HZPPOCAMPUS, VOL. 2, NO. 4, PAGES 389-396, OCTOBER 1992
Maintained Saturation of Hippocampal Long-term Potentiation Does Not Disrupt Acquisition of the Eight-arm Radial Maze Gilbert B. Robinson Department of Psychology, University of New Brunswick, Fredericton, New Brunswick, Canada
ABSTRACT The present experiment examined the anterograde effect of bilateral saturation of long-term potentiation (LTP) of the rat perforant path-granule cell response on acquisition of the eightarm radial maze. To ensure maintained saturation, high-frequency stimulation was applied to the perforant path immediately prior to each trial. LTP did not significantly increase the number of trials required for acquisition of the standard eight-arm radial maze task. Furthermore, the magnitude of LTP did not correlate with the rate of acquisition. LTP also did not significantly affect the number of either working or reference memory errors during subsequent training with only four of the eight arms baited; both control and LTP rats made significantly more reference memory than working memory errors, with the number of both types of errors decreasing as training progressed. These results indicate that prior saturation of LTP within the perforant pathdentate granule cell circuit does not affect acquisition of either the reference or working memory components of the radial maze task. The results are discussed in relation to the role of LTP in acquisition of the eight-arm radial maze. Key words: rat,
dentate gyrus, perforant path, working memory, reference memory
The possibility that long-term potentiation (LTP) of hippocampal synapses is involved in the acquisition andlor retention of information has been suggested by a number of investigators (Berger, 1984; Teyler and Discenna, 1984; McNaughton and Morris, 1987; Skelton et al., 1987; Barnes, 1988; Laroche et al., 1991). Several lines of evidence revolve around the relationship between LTP and spatial learning, a hippocampal-dependent task (O’Keefe and Nadel, 1978; Moms et al., 1982). First, high-frequency activation of the perforant path, resulting in LTP of both the perforant path-hippocampal dentate granule cell population spike and excitatory postsynaptic potential (EPSP), disrupts acquisition of new spatial information on the Barnes circular maze (Barnes, 1979) as well as recently established, but not wellestablished, spatial information (McNaughton et al., 1986). Second, it appears that rats may be capable of learning a spatial location (Morris water maze) only once LTP of the perforant path-granule cell response has decayed (Castro et al., 1989). Third, various correlations/similarities between LTP and spatial learning or memory have been demonstrated, including (1) similarities in LTP decay and the forgetting of spatial information (Barnes, 1979; Barnes and McNaughton, 1985; Deupree et al., 1991); (2) both LTP induction and the acquisition of spatial information are disrupted by antagonists Correspondence and reprint requests to G. B. Robinson, Department of Psychology, University of New Brunswick, Fredericton, New Brunswick, E3B 6E4 Canada.
of N-methyl-D-aspartate (NMDA)-mediated activity (Morris, 1989; Shapiro and Caramanos, 1990;Ward et al., 1990;Lyford and Jarrard, 1991); and (3) the maintenance and retention of LTP and spatial memory, respectively, are not disrupted by NMDA antagonists (Robinson et al., 1989). Together these studies raise the possibility that an LTP-like process (synaptic modifiability leading to increased synaptic strength) (McNaughton and Moms, 1987) occurs during the acquisition of spatial information (e.g., Sharp et al., 1985). The Olton eight-arm radial maze (Olton and Samuelson, 1976) is a potential spatial task with both working and reference memory components. Trial-specific working memory allows the animal to recall arms previously entered on the current trial and therefore avoid reentries. Reference memory, on the other hand, refers to stored representations that are useful for all trials (e.g., spatial representations of the environment, there is food at the end of the arms). McNaughton et al. (1986) previously reported that LTP saturation did not affect subsequent working memory. The effect on reference memory acquisition, however, was not determined, as that component of the task was acquired prior to LTP saturation. Training in the McNaughton et al. (1986) study began 24 hours following LTP saturation and continued for a number of days. As a result, considerable decay of synaptic strength likely would have occurred. Short-term potentiation, for example, decays with a time constant of approximately 1-6 minutes (McNaughton, 1982; Racine and Milgram, 1983), whereas the short- and long-term components of LTP decay
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with time constants of 1.5 hours and 5 days, respectively (Racine et al., 1983). If modifiable synapses are required for spatial learning, training that occurs soon after an intense burst of high-frequency stimulation should produce near maximal levels of impairment. This possibility was addressed in the following experiment. In addition, the anterograde effect of LTP saturation on both the reference and working memory components of the eight-arm radial maze was examined.
MATERIALS AND METHODS Subjects Sixteen male Long-Evans hooded rats were initially prepared as subjects for these experiments, The rats were housed individually with ad lib access to food and water and placed on a 12 hour 1ight:dark cycle. All testing took place within the initial 3 hours of the light cycle. Three of the 16 animals were not used in the analysis (one developed both electrographic and behavioral seizures during training on the eightarm maze, one failed to acquire the eight-arm maze, even after 61 trials, and the headcap was lost from the third animal early in training).
Surgery The rats (400-450 g) were deeply anesthetized with sodium pentobarbitol (65 mg/kg) and placed on a Kopf stereotaxic apparatus. With the upper incisor bar set 3 mm below the interaural line, a recording electrode (a single Teflon-coated stainless steel wire, 0.28 mm diameter) was implanted in the granule cell layer of the dentate gyrus of each hemisphere. The coordinates, relative to bregma, were 3.8 mm posterior and 2.3 mm lateral. Bipolar stimulating electrodes (two Teflon-coated stainless steel wires twisted together and separated horizontally at the tips) were lowered to the perforant path of each hemisphere. The coordinates, relative to bregma, were 7.8 mm posterior and 4.4 mm lateral. In addition to stereotaxic coordinates, electrophysiological monitoring of perforant path-granule cell field potentials was used to maximize final electrode sites. Four stainless steel anchoring screws were also placed in the skull. Two of these screws, located anterior to bregma and lateral to one another, served as ground and reference. The electrodes were anchored to the skull using dental acrylic once the optimal sites were located (i.e., sites that evoked the maximum response amplitude). The electrodes and grounds then were attached to a nine-pin headcap, which was also fixed to the skull using dental acrylic. The rats then were administered 100,000 units of penicillin (i.m.), placed under a heat lamp, and closely monitored during recovery.
Electrophysiology Two to 3 weeks after surgery, the animals were put on a food-deprivation schedule. When an animal reached 90% of its body weight, an input/output (UO) response was collected. This consisted of the application of 50 electrical pulses to the perforant path at 0.1 Hz. The stimulation intensity was increased after every tenth pulse. The intensities for each animal were chosen to evoke a range of response amplitudes from threshold for spike discharge to the maximum amplitude
spike. All stimulation and recording took place within a shielded recording chamber. The above pretetanus 1/0 response was obtained once per day for a minimum of 3 days. Once responses were stable, animals were randomly placed in either the control (CON; n = 6) or experimental group (LTP; n = 7). Ten high-frequency trains of electrical pulses (400 Hz, 50 ms) were then applied to both the right and left perforant path (1 train every 10 s) of animals in the LTP group. The intensity of pulses within each train was twice that necessary to evoke the maximum amplitude population spike to a single pulse (range, 16-30 V). The set of high-frequency trains was repeated once per day for each of the next 4 days (total, 50 trains). Animals were closely monitored for both electrographic and behavioral seizures, both during and following all train stimulation. Except for the high-frequency stimulation, CON subjects received identical treatment. At the end of this period, animals had attained 80% of their ad lib body weight. To ensure the longer-lasting component of LTP was saturated prior to the start of behavioral training, an I/O response was obtained 24 hours after application of each of the first four train sets (i.e., prior to application of train sets two to five). To monitor the shorter-lasting component of LTP (that present at the time of behavioral training), an I/O response was collected 15-25 minutes after application of each of the first five train sets. An I/O response was also obtained 15-25 minutes after criterion was reached on the standard eight-arm task. Inputloutput responses were obtained from CON animals at comparable time points. The perforant path-granule cell responses were bandpass filtered (1 Hz and 3 kHz), sampled by a 12-bit AID converter at 10 kHz for 20 ms, and saved to disk for later analysis of both population spike amplitude and EPSP slope. The EPSP slope was determined between the start of the positivity and a point approximately 1 ms later (but prior to onset of the population spike). Spike amplitude was measured as the height of a line drawn from the peak of the negativity to a tangent joining spike onset and offset.
Apparatus and training The test apparatus was a grey, eight-arm radial maze constructed of wood and elevated 95 cm from the floor. The octagonal central platform was 40 cm in diameter. Each arm was 60 cm long and 9 cm wide with edges 2 cm high. A food cup, 2.5 cm in diameter and 1 cm deep, was located 3 cm from the outer end of each arm. Surrounding the maze were numerous distal cues (e.g., mobiles, posters, tables, chairs, cupboards, filing cabinets) that remained in a constant location from trial to trial. The room was lit with a 60 W bulb. The first trial occurred immediately following collection of the I/O response that was obtained after application of the fifth set of 10 high-frequency trains. All eight arms of the maze were baited with half a piece of Froot Loops cereal. The rat then was placed on the central platform and allowed to explore the maze (i.e., there was no maze adaptation) until all food was consumed. A maximum of 20 minutes was allowed for each trial. Each rat received one trial per day. The set of high-frequency trains was applied to the perforant path immediately prior to the start of all subsequent trials. The maze
HIPPOCAMPAL LTP AND LEARNING / Robinson 391 was carefully cleaned with a damp sponge and then wiped dry following each trial. The number of errors (arm reentries), the order in which arms were entered, and the time to visit all arms were recorded for each trial. An arm entry was scored whenever a rat went more then two thirds of the way down its length. Training continued until a rat made no more than three errors over three consecutive trials with no more than two errors on any given trial. Training on the 418 task began the day after the above criterion was met. Four of the eight arms were baited for each trial. The baited and nonbaited arms remained constant throughout training. Two types of errors were recorded for this task. Entry into an unbaited arm was scored a reference memory error; arm reentries were scored as working memory errors. Training on the 418 task continued for 30 days. The set of high-frequency trains was applied to the perforant path of rats in the LTP group immediately prior to each trial. The headcap of one animal in the LTP group was lost during train-
A
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CON
ing. Thus, the results of the 4/8task are based on six CON and six LTP animals.
RESULTS
Elect rophysiology Figure 1 illustrates changes in both the EPSP and spike component of the perforant path-granule cell response in both the CON (Figs. 1A and lC, respectively) and LTPgroup (Figs. 1B and lD, respectively). There was no significant change in either the EPSP [F(1,5) = .41, P = 3 1 1 or spike [F(1,5) = .75, P = .426] of the CON group, from the baseline I/O to the I/O obtained 15-25 minutes following criterion on the standard eight-arm task. In the LTP group, application of the first train set resulted in a significant increase in the amplitude of both the EPSP [F(1,6) = 47.02, P < .001] and population spike [F(1,6) = 55.93, P < .001]. Subsequent analyses examined whether each additional train set, applied prior to the start of training, resulted in a greater magnitude of the
B
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3
3.0-
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0 l .0. O I - e ! ? f c D
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STIMULATION INTENSITY (Arbitrary Units) Fig. 1 . Changes in the inputloutput response of perforant path-granule cell excitatory postsynaptic potentials (EPSP) and population spike in control (CON) rats (A and C, respectively) and in long-term potentiation (LTP) rats receiving highfrequency stimulation of the perforant path (B and D, respectively). There was significant long-term potentiation of both the EPSP and population spike in the LTP group (n = 7) but not in the CON group (n = 6). Illustrated are average baseline I/O responses obtained prior to tetanization (=), and I/O responses obtained between 15 and 25 minutes after application of the first ( 0 ) and fifth ( 0 ) set of high-frequency trains, and 15-25 minutes after criterion was met for the eight-arm radial maze (0). Due to normal variations in electrode placements, the five stimulation intensities were not identical for all animals. Thus, the arbitrary units of stimulation intensity refer only to each of the five intensities that were tested within each animal (actual stimulation intensities ranged from 2 to 15 V).
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Fig. 2 . Examples of evoked perforant path-granule cell responses in one animal from the long-term potentiation (LTP) group, prior to tetanization (solid lines) and 15-25 minutes following application of the fifth train set (dashed lines). Each response is the average granule cell response to 10 perforant path pulses (0.1 Hz) applied at the low, middle, and high intensity. Calibration bars, 5 mV and 5 ms.
shorter-lasting component of LTP than that induced by the previous train set. The application of additional sets of trains, following the second set, did not result in additional significant increases in either the EPSP [F(1,6) = 3.08, P = .130] or spike [F(1,6) = 4.65, P = .075]. This indicates that saturation of the shorter-lasting component of LTP had occurred prior to the start of training. The average percent increase from baseline (in response to the highest test pulse stimulation intensity) in EPSP slope and spike amplitude at the start of training was 32.0 ? 5.2% and 66.3 ? 8.0%, respectively. Figure 2 compares, for one animal, average responses evoked by the first, third, and fifth stimulation intensity prior to tetanization and 15-25 minutes following application of the fifth train set. The longer-lasting component of LTP also was saturated prior to the start of training. The average percent increment, over the pretetanus amplitude, observed at the highest test pulse intensity for both the EPSP and spike are illustrated in Figure 3. On the first day of training, the average magnitude of the longer-lasting component of LTP of the EPSP and spike was 20.4% and 59.4%, respectively.
the time to complete the trial [F(l,ll) = .97, P = .345]. There was, however, a significant decrease in both the number of errors [F(2,22) = 3.65, P < .043] and the time taken to complete a trial [F(2,22) = 9.19, P < .001], indicating that both groups were learning. Neither the group by error [F(2,22) = 2.62, P = .095] nor the group by time [F(1,6) = .26, P = .773] interactions were significant.
4
30.01
Effect of LTP on acquisition of eight-arm radial maze
T
-
r
I 2 4 hrs post-train set 1 24 hrs post-train set 2 L13 24 hrs Dost-train set 3 24 hrs post-train set 4 ~
The CON and LTP groups did not differ significantly in the number of trials to criterion (see Fig. 4; t-test, t = 1.56, df = 11, P < .147). Control rats required an average of 7.2 ? 1.1 trials, compared to 10.3 k 1.6 for the LTP animals. Both the EPSP [F(1,6) = 11.37, P < .015] and spike [F( 1,6) = 55.07, P < .001] were still significantly potentiated (relative to the pretetanus response) following criterion. The additional train sets applied prior to each training session did not result in significantly larger responses than those obtained following the fifth train set (i.e., prior to the first trial). In fact, the magnitude of LTP of both the EPSP and spike, following criterion, was not significantly different from that observed 15-25 minutes following application of the third train set. Thus, LTP saturation occurred prior to the start of training and was maintained throughout training. All animals received at least three training trials. A repeated measure analysis of variance (ANOVA) did not reveal any group differences in either the number of errors made during the initial three trials [F(l,ll) = 0.84, P = .378], or
80.01 I
Fig. 3 . Saturation of the longer-lasting component of LTP. Illustrated are the average percent increment in excitatory postsynaptic potential (EPSP) (upper panel) and population spike (lower panel), at the highest test pulse intensity, obtained 24 hours after application of each of the first four train sets.
HIPPOCAMPAL LTP AND LEARNING / Robinson l2
1
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related negatively with trials to criterion (range of r values, - .75- - .09). Only the correlation of the fourth spike amplitude with days to criterion was significant (r = - .752, twotailed test, P < .05), indicating the trials to criterion decreased as the magnitude of spike LTP increased.
7-
Effect of LTP on reference and working memory There was no significant group difference [F(1,10) = 2.87, P = .121] in the number of reference and working memory errors (Fig. 5). There was, however, a significant difference CON
LTP
Fig. 4. Average number of trials to criterion for acquisition of the eight-arm radial maze task in the control (CON) (n = 6) and long-term potentiation (LTP) (n = 7) groups. Error bars, standard error of the mean.
between the number of working and reference memory errors [F(1,10) = 256.33, P < .OOl] and a significant interaction effect between working and reference memory errors [F(9,90) = 3.50, P < .001], indicating a significant difference in the decrease in errors across trials. Subsequent analysis, however, showed both working [F(9,90) = 2.20, P < .029] and reference memory errors [F(9,90) = 7.76, P < .001] decreased significantly over training.
Correlation of LTP magnitude with learning rate If LTP saturation retards subsequent acquisition of the eight-arm radial maze, there should be a positive correlation between the magnitude of LTP (determined 15-25 minutes after tetanization) and trials to criterion. Averaging may obscure this relation (i.e., a rat in the LTP group may have exhibited only a small magnitude of LTP and therefore little disruption of spatial learning would occur). To examine this possibility, the magnitude of LTP (at each of the five IIO intensities), both at the start of training and criterion, was correlated with acquisition rate. There was no correlation between the magnitude of LTP of the EPSP and the rate of acquisition (range of r values, .23-S6). Nevertheless, the positive correlation values indicate a tendency for the trials to criterion to increase as the magnitude of LTP increased. Except for the third stimulation intensity (r = .57 at the start of training and r = 5 8 at criterion) the magnitude of LTP of the population spike cor4 CON 4 CLTP
1
-.
reference memory
w I32
$
workingmemory
0-r 0-
-
.
1
- 0 0 2 2 4 4 6 6 88 1i o0 TRAINING BLOCK (3 mals/block)
Fig. 5. Average number of reference and working memory errors in the 4/8 task in the control (CON) (n = 6; open squares) and long-term potentiation (LTP) (n = 6; closed circles) group. Each block represents the average number of errors over three trials.
DISCUSSION The above results demonstrate that saturation of LTP at perforant path-granule cell synapses does not disrupt acquisition of either the reference or working memory components of the eight-arm radial maze task. Control and LTP animals did not differ in either their rate of acquisition or performance on the standard version of the task (i.e., all eight arms baited) or in the number of either working or reference memory errors during subsequent testing on the 4J8 task. Together these findings imply that LTP saturation affected neither the reference nor working memory components of the tasks. These results therefore extend the results of McNaughton et al. (1986); maintained LTP saturation not only leaves working memory performance intact but also does not affect acquisition of the reference memory components of the eight-arm radial maze. In agreement with the above, there was not a consistent relation between LTP magnitude and learning. If LTP had facilitated or disrupted acquisition, a negative or positive correlation, respectively, should exist between LTP magnitude and trials to criterion. Significant correlations, however, appear to exist between LTP decay and retention of a spatial task (Barnes, 1979; Barnes and McNaughton, 1985; Deupree et al., 1991). Possible relations between LTP decay and retention were not examined in the present study. Acquisition of the radial maze tasks, in the present study, were not disrupted even though training occurred while synapses were maximally potentiated. The eight-arm maze is thought to be a spatial learning task and it is commonly believed that synaptic modifiability within the hippocampus is involved in spatial learning (McNaughton and Morris, 1987; Barnes, 1988). Thus, a number of possible explanations for the present results were considered. First, there may be inherent differences in the nature of the spatial task on the eight-arm maze, compared to either the Barnes circular maze or the Morris water maze. LTP saturation disrupts acquisition of spatial reference memory on both the Barnes circular maze (McNaughton et al., 1986) and the Moms water maze (Castro et al., 1989) but does not disrupt working memory on the eight-arm maze (McNaughton et al., 1986). Sutherland et al.
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(1991), however, recently reported that LTP saturation did not affect subsequent spatial learning in the Morns water maze task. In that study, training occurred soon after tetanization as opposed to 24 hours later (Castro et al., 1989). A second consideration arises from the latter point; the effects of high-frequency stimulation on behavior may differ depending on whether behavioral testing is immediate or delayed by 24 hours. Seizures that are evoked via perforant path stimulation, for example, do not disrupt subsequent acquisition of the eight-arm radial maze if training begins 24 hours after the last seizure is evoked (G.B. Robinson, unpublished observations). In contrast, recent experiments have raised the possibility that training 30-45 minutes after each kindling stimulation (whether or not behavioral seizures are evoked) will disrupt spatial learning on the Morns water maze (Kirkby et al., 1991). The effect of recent seizure activity on acquisition of the eight-arm maze is currently under investigation. Whether or not the time between stimulation and test underlies these differences, if synaptic modifiability is required for spatial learning, testing immediately after tetanization should theoretically produce the greatest disruption of spatial learning. Third, LTP induction within perforant path-dentate synapses, prior to familiarization with the task, may have resulted in the adoption of alternate response strategies, or spatial processing, that was dependent on neural circuits other than those of the hippocampal formation (Kolb et al., 1983). The animals in the present study did not appear to develop a response strategy, in terms of the order in which the arms were entered. Furthermore, as noted above, perforant path kindling results in transynaptic potentiation effects but does not disrupt learning. The induction of LTP in animals familiar with a spatial task, however, may alter subsequent processing. The changed conditions between initial acquisition and retest may impair the retrieval of information necessary for learning to occur. Prior familiarity with the task may also disrupt the ability of other neural structures to assume processing that had previously occurred in the hippocampus. Finally, and most critical, is concern with how LTP saturation is defined. The present results, for example, only demonstrate LTP saturation at those synapses accessible to the recording electrodes. It is possible that LTP either did not occur at all synapses (e.g., in the ventral blade of the dentate gyrus) or may not have saturated throughout the dentate gyrus. However, the magnitude of the longer-lasting component of LTP, of both the EPSP and population spike, was similar to that reported to disrupt the acquisition of spatial information on the Barnes circular maze (McNaughton et al., 1986). Moreover, each train set was applied at an intensity twice that necessary to evoke the maximum spike amplitude to a single pulse. Thus, tetanization procedures should have maximized the number of affected synapses. Furthermore, behavioral testing only occurred when potentiation effects would be near maximal (Racine et al., 1983). Nevertheless, it is possible that additional synaptic modifiability occurred as a result of learning. Short-term exploratory modulation of dentate synapses, for example, is not blocked by electrically-induced LTP (McNaughton et al., 1991). In agreement with the proposed role of hippocampal LTP in spatial learning, several studies have demonstrated that
NMDA antagonists, which block hippocampal dentate synaptic modifiability in behaving animals (Gilbert and Mack, 1990; Robinson and Reed, 1992), disrupt spatial learning on the eight-arm radial maze (Robinson et al., 1989; Shapiro and Caramanos, 1990; Ward et al., 1990; Lyford and Jarrard, 1991). Sutherland et al. (1991), however, found that the NMDA antagonist MK8Ol did not affect spatial learning providing animals were sufficiently familiar with the task (Morris water maze) prior to testing. Furthermore, attributing spatial learning deficits to the blockade of hippocampal NMDA-mediated activity is controversial (Keith and Rudy, 1990; Lyford and Jarrard, 1991). As shown in the present study, LTP saturation, which should block learning-related synaptic modifiability (and limit that blockade to the activated synapses; however, see McNaughton et al., 1991), fails to retard learning on the eight-arm radial maze. The potential discrepancy between the effect of LTP with the effect of a LTP antagonist on spatial learning is not the first reported. Warren et al. (1991) recently reported that inescapable shock, which blocks LTP in area CA1 of the hippocampal formation (Shors et al., 1989), does not block spatial learning. In summary, LTP saturation within perforant path-dentate synapses does not disrupt learning on the eight-arm radial maze. The proposed role of LTP in learning, however, is difficult to disprove (Keith and Rudy, 1990). There could be any number of explanations for negative findings, including the possibilities that LTP either did not occur or was not saturated outside of the region monitored. It also is possible that information processing within the hippocampus may only be disrupted by certain changes in the input/output relationship (E-S potentiation; Wilson, 1981) or by the possible contribution of LTP at perforant path-CA3 and -CA1 synapses (Yeckel and Berger, 1990), both of which may vary across studies. Nevertheless, if acquisition of the reference and working memory components of the eight-arm radial maze requires widespread synaptic modifiability within the dentate gyrus, the procedures utilized here should have resulted in impaired performance.
ACKNOWLEDGMENTS Supported by grants from the Natural Sciences and Engineering Research Council of Canada and from the University of New Brunswick Research Fund.
References Barnes, C. A. (1979) Memory deficits associated with senescence: A neurophysiological and behavioral study in the rat. J . Comp. Physiol. Psychol. 93:74-104. Barnes, C. A. (1988) Spatial learning and memory processes: The search for their neurobiological mechanisms in the rat. Trends Neurosci. 11:163-169. Barnes, C. A , , and B. L. McNaughton (1985) An age comparison of the rates of acquisition and forgetting of spatial information in relation to long-term enhancement of hippocampal synapses. Behav. Neurosci. 99: 1040-1048. Berger, T. W. (1984) Long-term potentiation of hippocampal synaptic transmission affects rate of behavioral learning. Science 224:627630. Castro, C. A., L. H. Silbert, B. L. McNaughton, and C. A. Barnes (1989) Recovery of spatial learning deficits after decay of electri-
HIPPOCAMPAL LTP AND LEARNING / Robinson 395 cally induced synaptic enhancement in the hippocampus. Nature 342~545-548. Deupree, D. L., D. A. Turner, and C. L. Watters (1991) Spatial performance correlates with in vitro potentiation in young and aged Fischer 344 rats. Brain Res. 554:l-9. Gilbert, M. E., and C. M. Mack (1990) The NMDA antagonist, MK801, suppresses long-term potentiation, kindling, and kindling-induced potentiation in the perforant path of the unanesthetized rat. Brain Res. 519:89-96. Keith, J. R., and J. W. Rudy (1990) Why NMDA-receptor-dependent long-term potentiation may not be a mechanism of learning and memory: Reappraisal of the NMDA-receptor blockade strategy. Psychobiology 18:251-257. Kirkby, R. D., R. K. McNamara, R. W. Skelton, andM. E. Corcoran (1991) Limbic seizures, but not kindling, impair place learning in the Morris water maze: Retrograde and anterograde deficits. SOC. Neurosci. Abstr. 17:482. Kolb, B., R. J. Sutherland, and I. Q. Whishaw (1983) A comparison of the contributions of the frontal and parietal association cortex to spatial localization in rats. Behav. Neurosci. 97: 13-27. Laroche, S., V. Doyere, and C. Redini Del Negro (1991) What role for long-term potentiation in learning and the maintenance of memories? In Long-term Potentiation: A Debate of Current Issues, M. Baudry and J. L. Davis, eds., pp. 301-316, MIT Press, Cambridge, MA. Lyford, G. L., and L. E. Jarrard (1991) Effects of the competitive NMDA antagonist CPP on performance of a place and cue radial maze task. Psychobiology 19:157-160. McNaughton, B. L. (1982) Long-term synaptic enhancement and short-term potentiation in rat fascia dentata act through different mechanisms. J. Physiol. (Lond.) 324:249-269. McNaughton, B. L., C. A. Barnes, G. Rao, J. Baldwin, and M. Rasmussen (1986) Long-term enhancement of hippocampal synaptic transmission and the acquisition of spatial information. J. Neurosci. 6:563-571. McNaughton, B . L., C. A. Erickson, C. A. Barnes, and G. D. Stevenson (1991) Additive relationship between perforant path LTEI LTP and STEM suggests involvement of different synaptic populations. SOC.Neurosci. Abstr. 17:1395. McNaughton, B. L., and R. G. M. Morris (1987) Hippocampal synaptic enhancement and information storage within a distributed memory system. Trends Neurosci. 10:408-415. Morris, R. G. M. (1989) Synaptic plasticity and learning: Selective impairment of learning in rats and blockade of long-term potentiation in vivo by the N-methyl-D-aspartate receptor antagonist AP5. J. Neurosci. 9:3040-3057. Morris, R. G. M., P. Garrud, J. N. P. Rawlins, and J. O’Keefe (1982) Place navigation impaired in rats with hippocampal lesions. Nature 297 :681-683.
O’Keefe, J., and L. Nadel (1978) The Hippocampus as a Cognitive Map, Oxford University Press, London. Olton, D. S . , and R. J. Samuelson (1976) Remembrance of places passed: Spatial memory in rats. J. Exp. Psychol. (Animal Behav.) 2~97-116. Racine, R. J., and N. W. Milgram (1983) Short-term potentiation phenomena in the rat limbic forebrain. Brain Res. 260:201-216. Racine, R. J., N. W. Milgram, and S. Hafner (1983) Long-term potentiation phenomena in the rat limbic forebrain. Brain Res. 260: 217-231. Robinson, G. B., and G. D. Reed (1992) Effect of MK-801 on the induction and subsequent decay of long-term potentiation in the unanesthetized rabbit hippocampal dentate gyrus. Brain Res. 569: 78-85. Robinson, G. S . , Jr., G . B. Crooks, Jr., P. G. Shinkman, and M. Gallagher (1989) Behavioral effects of MK-801 mimic deficits associated with hippocampal damage. Psychobiology 17:156-164. Shapiro, M. L., and Z. Caramanos (1990) NMDA antagonist MK801 impairs acquisition but not performance of spatial working and reference memory. Psychobiology 18:231-243. Sharp, P. E., B. L. McNaughton, and C. A. Barnes (1985) Enhancement of hippocampal field potentials in rats exposed to a novel, complex environment. Brain Res. 339:361-365. Shors, T. J., T. B. Seib, S. Levine, and R. F. Thompson (1989) Inescapable versus escapable shock modulates long-term potentiation in the rat hippocampus. Science 244:224-226. Skelton, R. W., A. S. Scarth, D. M. Wilkie, J. J. Miller, and A. G. Phillips (1987) Long-term increases in dentate granule cell responsivity accompany operant conditioning. J. Neurosci. 7:3081-3087. Sutherland, R. J., H. C. Dringenberg, J. M. Hoesing, and R. W. Skelton (1991) Is LTE in the hippocampus necessary for place learning? SOC.Neurosci. Abstr. 17:483. Teyler, T. J., and P. Discenna (1984) Long-term potentiation as a candidate mnemonic device. Brain Res. Rev. 7: 15-28. Ward, L., S. E. Mason, and W. C. Abraham (1990) Effects of the NMDA antagonists CPP and MK-801 on radial arm maze performance in rats. Pharmacol. Biochem. Behav. 35785-790. Warren, D. A., C. A. Castro, J. W. Rudy, and S. F. Maier (1991) No spatial learning impairment following exposure to inescapable shock. Psychobiology 19: 127-134. Wilson, R. C. (1981) Changes in translation of synaptic excitation to dentate granule cell discharge accompanying long-term potentiation. I: Differences between normal and reinnervated dentate gyrus. J. Neurophysiol. 46:324-338. Yeckel, M. F., and T. W. Berger (1990) Feedforward excitation of the hippocampus by afferents from the entorhinal cortex: Redefinition of the role of the trisynaptic pathway. Proc. Natl. Acad. Sci. U. S.A. 87:5832-5836.
Maintained Saturation of Hippocampal Long-term Potentiation Does Not Disrupt Acquisition of the Eight-arm Radial Maze Gilbert B. Robinson Department of Psychology, University of New Brunswick, Fredericton, New Brunswick, Canada
ABSTRACT The present experiment examined the anterograde effect of bilateral saturation of long-term potentiation (LTP) of the rat perforant path-granule cell response on acquisition of the eightarm radial maze. To ensure maintained saturation, high-frequency stimulation was applied to the perforant path immediately prior to each trial. LTP did not significantly increase the number of trials required for acquisition of the standard eight-arm radial maze task. Furthermore, the magnitude of LTP did not correlate with the rate of acquisition. LTP also did not significantly affect the number of either working or reference memory errors during subsequent training with only four of the eight arms baited; both control and LTP rats made significantly more reference memory than working memory errors, with the number of both types of errors decreasing as training progressed. These results indicate that prior saturation of LTP within the perforant pathdentate granule cell circuit does not affect acquisition of either the reference or working memory components of the radial maze task. The results are discussed in relation to the role of LTP in acquisition of the eight-arm radial maze. Key words: rat,
dentate gyrus, perforant path, working memory, reference memory
The possibility that long-term potentiation (LTP) of hippocampal synapses is involved in the acquisition andlor retention of information has been suggested by a number of investigators (Berger, 1984; Teyler and Discenna, 1984; McNaughton and Morris, 1987; Skelton et al., 1987; Barnes, 1988; Laroche et al., 1991). Several lines of evidence revolve around the relationship between LTP and spatial learning, a hippocampal-dependent task (O’Keefe and Nadel, 1978; Moms et al., 1982). First, high-frequency activation of the perforant path, resulting in LTP of both the perforant path-hippocampal dentate granule cell population spike and excitatory postsynaptic potential (EPSP), disrupts acquisition of new spatial information on the Barnes circular maze (Barnes, 1979) as well as recently established, but not wellestablished, spatial information (McNaughton et al., 1986). Second, it appears that rats may be capable of learning a spatial location (Morris water maze) only once LTP of the perforant path-granule cell response has decayed (Castro et al., 1989). Third, various correlations/similarities between LTP and spatial learning or memory have been demonstrated, including (1) similarities in LTP decay and the forgetting of spatial information (Barnes, 1979; Barnes and McNaughton, 1985; Deupree et al., 1991); (2) both LTP induction and the acquisition of spatial information are disrupted by antagonists Correspondence and reprint requests to G. B. Robinson, Department of Psychology, University of New Brunswick, Fredericton, New Brunswick, E3B 6E4 Canada.
of N-methyl-D-aspartate (NMDA)-mediated activity (Morris, 1989; Shapiro and Caramanos, 1990;Ward et al., 1990;Lyford and Jarrard, 1991); and (3) the maintenance and retention of LTP and spatial memory, respectively, are not disrupted by NMDA antagonists (Robinson et al., 1989). Together these studies raise the possibility that an LTP-like process (synaptic modifiability leading to increased synaptic strength) (McNaughton and Moms, 1987) occurs during the acquisition of spatial information (e.g., Sharp et al., 1985). The Olton eight-arm radial maze (Olton and Samuelson, 1976) is a potential spatial task with both working and reference memory components. Trial-specific working memory allows the animal to recall arms previously entered on the current trial and therefore avoid reentries. Reference memory, on the other hand, refers to stored representations that are useful for all trials (e.g., spatial representations of the environment, there is food at the end of the arms). McNaughton et al. (1986) previously reported that LTP saturation did not affect subsequent working memory. The effect on reference memory acquisition, however, was not determined, as that component of the task was acquired prior to LTP saturation. Training in the McNaughton et al. (1986) study began 24 hours following LTP saturation and continued for a number of days. As a result, considerable decay of synaptic strength likely would have occurred. Short-term potentiation, for example, decays with a time constant of approximately 1-6 minutes (McNaughton, 1982; Racine and Milgram, 1983), whereas the short- and long-term components of LTP decay
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with time constants of 1.5 hours and 5 days, respectively (Racine et al., 1983). If modifiable synapses are required for spatial learning, training that occurs soon after an intense burst of high-frequency stimulation should produce near maximal levels of impairment. This possibility was addressed in the following experiment. In addition, the anterograde effect of LTP saturation on both the reference and working memory components of the eight-arm radial maze was examined.
MATERIALS AND METHODS Subjects Sixteen male Long-Evans hooded rats were initially prepared as subjects for these experiments, The rats were housed individually with ad lib access to food and water and placed on a 12 hour 1ight:dark cycle. All testing took place within the initial 3 hours of the light cycle. Three of the 16 animals were not used in the analysis (one developed both electrographic and behavioral seizures during training on the eightarm maze, one failed to acquire the eight-arm maze, even after 61 trials, and the headcap was lost from the third animal early in training).
Surgery The rats (400-450 g) were deeply anesthetized with sodium pentobarbitol (65 mg/kg) and placed on a Kopf stereotaxic apparatus. With the upper incisor bar set 3 mm below the interaural line, a recording electrode (a single Teflon-coated stainless steel wire, 0.28 mm diameter) was implanted in the granule cell layer of the dentate gyrus of each hemisphere. The coordinates, relative to bregma, were 3.8 mm posterior and 2.3 mm lateral. Bipolar stimulating electrodes (two Teflon-coated stainless steel wires twisted together and separated horizontally at the tips) were lowered to the perforant path of each hemisphere. The coordinates, relative to bregma, were 7.8 mm posterior and 4.4 mm lateral. In addition to stereotaxic coordinates, electrophysiological monitoring of perforant path-granule cell field potentials was used to maximize final electrode sites. Four stainless steel anchoring screws were also placed in the skull. Two of these screws, located anterior to bregma and lateral to one another, served as ground and reference. The electrodes were anchored to the skull using dental acrylic once the optimal sites were located (i.e., sites that evoked the maximum response amplitude). The electrodes and grounds then were attached to a nine-pin headcap, which was also fixed to the skull using dental acrylic. The rats then were administered 100,000 units of penicillin (i.m.), placed under a heat lamp, and closely monitored during recovery.
Electrophysiology Two to 3 weeks after surgery, the animals were put on a food-deprivation schedule. When an animal reached 90% of its body weight, an input/output (UO) response was collected. This consisted of the application of 50 electrical pulses to the perforant path at 0.1 Hz. The stimulation intensity was increased after every tenth pulse. The intensities for each animal were chosen to evoke a range of response amplitudes from threshold for spike discharge to the maximum amplitude
spike. All stimulation and recording took place within a shielded recording chamber. The above pretetanus 1/0 response was obtained once per day for a minimum of 3 days. Once responses were stable, animals were randomly placed in either the control (CON; n = 6) or experimental group (LTP; n = 7). Ten high-frequency trains of electrical pulses (400 Hz, 50 ms) were then applied to both the right and left perforant path (1 train every 10 s) of animals in the LTP group. The intensity of pulses within each train was twice that necessary to evoke the maximum amplitude population spike to a single pulse (range, 16-30 V). The set of high-frequency trains was repeated once per day for each of the next 4 days (total, 50 trains). Animals were closely monitored for both electrographic and behavioral seizures, both during and following all train stimulation. Except for the high-frequency stimulation, CON subjects received identical treatment. At the end of this period, animals had attained 80% of their ad lib body weight. To ensure the longer-lasting component of LTP was saturated prior to the start of behavioral training, an I/O response was obtained 24 hours after application of each of the first four train sets (i.e., prior to application of train sets two to five). To monitor the shorter-lasting component of LTP (that present at the time of behavioral training), an I/O response was collected 15-25 minutes after application of each of the first five train sets. An I/O response was also obtained 15-25 minutes after criterion was reached on the standard eight-arm task. Inputloutput responses were obtained from CON animals at comparable time points. The perforant path-granule cell responses were bandpass filtered (1 Hz and 3 kHz), sampled by a 12-bit AID converter at 10 kHz for 20 ms, and saved to disk for later analysis of both population spike amplitude and EPSP slope. The EPSP slope was determined between the start of the positivity and a point approximately 1 ms later (but prior to onset of the population spike). Spike amplitude was measured as the height of a line drawn from the peak of the negativity to a tangent joining spike onset and offset.
Apparatus and training The test apparatus was a grey, eight-arm radial maze constructed of wood and elevated 95 cm from the floor. The octagonal central platform was 40 cm in diameter. Each arm was 60 cm long and 9 cm wide with edges 2 cm high. A food cup, 2.5 cm in diameter and 1 cm deep, was located 3 cm from the outer end of each arm. Surrounding the maze were numerous distal cues (e.g., mobiles, posters, tables, chairs, cupboards, filing cabinets) that remained in a constant location from trial to trial. The room was lit with a 60 W bulb. The first trial occurred immediately following collection of the I/O response that was obtained after application of the fifth set of 10 high-frequency trains. All eight arms of the maze were baited with half a piece of Froot Loops cereal. The rat then was placed on the central platform and allowed to explore the maze (i.e., there was no maze adaptation) until all food was consumed. A maximum of 20 minutes was allowed for each trial. Each rat received one trial per day. The set of high-frequency trains was applied to the perforant path immediately prior to the start of all subsequent trials. The maze
HIPPOCAMPAL LTP AND LEARNING / Robinson 391 was carefully cleaned with a damp sponge and then wiped dry following each trial. The number of errors (arm reentries), the order in which arms were entered, and the time to visit all arms were recorded for each trial. An arm entry was scored whenever a rat went more then two thirds of the way down its length. Training continued until a rat made no more than three errors over three consecutive trials with no more than two errors on any given trial. Training on the 418 task began the day after the above criterion was met. Four of the eight arms were baited for each trial. The baited and nonbaited arms remained constant throughout training. Two types of errors were recorded for this task. Entry into an unbaited arm was scored a reference memory error; arm reentries were scored as working memory errors. Training on the 418 task continued for 30 days. The set of high-frequency trains was applied to the perforant path of rats in the LTP group immediately prior to each trial. The headcap of one animal in the LTP group was lost during train-
A
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ing. Thus, the results of the 4/8task are based on six CON and six LTP animals.
RESULTS
Elect rophysiology Figure 1 illustrates changes in both the EPSP and spike component of the perforant path-granule cell response in both the CON (Figs. 1A and lC, respectively) and LTPgroup (Figs. 1B and lD, respectively). There was no significant change in either the EPSP [F(1,5) = .41, P = 3 1 1 or spike [F(1,5) = .75, P = .426] of the CON group, from the baseline I/O to the I/O obtained 15-25 minutes following criterion on the standard eight-arm task. In the LTP group, application of the first train set resulted in a significant increase in the amplitude of both the EPSP [F(1,6) = 47.02, P < .001] and population spike [F(1,6) = 55.93, P < .001]. Subsequent analyses examined whether each additional train set, applied prior to the start of training, resulted in a greater magnitude of the
B
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STIMULATION INTENSITY (Arbitrary Units) Fig. 1 . Changes in the inputloutput response of perforant path-granule cell excitatory postsynaptic potentials (EPSP) and population spike in control (CON) rats (A and C, respectively) and in long-term potentiation (LTP) rats receiving highfrequency stimulation of the perforant path (B and D, respectively). There was significant long-term potentiation of both the EPSP and population spike in the LTP group (n = 7) but not in the CON group (n = 6). Illustrated are average baseline I/O responses obtained prior to tetanization (=), and I/O responses obtained between 15 and 25 minutes after application of the first ( 0 ) and fifth ( 0 ) set of high-frequency trains, and 15-25 minutes after criterion was met for the eight-arm radial maze (0). Due to normal variations in electrode placements, the five stimulation intensities were not identical for all animals. Thus, the arbitrary units of stimulation intensity refer only to each of the five intensities that were tested within each animal (actual stimulation intensities ranged from 2 to 15 V).
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Fig. 2 . Examples of evoked perforant path-granule cell responses in one animal from the long-term potentiation (LTP) group, prior to tetanization (solid lines) and 15-25 minutes following application of the fifth train set (dashed lines). Each response is the average granule cell response to 10 perforant path pulses (0.1 Hz) applied at the low, middle, and high intensity. Calibration bars, 5 mV and 5 ms.
shorter-lasting component of LTP than that induced by the previous train set. The application of additional sets of trains, following the second set, did not result in additional significant increases in either the EPSP [F(1,6) = 3.08, P = .130] or spike [F(1,6) = 4.65, P = .075]. This indicates that saturation of the shorter-lasting component of LTP had occurred prior to the start of training. The average percent increase from baseline (in response to the highest test pulse stimulation intensity) in EPSP slope and spike amplitude at the start of training was 32.0 ? 5.2% and 66.3 ? 8.0%, respectively. Figure 2 compares, for one animal, average responses evoked by the first, third, and fifth stimulation intensity prior to tetanization and 15-25 minutes following application of the fifth train set. The longer-lasting component of LTP also was saturated prior to the start of training. The average percent increment, over the pretetanus amplitude, observed at the highest test pulse intensity for both the EPSP and spike are illustrated in Figure 3. On the first day of training, the average magnitude of the longer-lasting component of LTP of the EPSP and spike was 20.4% and 59.4%, respectively.
the time to complete the trial [F(l,ll) = .97, P = .345]. There was, however, a significant decrease in both the number of errors [F(2,22) = 3.65, P < .043] and the time taken to complete a trial [F(2,22) = 9.19, P < .001], indicating that both groups were learning. Neither the group by error [F(2,22) = 2.62, P = .095] nor the group by time [F(1,6) = .26, P = .773] interactions were significant.
4
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Effect of LTP on acquisition of eight-arm radial maze
T
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I 2 4 hrs post-train set 1 24 hrs post-train set 2 L13 24 hrs Dost-train set 3 24 hrs post-train set 4 ~
The CON and LTP groups did not differ significantly in the number of trials to criterion (see Fig. 4; t-test, t = 1.56, df = 11, P < .147). Control rats required an average of 7.2 ? 1.1 trials, compared to 10.3 k 1.6 for the LTP animals. Both the EPSP [F(1,6) = 11.37, P < .015] and spike [F( 1,6) = 55.07, P < .001] were still significantly potentiated (relative to the pretetanus response) following criterion. The additional train sets applied prior to each training session did not result in significantly larger responses than those obtained following the fifth train set (i.e., prior to the first trial). In fact, the magnitude of LTP of both the EPSP and spike, following criterion, was not significantly different from that observed 15-25 minutes following application of the third train set. Thus, LTP saturation occurred prior to the start of training and was maintained throughout training. All animals received at least three training trials. A repeated measure analysis of variance (ANOVA) did not reveal any group differences in either the number of errors made during the initial three trials [F(l,ll) = 0.84, P = .378], or
80.01 I
Fig. 3 . Saturation of the longer-lasting component of LTP. Illustrated are the average percent increment in excitatory postsynaptic potential (EPSP) (upper panel) and population spike (lower panel), at the highest test pulse intensity, obtained 24 hours after application of each of the first four train sets.
HIPPOCAMPAL LTP AND LEARNING / Robinson l2
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related negatively with trials to criterion (range of r values, - .75- - .09). Only the correlation of the fourth spike amplitude with days to criterion was significant (r = - .752, twotailed test, P < .05), indicating the trials to criterion decreased as the magnitude of spike LTP increased.
7-
Effect of LTP on reference and working memory There was no significant group difference [F(1,10) = 2.87, P = .121] in the number of reference and working memory errors (Fig. 5). There was, however, a significant difference CON
LTP
Fig. 4. Average number of trials to criterion for acquisition of the eight-arm radial maze task in the control (CON) (n = 6) and long-term potentiation (LTP) (n = 7) groups. Error bars, standard error of the mean.
between the number of working and reference memory errors [F(1,10) = 256.33, P < .OOl] and a significant interaction effect between working and reference memory errors [F(9,90) = 3.50, P < .001], indicating a significant difference in the decrease in errors across trials. Subsequent analysis, however, showed both working [F(9,90) = 2.20, P < .029] and reference memory errors [F(9,90) = 7.76, P < .001] decreased significantly over training.
Correlation of LTP magnitude with learning rate If LTP saturation retards subsequent acquisition of the eight-arm radial maze, there should be a positive correlation between the magnitude of LTP (determined 15-25 minutes after tetanization) and trials to criterion. Averaging may obscure this relation (i.e., a rat in the LTP group may have exhibited only a small magnitude of LTP and therefore little disruption of spatial learning would occur). To examine this possibility, the magnitude of LTP (at each of the five IIO intensities), both at the start of training and criterion, was correlated with acquisition rate. There was no correlation between the magnitude of LTP of the EPSP and the rate of acquisition (range of r values, .23-S6). Nevertheless, the positive correlation values indicate a tendency for the trials to criterion to increase as the magnitude of LTP increased. Except for the third stimulation intensity (r = .57 at the start of training and r = 5 8 at criterion) the magnitude of LTP of the population spike cor4 CON 4 CLTP
1
-.
reference memory
w I32
$
workingmemory
0-r 0-
-
.
1
- 0 0 2 2 4 4 6 6 88 1i o0 TRAINING BLOCK (3 mals/block)
Fig. 5. Average number of reference and working memory errors in the 4/8 task in the control (CON) (n = 6; open squares) and long-term potentiation (LTP) (n = 6; closed circles) group. Each block represents the average number of errors over three trials.
DISCUSSION The above results demonstrate that saturation of LTP at perforant path-granule cell synapses does not disrupt acquisition of either the reference or working memory components of the eight-arm radial maze task. Control and LTP animals did not differ in either their rate of acquisition or performance on the standard version of the task (i.e., all eight arms baited) or in the number of either working or reference memory errors during subsequent testing on the 4J8 task. Together these findings imply that LTP saturation affected neither the reference nor working memory components of the tasks. These results therefore extend the results of McNaughton et al. (1986); maintained LTP saturation not only leaves working memory performance intact but also does not affect acquisition of the reference memory components of the eight-arm radial maze. In agreement with the above, there was not a consistent relation between LTP magnitude and learning. If LTP had facilitated or disrupted acquisition, a negative or positive correlation, respectively, should exist between LTP magnitude and trials to criterion. Significant correlations, however, appear to exist between LTP decay and retention of a spatial task (Barnes, 1979; Barnes and McNaughton, 1985; Deupree et al., 1991). Possible relations between LTP decay and retention were not examined in the present study. Acquisition of the radial maze tasks, in the present study, were not disrupted even though training occurred while synapses were maximally potentiated. The eight-arm maze is thought to be a spatial learning task and it is commonly believed that synaptic modifiability within the hippocampus is involved in spatial learning (McNaughton and Morris, 1987; Barnes, 1988). Thus, a number of possible explanations for the present results were considered. First, there may be inherent differences in the nature of the spatial task on the eight-arm maze, compared to either the Barnes circular maze or the Morris water maze. LTP saturation disrupts acquisition of spatial reference memory on both the Barnes circular maze (McNaughton et al., 1986) and the Moms water maze (Castro et al., 1989) but does not disrupt working memory on the eight-arm maze (McNaughton et al., 1986). Sutherland et al.
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(1991), however, recently reported that LTP saturation did not affect subsequent spatial learning in the Morns water maze task. In that study, training occurred soon after tetanization as opposed to 24 hours later (Castro et al., 1989). A second consideration arises from the latter point; the effects of high-frequency stimulation on behavior may differ depending on whether behavioral testing is immediate or delayed by 24 hours. Seizures that are evoked via perforant path stimulation, for example, do not disrupt subsequent acquisition of the eight-arm radial maze if training begins 24 hours after the last seizure is evoked (G.B. Robinson, unpublished observations). In contrast, recent experiments have raised the possibility that training 30-45 minutes after each kindling stimulation (whether or not behavioral seizures are evoked) will disrupt spatial learning on the Morns water maze (Kirkby et al., 1991). The effect of recent seizure activity on acquisition of the eight-arm maze is currently under investigation. Whether or not the time between stimulation and test underlies these differences, if synaptic modifiability is required for spatial learning, testing immediately after tetanization should theoretically produce the greatest disruption of spatial learning. Third, LTP induction within perforant path-dentate synapses, prior to familiarization with the task, may have resulted in the adoption of alternate response strategies, or spatial processing, that was dependent on neural circuits other than those of the hippocampal formation (Kolb et al., 1983). The animals in the present study did not appear to develop a response strategy, in terms of the order in which the arms were entered. Furthermore, as noted above, perforant path kindling results in transynaptic potentiation effects but does not disrupt learning. The induction of LTP in animals familiar with a spatial task, however, may alter subsequent processing. The changed conditions between initial acquisition and retest may impair the retrieval of information necessary for learning to occur. Prior familiarity with the task may also disrupt the ability of other neural structures to assume processing that had previously occurred in the hippocampus. Finally, and most critical, is concern with how LTP saturation is defined. The present results, for example, only demonstrate LTP saturation at those synapses accessible to the recording electrodes. It is possible that LTP either did not occur at all synapses (e.g., in the ventral blade of the dentate gyrus) or may not have saturated throughout the dentate gyrus. However, the magnitude of the longer-lasting component of LTP, of both the EPSP and population spike, was similar to that reported to disrupt the acquisition of spatial information on the Barnes circular maze (McNaughton et al., 1986). Moreover, each train set was applied at an intensity twice that necessary to evoke the maximum spike amplitude to a single pulse. Thus, tetanization procedures should have maximized the number of affected synapses. Furthermore, behavioral testing only occurred when potentiation effects would be near maximal (Racine et al., 1983). Nevertheless, it is possible that additional synaptic modifiability occurred as a result of learning. Short-term exploratory modulation of dentate synapses, for example, is not blocked by electrically-induced LTP (McNaughton et al., 1991). In agreement with the proposed role of hippocampal LTP in spatial learning, several studies have demonstrated that
NMDA antagonists, which block hippocampal dentate synaptic modifiability in behaving animals (Gilbert and Mack, 1990; Robinson and Reed, 1992), disrupt spatial learning on the eight-arm radial maze (Robinson et al., 1989; Shapiro and Caramanos, 1990; Ward et al., 1990; Lyford and Jarrard, 1991). Sutherland et al. (1991), however, found that the NMDA antagonist MK8Ol did not affect spatial learning providing animals were sufficiently familiar with the task (Morris water maze) prior to testing. Furthermore, attributing spatial learning deficits to the blockade of hippocampal NMDA-mediated activity is controversial (Keith and Rudy, 1990; Lyford and Jarrard, 1991). As shown in the present study, LTP saturation, which should block learning-related synaptic modifiability (and limit that blockade to the activated synapses; however, see McNaughton et al., 1991), fails to retard learning on the eight-arm radial maze. The potential discrepancy between the effect of LTP with the effect of a LTP antagonist on spatial learning is not the first reported. Warren et al. (1991) recently reported that inescapable shock, which blocks LTP in area CA1 of the hippocampal formation (Shors et al., 1989), does not block spatial learning. In summary, LTP saturation within perforant path-dentate synapses does not disrupt learning on the eight-arm radial maze. The proposed role of LTP in learning, however, is difficult to disprove (Keith and Rudy, 1990). There could be any number of explanations for negative findings, including the possibilities that LTP either did not occur or was not saturated outside of the region monitored. It also is possible that information processing within the hippocampus may only be disrupted by certain changes in the input/output relationship (E-S potentiation; Wilson, 1981) or by the possible contribution of LTP at perforant path-CA3 and -CA1 synapses (Yeckel and Berger, 1990), both of which may vary across studies. Nevertheless, if acquisition of the reference and working memory components of the eight-arm radial maze requires widespread synaptic modifiability within the dentate gyrus, the procedures utilized here should have resulted in impaired performance.
ACKNOWLEDGMENTS Supported by grants from the Natural Sciences and Engineering Research Council of Canada and from the University of New Brunswick Research Fund.
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