prior to making its response. In the example shown in Figure 4, maximal non- burst activity followed maximal burst activity by approxi- mately 250 ms.

Hippocampal EEG was successfully recorded along with activity from 12 of the 22 cells that showed significant burst- ing. For 11 of these cells the bursts occurred reliably (all P

< .05) at a preferred phase of the ongoing theta rhythm (see panel C of Fig. 2). Furthermore, the preferred phase of burst- ing was remarkably similar for these 11 cells. The mean pref- erence was 312.6" from the positive peak of dentate theta (SEM, 9.85; range, 224"-337").

Ten cue-sampling cells (42%) exhibited a significant pref- erence for the occurrence of unit activity preferentially at 80- 140 ms prior to individual identified bursts (see panel D in Fig. 2 , panel C in Figs. 3, 4). For each cell there was a unique preferred latency of prior activity. In the cell shown in Figure 2, prior activity occurred maximally at 130 ms (cor- responding to a theta frequency of 7.7 Hz). In the cell shown in Figure 3, the peak latency of activity prior to bursts was at 90 ms (1 1.1 Hz). In the cell shown in Figure 4, peak prior spike activity was observed sharply at 120 ms (8.3 Hz). In several cases a secondary peak of activity was also observed at a period corresponding to twice that of the first peak prior

LEARNING-RELATED PATTERNS OF HIPPOCAMPAL SINGLE CELL ACTIVITY i Otto et aI . 1 87

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to the burst (Figs. 3, 4), suggesting that strings of 3 or more bursts occurred with some regularity. Seven additional cells exhibited peak prior activity within the 80-140 ms interval, but this apparent preference did not reach statistical signif- icance. Bursts in the remaining 5 cells showed no tendency to be preceded preferentially by activity within this interval.

Goal-approach cells

The firing patterns of 18 goal-approach cells were analyzed for theta-bursting. An example of a goal-approach cell that fired in a theta-bursting pattern appears in Figure 5.

Twelve (67%) goal-approach cells burst on at least 10% of the trials (mean, 35.4; SEM, 5.7). The remaining 6 cells burst on fewer than 9% of the trials, and bursting appeared un- correlated with behavioral and trial events. The average modal interspike interval for spikes within bursts was 6.4 ms (SEM, 0.5; range, 4-9 ms).

Hippocampal EEG was recorded along with unit activity for 9 goal approach cells; only 4 of these cells burst reliably

in phase with the theta rhythm. The average phase of bursting for these 4 cells was 275" (SEM, 47.4; range, 134-337) from the positive peak of dentate theta.

Analysis of unit activity prior to identified bursts indicated that 7 (38%) showed statistically significant spike or burst activity 80-140 ms before a burst. Five additional cells ex- hibited peak prior activity within the theta interval, but this preference failed to reach statistical significance.

Place cells

The results of analyses of a typical place cell are illustrated in Figure 6; as this case illustrates, bursting was in most cases confined to a small portion of the place field. Of 23 cells with significant place correlates, 21 (92%) also exhibited bursting within the place field. The average modal interspike interval for spikes within bursts was 5.1 (SEM, 0.42).

Of the 19 place cells for which hippocampal EEG was suc- cessfully recorded, 16 (84%) burst reliably in phase with the

188 HIPPOCAMPUS VOL. 1, NO. 2, APRIL 1991

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Fig. 5. (A) EEG, movement, and unit activity across trials time-locked to the discriminative response. (B) Distribution of within-burst interspike intervals. (C) Distribution of the incidence of burst onset across phases of the theta cycle. Bursts did not occur at a statistically reliable phase of the theta rhythm for the cell. (D) Distribution of latencies of neural activity prior to identified bursts. See Figure 2 legend for details.

ongoing theta rhythm. The phase at which these bursts oc- curred was consistent across cells (mean, 300", SEM, 14.2).

Analysis of the incidence and latency of activity prior to identified bursts indicated that 15 cells (65%) exhibited a sta- tistically significant preference for prior activity within the theta interval. Two additional cells exhibited peak prior ac- tivity within the theta interval, but this apparent preference did not reach statistical significance.

Variations in theta-bursting associated with different behavioral correlates

The proportion of each cell type meeting the previously described criteria for each aspect of theta-bursing appears in Table 1 . Pairwise chi-square tests were conducted to deter- mine whether the behaviorally defined cell types studied here differed in their tendency to exhibit each characteristic of theta-bursting.

Goal-approach cells were significantly less likely to burst compared to both cue-sampling cells (xz = 4.17, P < .05) and

place cells (x2 = 3.9, P < .05). Furthermore, bursting in goal- approach cells was less likely to be phase-locked to the on- going theta rhythm than that in both cue-sampling cells (xz = 5.62, P < .05) and place cells ( x 2 = 4.73, P < -05). Cue- sampling and place cells did not differ in their tendency to burst (x2 = 0.01, ns), in their incidence of phase-locked ac- tivity (xZ = .36, ns), or with respect to the specific phase of theta at which they burst (t25 = 0.66, ns). Thus fewer goal- approach cells burst, and when they did burst they did so in phase with the theta rhythm less often than the other cell types. Finally, there was a trend for fewer goal-approach cells to exhibit preferred activity prior to a burst compared to both cue-sampling and place cells.

DISCUSSION

The 3 primary findings presented herein reveal compelling similarities between the firing patterns of single neurons in CAI and the neural activation parameters optimal for hip- pocampal LTP induction. First, CAI neurons were observed

LEARNING-RELATED PATTERNS OF HIPPOCAMPAL SINGLE CELL ACTIVITY / Otto et a1 . 1 89

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- 3 0 0 - 2 0 0 - 1 0 0 burs t 0 360

PHASE (degrees) LATENCY (ms)

within the behavioral chamber during performance of a spatial task. See text for details of place field mapping. (B) Localization of bursts within the behavioral chamber during performance of the spatial task. (C) Distribution of interspike intervals for identified bursts. (D) Distribution of the incidence of burst onset across phases of the theta cycle. (E) Distribution of latencies of neural activity prior to identified bursts.

to discharge in high-frequency bursts. Second, the occur- rence of these bursts was well phase-locked to the ongoing hippocampal theta rhythm. Finally, individual bursts were frequently preceded by neural activity at latencies that par- allel the theta frequency. The emergence of neural activity with these 3 aspects of temporal patterning, collectively re- ferred to as “theta-bursting,” was well time-locked to rele-

vant behavioral events in the olfactory discrimination task and to visits to a particular location within the behavioral chamber in the spatial memory task. Further, in the olfactory task, these patterns emerged preferentially in cells that fired during stimulus analysis and response selection, suggesting a specific association between theta-bursting and mnemonic processing.

CA1 cells burst during task-relevant behaviors

cells observed in this study is their tendency to discharge in rapid succession (i.e., burst), suggesting a strong similarity between an endogenous CA1 firing repertoire and the re- Behavioral Theta Phase

Correlate Bursting Preference ms quirement of high-frequency stimulation for LTP induction.

Table 1 . Proportion of Each Behaviorally Defined Cell Type The most conspicuous feature of the activity of the CAI

Exhibiting Theta-bursting Characteristics Prior Activity

at 80-140 ~

Within the hippocampus of the behaving rat, there are 2 cell Cue-sampling (22124) 92% ”12) 92% 42% types whose firing characteristics fit this description. Com-

plex-spike cells fire at low rates overall, and only very oc- Goal-approach (12118) 67% (419) 44%

- casionally in high-frequency bursts of 2-11 spikes of pro- Place

(7’18) 39% (21123) 91% (16119) 84% (1.5123) 65%

The proportion each behaviorally defined cell type exhibiting theta- burstingcharacteristics, expressed in terms of its incidencekotal num- ber of cells analyzed (in parentheses) and as a percent. Bursting in- cidence reflects the number of cells in each class exhibiting significant bursting relative to the total number of cells with that correlate. Thera Phase incidence reflects the proportion of cells with reliable theta phase relationships relative to the total number of cells within that class for which theta activity was recorded. Prior Activity reflects the proportion of the total number ofcells in each class with preferred activity 80-140 ms prior to a burst.

gressively decreasing -amplitude (Ranck, 1973). Complex- spike place in an environment (o?Keefe, 1976) or othemise actively inves- tigating a particular configuration of specific stimuli (Wiener et al., 1989). These cells are localized to the CA1 pyramidal

(Fox and Ranck, 1975). The other major type of cell observed in CAI of the behaving rat, the “theta cells,” also exhibit bursting. However, in contrast to complex-spike cells, theta

fire on]y when the rat is in a

layer, and are therefore thought to be pyramidal

190 HZPPOCAMPUS VOL. 1, NO. 2, APRIL 1991

cells burst much more frequently and are considerably less selective in the behavioral conditions during which they fire; these cells discharge in synchrony with each phase of the hippocampal theta rhythm during a variety of exploratory or locomotory activities (Ranck, 1973). Theta cells are found mainly in the layers of CAI in which interneurons are prev- alent (Fox and Ranck, 1975).

Two lines of evidence suggest that the theta-bursting cells identified in the present study were complex-spike cells. First, these cells were highly specific with regard to the be- havioral events during which they increased activity and burst. Hippocampal theta was prevalent throughout perfor- mance of discrimination trials, so theta cells would have been recognized by nearly continuous burst activity. Second, both on-line waveform characteristics and post-hoc histological analyses of electrode location confirmed that these cells were recorded in the CAI pyramidal cell layer.

It should be noted that an additional characteristic of com- plex-spike cells is a progressive decline in amplitude of suc- cessive action potentials within a complex spike discharge (Ranck, 1973). Since our method of unit discrimination was based on spike amplitude, it is likely that the number of spikes within a complex-spike burst, and perhaps the occurrence of bursting, are undercounted.

Bursts occur just prior to the positive peak of the dentate theta rhythm

Our observation that CAI complex-spike cells burst pref- erentially at a particular phase of the ongoing theta rhythm suggests a second similarity between the endogenous firing repertoires of these cells and the optimal induction param- eters for LTP. Pavlides e t al. (1988) found that in dentate gyrus, LTP is preferentially induced when bursts of perforant path stimulation are time-locked to the positive peak of den- tate theta; thus, brief bouts of high-frequency activity are most effective in inducing LTP when they are appropriately phase-locked to the ongoing hippocampal theta rhythm. The theta rhythm in the CAI stratum pyramidale is phase-re- versed with respect to that in dentate gyrus (Winson, 1974, 1976), and the phase of CAI theta rhythm at which bursts applied to afferent fibers optimally induce LTP in that sub- field has not been examined. Thus, while the present data d o not directly address whether complex spikes are occurring at the optimal phase of theta to induce LTP in CA1, these bursts do occur reliably at the same theta phase associated with optimal LTP induction 2 synapses earlier (i.e., in dentate gyrus). Assuming a transmission time of 10-20 ms from den- tate gyrus to CA1, excitatory inputs through the trisynaptic circuit would arrive in CAI only slightly later in the theta cycle. A testable prediction based on this account is that LTP in CA1 will also be optimally induced by bursts of stimulation applied to the Schaffer collaterals during the positive phase of the dentate theta rhythm.

Bursts are preceded by neural activity at the theta interval

The finding that bursts are commonly preceded by neural activity a t latencies corresponding to the hippocampal theta rhythm suggests a third similarity between the firing patterns

of CAI cells and the stimulation patterns optimal for inducing LTP. Trains of electrical stimulation mimicking single com- plex-spike discharges can induce LTP if those trains are pre- ceded, or “primed,” by a similar train (Larson and Lynch, 1986) or a single pulse (Rose and Dunwiddie, 1986; Green- stein e t al., 1988) at latencies corresponding to frequencies of 5-10 Hz. In the present study, many bursting cells exhib- ited a strong tendency for individual bursts to be preceded by neural activity at latencies corresponding to the theta range of 7-12.5 Hz. Thus, the present data argue strongly that the activation patterns accounting for this priming effect are represented in the endogenous firing repertoires of CAI pyramidal cells.

Relationship between theta-bursting and mnemonic processing in the olfactory and spatial tasks

The functional significance of theta bursting may be re- vealed in part by comparisons among the 3 cell types with regard to the differential relevance of their behavioral cor- relates to task-specific memory demands and their relative incidence of theta bursting. Within the odor task, the 2 cell types observed differ considerably in their likely association with mnemonic processing. Goal-approach cells fired during movements toward the cul-de-sac or the water cup and were not active during periods critical to the odor discrimination judgement. In contrast, cue-sampling cells fired selectively during the period of stimulus analysis and response genera- tion. Thus the firing of cue-sampling cells reflects hippocam- pal information processing more directly relevant to the mem- ory demands of the odor discrimination task. In the place task, the firing of CAI cells likely reflects the processing or storage of spatial information directly relevant to memories for places (Bostock et al., 1986; Quirk et al., 1987: Pavlides and Winson, 1989).

Comparisons between these cell types with regard to their incidence of theta bursting indicated that both cue-sampling cells and place cells were more likely than goal-approach cells to burst during relevant periods and were more likely than goal-approach cells to burst in phase with the ongoing dentate theta rhythm and that, bursts in both cue-sampling and place cells were more likely to be “primed” by prior neural activity at the theta interval. These findings suggest, therefore, that theta-bursting occurs preferentially in association with anal- ysis, storage, o r retrieval of cues critical to memory forma- tion.

Theta-bursting and memory formation: Theoretical considerations

The present data suggest that all 3 aspects of patterned neural activity associated with optimal LTP induction, that is, high-frequency activity, bursting phase-locked to the hip- pocampal theta rhythm, and prior activity a t the theta inter- val, are reflected in the firing patterns of CA I pyramidal cells of behaving rats. Moreover, these patterns emerge prefer- entially during behavioral events associated with likely epi- sodes of memory formation or retrieval. While these data d o not address directly the functional significance of other nat- urally occurring patterns of neural activity to LTP (Buzsaki,

LEARNING-RELATED PATTERNS OF HIPPOCAMPAL SINGLE CELL ACTIVITY i Otto et aI. 191

1987), they do suggest several avenues that must be pursued in order to gain a broader understanding of the role of theta- bursting and LTP in learning and memory.

The theta-bursting patterns described in this report were observed in rats that had been well-trained on the olfactory and spatial tasks they were performing during data acquisi- tion; therefore, these subjects had learned the information required for successful performance prior to the behavioral sessions in which the theta-bursting phenomenon was ob- served. Initially it might be surprising that the neural activity patterns associated with the induction of LTP, and by hy- pothesis, memory formation, persist well after the critical in- formation has been learned. There are, however, several rea- sons why this might be the case. First, it is possible that theta- bursting serves to strengthen both LTP and memory. Con- sistent with this view is the observation that the magnitude of both induced LTP and behaviorally assessed memory are greater following repetitive stimulation sessions and over- training, respectively (Barnes, 1979). Second, theta-bursts may serve in the storage of “working memory,” a form of short-term memory critically dependent on hippocampal function (Olton et al., 1979) and reflected in the activity of hippocampal neurons in a similar odor discrimination task (Eichenbaum et al., 1987). Third, the present data do not exclude the possibility that theta-bursting patterns represent the “readout” of previously potentiated pathways rather than, or perhaps in addition to, the memory storage event it self.

Assuming for a moment that LTP does represent the phys- iological substrate of some form of memory, a second related question concerns the neuroanatomical locus of LTP induced by theta-bursts within hippocampus, given that long-term storage of learned information is believed to occur not in hip- pocampus but in the neocortex (Squire et al., 1984). One pos- sibility is that theta-bursting by CA1 pyramids results in the induction of LTP in efferent cortical targets of the hippocam- pus that are themselves responsible for long-term storage. Consistent with this hypothetical account is the observation of LTP in frontal cortex following electrical stimulation of CAI (LaRoche et al., 1989). Alternatively, or perhaps in ad- dition, theta-bursting in CAI might reflect the induction of LTP in CAI cells resulting from afferent activation by cells in CA3. By this account, LTP in CA1 may act as a temporary memory store, or “buffer,” prior to permanent storage of that information elsewhere (Rawlins, 1985), as a working memory store (see above), or as an “index” to loci of information stored permanently in cortex (Teyler and DiScenna, 1987). To test the hypothesis that the theta-bursting patterns ob- served in this study reflect the afferent firing pattern driving these CA1 cells, it would be necessary to determine first whether the cells comprising the primary course of afferent input to CAI, the CA3 pyramids, also exhibit theta-bursting, and second whether the CAI cells exhibit potentiation (i.e., become more excitable) as a consequence of theta-bursting.

It is of ultimate interest to determine the extent to which hippocampal LTP serves as a physiological mnemonic de- vice. Several studies have shown that explicit training (Ruth- rich et al., 1982; Weisz et al., 1984; Skelton et al., 1987) or exploratory behavior (Sharp et al., 1985; Green et al., 1990) result in increased excitability in dentate gyrus, suggesting

that hippocampal function is altered as a function of recent experience. It remains to be determined whether behaviorally related theta-bursting in CAI can be linked to consequent changes in hippocampal or cortical excitability, and if so, the extent to which these changes are functionally significant.

ACKNOWLEDGMENTS

This research was supported by PHS grant NS26402 and NSF grants BNS8721157 and BNS8810095. The authors thank Drs. Greg Rose and John Larson for their helpful com- ments on an earlier version of the manuscript.

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