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Stability of long temporal gradients of retrograde amnesia in mice
BEHAVIORAL AND NEURAL BIOLOGY
48, 237-245 (1987)
Stability of Long Temporal Gradients of Retrograde Amnesia in Mice 1 CURT W.
SPANIS* AND LARRY R.
S Q U I R E t "2
*Department of Biology, University of San Diego, San Diego, California 92110; and kVeterans Administration Medical Center, San Diego, and Department of Psychiatry, School of Medicine, University of California, San Diego, San Diego, California 92161 Mice were given a single training trial and then received a series of four electroconvulsive shocks (ECS), 1 h apart, at one of several times after training (1-180 days). Retention was then tested at one of three times after ECS: 7, 14, or 28 days. Control animals that received sham treatment exhibited gradual forgetting with increasing training-retention intervals. Mice given ECS exhibited temporally graded retrograde amnesia, which affected memories acquired up to about 14 days before treatment. The retrograde amnesia was relatively stable. maintaining its temporally graded appearance for at least 28 days after ECS. Some recovery may have occun-ed in the case of memories acquired 7 days or longer before ECS, but memories acquired only 1 or 5 days before ECS did not recover. These findings extend the parallel between experimental amnesia in laboratory animals and human amnesia. © 1987AcademicPress,Inc.
Memory is not fixed at the moment of learning. It appears to change, or consolidate, as time passes. The best support for this idea comes from experimental studies of amnesia, which show that memory remains susceptible to disruption by convulsive stimulation (or other agents) for a period of time after learning. Eventually memory becomes resistant to disruption (Glickman, 1961 ; McGaugh & Herz, 1972). Nearly all previous studies of experimental amnesia following electroconvulsive shock (ECS) in mice and rats have involved a single treatment, which typically produces brief retrograde amnesia, That is, memory has been found to be susceptible to disruption from a few seconds up to several minutes after learning. This retrograde amnesia appears to be long lasting, if not permanent; it does not seem to recover spontaneously (Chevalier, 1965; King & Glasser, This work was supported by the Medical Research Service of the Veterans Administration, by Grant MH24600 from the National Institute of Mental Health, and by the Office of Naval Research. 2 We thank Arthur Shimamura for his comments on the manuscript. Correspondence and reprint requests should be sent to Larry R. Squire, Veterans Admimstration Medical Center, 3350 La Jolla Village Dr. (V116A). San Diego, CA 92161. 237 0163-1047/87 $3.00 Copyright © 1987 by Academic Press, lnc All rights of reproduction in any form reserved
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SPANIS AND SQUIRE
1970; Luttges & McGaugh, 1967; for review, see McGaugh & Herz, 1972). Studies of patients receiving a prescribed course of electroconvulsive therapy (ECT) showed that retrograde amnesia can be extensive (Squire & Cohen, 1979; Squire, Slater, & Chace, 1975). Patients sometimes forgot events that occurred a few years prior to the onset of amnesia without forgetting more remote events. Much of this retrograde amnesia recovered during the months following ECT (Squire, Slater & Miller, 1981). Recently, this finding that retrograde amnesia following convulsive stimulation is sometimes extensive rather than brief was confirmed in experimental animals. Four ECS treatments, spaced 1 h apart, disrupted memories acquired from 1 day to 3 weeks previously (Squire and Spanis, 1984). The retrograde amnesia was temporally graded across this time period. Temporally graded retrograde amnesia covering several days was also found in rats following a combination of locus coeruleus damage and ECS (Zornetzer, Abraham, & Appleton, 1978). There have been no studies in experimental animals of the stability of long temporal gradients of retrograde amnesia. The present study assessed the stability of retrograde amnesia in mice during 28 days following ECS.
METHOD Male Swiss-Webster albino mice (n = 1410, 35-50 g; Simonsen Laboratories, Gilroy, CA) were trained in a one-trial, step-through passive avoidance task, a standardized and much used method for the study of memory in rodents (Jarvik & Kopp, 1967). The apparatus was a troughshaped, two-compartment box, consisting of an outer lighted area (3 cm wide x 7 cm long x 12.5 cm high) and an inner dark area (3 cm x 23 cm x 12.5 cm high). Each mouse was trained and tested only once. For training, a mouse was placed in the start compartment, facing an opaque door to the dark inner compartment. After 10 s, the door to the inner compartment was raised. If the mouse turned to face away from the door to the inner compartment, either before or (more rarely) after the door was raised, the mouse was gently turned to face the dark compartment. When the mouse entered the inner compartment and touched the rear footplates, it received footshock (0.65 mA for 5 s) and then was allowed to escape back to the start compartment. Later, memory was tested in a second trial in which no footshock was delivered. This retest trial was otherwise identical to the training trial, except that the mouse was not turned around once the door was raised. After the door was raised, the time taken to enter the inner compartment and touch the rear plates was recorded automatically (step-through latency). Mice not entering the dark compartment within 600 s were given the maximum score of 600. All training and testing were scheduled between 1100 and 1800 hs. Mice were trained, given ECS at one of several intervals after training
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(ranging from 1 day to 180 days), and then retested either 7, 14, or 28 days after treatment. The results at 14 days after ECS were reported previously (Squire & Spanis, 1984) but are also included here. ECS was always administered four times at 1-h intervals. ECS was delivered transcorneally (60 Hz constant current, sinusoidal waveform, 35 mA for 200 ms), using small metal electrodes covered with saline-soaked cotton pledgets. Clonic-tonic seizures occurred in each case. Control mice, housed together with each ECS-treated group, were handled in an identical way except that no current was passed through the electrodes. Each ECS group consisted of 26-59 mice (mean = 37.9). Each control group consisted of 18-48 mice (mean = 29.2). In summary, there were two treatment groups (mice given ECS and mice given sham treatment), and there were three treatment-retest intervals (7, 14, and 28 days). Within each of these three conditions, there were six to eight different training-treatment intervals (ranging from 1 day to 180 days). The three treatment-retest conditions were employed in three separate experiments conducted at different times. However, within each condition, all training-treatment intervals and both groups (ECS and sham treatment) were tested concurrently.
RESULTS In previous studies involving this task, the response latencies at retest were often not normally distributed, and nonparametric statistics were typically employed. The distribution was examined by subtracting each of the 1410 raw scores from the mean of the group to which each score belonged. This made it possible to inspect the distribution of all 1410 scores around a single mean. The distribution had a single peak and was approximately symmetrical with a slight leftward skew. Only 7.2% of the animals obtained the maximum raw score of 600 s. Accordingly, parametric tests (all two-tailed) were used to analyze the data. However, in order to describe the findings fully, and to facilitate comparisons with previous studies of this task, the data have been presented separately as both mean scores and median scores. Moreover, all comparisons involving two groups were also evaluated with nonparametric tests. With two minor exceptions, noted below, nonparametric tests always yielded the same results as parametric tests. The mean step-through latency during original training was 7.4 s (median = 6 s). Figure 1 shows the results obtained in the retention tests for all ECS and control mice. The upper three panels show mean step-through latencies on the retention test for each of the three conditions (treatmentretest intervals of 7. 14, and 28 days), and for all training-treatment intervals. The standard error of the mean for the control groups ranged from 23 to 49 s (mean = 35 s). The standard error of the mean for the ECS groups ranged from 12 to 39 s (mean = 29 s). The lower three
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FIG. 1. Retention scores of mice given electroconvulsive shock (ECS, four treatments at hourly intervals) or sham treatment (Control) at one of several intervals after training on a one-trial passive avoidance task. Retention was always tested either 7 days (left), 14 days (middle), or 28 days (right) after ECS. The data are expressed both as mean stepthrough latencies on the retention test (upper panels) and as median step-through latencies (lower panels).
panels of Fig. 1 show the same data expressed as median step-through latencies. In all cases a high step-through latency indicates good retention of the training footshock, and a low step-through latency indicates poor retention. Control mice exhibited a forgetting curve, avoiding the inner compartment most strongly at short training-retest intervals. Mice given ECS exhibited marked retrograde amnesia, which diminished in severity as the trainingECS interval increased. Overall, the scores of the control mice and the ECS mice were different when the training-ECS interval was short, and the scores tended to converge when the training-ECS interval was long. Three separate analyses of variance were first done, using the data from each ECS-retest interval (in Figs. 1A-IC). Each analysis thus involved two factors: group (ECS and control) and training-ECS interval. In all three cases, the effect of group was highly significant (Fs > 10.0, ps < .002), showing that mice performed differently depending on whether they had received ECS or sham treatment. The effect of training-ECS
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interval was significant for the 7- and 14-day ECS-retest conditions (Fs > 2.2, ps < .05), but not for the 28-day ECS-retest condition (p > 0.1, Fig. 1C). This finding shows that retention was poorer overall when the training-ECS interval (and correspondingly, the training-retention interval) was long than when the training-ECS interval was short. (Mice in the 28-day condition were not tested at the longer intervals.) Finally, the interaction of group x training-ECS interval was highly significant (Fs > 3.4, ps < .003). This finding shows that ECS affected performance on the retention test differently depending on how long after training ECS was given. Memories formed from 1 day to about 1 or 2 weeks before ECS were disrupted. Memories acquired more than 14 days before ECS were not affected. The characteristics of the retrograde amnesia produced by ECS were explored further by individual comparisons within each ECS-retest condition. In Fig. 1A, comparisons involving corresponding ECS and control groups showed that memories formed 7 days or less before ECS were significantly disrupted (ts > 2.9, ps < .01). Memories formed 14 days or more before treatment were not affected (ts < 1.0, ps > 0.10). A single exception to this finding was that mice given ECS 28 days after training, and then tested 7 days later, performed more poorly than the corresponding control group (t(52) = 2.26, p < .05). However, this difference was not confirmed by a nonparametric test (Mann-Whitney test, p = .19, see Fig. 1D). Within the ECS group, retention was better for mice given ECS 14 days after training than for mice given ECS only 1 day after training (t(84) --- 3.7, p < .01). The results were similar when the interval between treatment and retest was extended to 14 days (Fig. 1B). Memories acquired 1 or 7 days before ECS were disrupted (ts > 2.8, ps < .01), but memories acquired 14 days or more before ECS were not affected (ts < 1.0, ps > 0.10). In addition, within the ECS group, retention was better for mice given ECS 14 days after training than for mice given ECS only 1 day after training (t(101) = 2.63, p < .01) (the nonparametric test for this comparison (Fig. 1E) yielded p = .06). When the interval between ECS and retest was extended to 28 days (Fig. 1C), temporally graded retrograde amnesia was also observed. However, unlike the findings for ECS-retest intervals of 7 and 14 days, in this condition memories acquired 7 days before ECS were not affected. Specifically, memories acquired 1 or 5 days before ECS were disrupted (ts > 2.16, ps < .05), but memories acquired 7 days or more before treatment were not affected (ts < 1.6, ps > 0.10). Within the ECS group, retention was better for mice given ECS 7 days after training than for mice given ECS only 1 day after training (t(62) = 2.01, p < .05). Taken together, these results show that ECS produced temporally graded retrograde amnesia and that amnesia persisted for at least 28 days
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after treatment. This conclusion seems valid, regardless whether the data are expressed as means or medians, and regardless whether the data are analyzed with parametric or nonparametric statistics. Comparisons across the three ECS-retest conditions (7, 14, and 28 days; Figs. 1A-1C) must be made with caution because the mice in these conditions were tested at different times. One way to begin to evaluate the effect of ECS-retest interval is to compare the scores of mice given ECS at the five training-ECS intervals (1, 7, 14, 21, and 28 days) that appeared in all three ECS-retest conditions. Accordingly, the data for the ECS mice were submitted to a two-way analysis of variance (3 E C S retest intervals x 5 training-ECS intervals). This analysis yielded the expected effect of training-ECS interval (F(4, 575) = 4.1, p < .003), confirming that ECS affected retention differently depending on how long after training ECS was given. In addition, there was a significant effect of ECS-retest interval (F(2, 575) = 3.6, p < .03) and a significant interaction between ECS-retest interval and training-ECS interval (F(8, 575) = 3.3, p < .002). The overall means for the ECS mice in the three ECS-retest conditions were 144 s (7 days), 198 s (14 days), and 202 s (28 days). The effect of ECS-retest interval shows that, despite the stability of temporally graded retrograde amnesia, some improvement in retention scores did occur as the interval between ECS and retest was extended. The significant interaction term suggests that the improvement in retention score was affected by the age of the memory at the time of ECS. If retention scores can improve as the ECS-retest interval is extended, one should not expect such improvement to occur equally across all training-ECS intervals. Indeed, it is difficult to see how improvement could occur, except at training-ECS intervals where memory was initially affected by ECS, i.e., training-ECS intervals of 1 to 14 days. The results confirm this expectation. Averaging across training-ECS intervals of 1, 7, and 14 days, the mean retention score improved from 120.2 s to 190.4 s to 213.6 s as the ECS-retest interval was extended from 7 to 14 to 28 days, respectively. For training-ECS intervals of 21 and 28 days, the corresponding retention scores were 204.8, 211.3, and 187.1 s for ECSretest intervals of 7, 14, and 28 days. Thus, to the extent that performance improved with increasing ECS-retest interval, the improvement seemed limited to mice given ECS from 1 to 14 days, but not longer, after training. One clear example of improvement occurred for mice given ECS 7 days after passive avoidance training. In tests done 7 and 14 days after the ECS (Figs. 1A and 1B), marked retrograde amnesia was present. In contrast, in the test done 28 days after ECS (Fig. 1C), memory for passive avoidance training was spared. To assess possible proactive effects of ECS on step-through latency, two additional groups of mice were given four ECS (n = 25) or four
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sham treatments (n = 19) 1 week prior to initial training and were retested 1 day after training. Two other groups were given four ECS (n = 24) or four sham treatments (n = 23) 2 weeks prior to training and were retested 1 day after training. The mean step-through latencies for the groups trained 1 week after treatment were 193 s (ECS) and 298 s (control) t(42) = 1.85, p = .07). For the groups trained 2 weeks after treatment, the scores were 262 s (ECS) and 250 s (control) t(45) = 0.2, p > 0.10). Thus at 1 week after ECS there was a marginal effect of ECS on stepthrough latencies, but by 2 weeks the effects of ECS had subsided.
DISCUSSION A series of four ECS, spaced 1 h apart, produced marked retrograde amnesia, which persisted for at least 28 days. The retrograde amnesia was temporally graded and diminished monotonically as the trainingECS interval increased. On average, memories acquired only a few days prior to treatment were severely disrupted by ECS, memories acquired 21 days or longer prior to ECS were not affected, and memories acquired 7 to 14 days prior to treatment were affected to an intermediate extent. These observations confirm the finding that retrograde amnesia in mice can be temporally graded across long time periods (Squire & Spanis, 1984). In addition, the results show that the phenomenon of temporally graded retrograde amnesia is relatively stable. It can be observed 7 days after ECS, and it is still present after 28 days. At the same time, the results raised the possibility that some recovery from retrograde amnesia may have occurred as time passed after ECS. For example, at 7 and 14 days after ECS, the retrograde amnesia gradient clearly extended to and included memories acquired 7 days before treatment. Yet at 28 days after ECS, only memories acquired 1 and 5 days before treatment were impaired; memories that were 7 days or older at the time of treatment were not significantly affected. Therefore, it is possible that 7-day-old memories were initially affected by ECS but that they spontaneously recovered within 28 days. According to this interpretation, 1-day-old memories were lost for at least 28 days following ECS, perhaps permanently; 7-day-old memories were lost but recovered by 28 days after ECS; memories older than 14 days were not affected, even at 7 days after ECS. This idea is consistent with the finding that retrograde amnesia in psychiatric patients following ECT is recoverable to a considerable extent (Squire, et al., 1981). It is also consistent with the finding that recovery occurs most readily for information acquired months and years before ECT, and less readily or not at all for information acquired only days or weeks before treatment. Finally, this idea also provides support for a widely held view of human retrograde amnesia (albeit one that is still incompletely documented with objective tests), namely, that recovery
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from retrograde amnesia follows a temporal sequence whereby oldest memories recover first and most recent memories recover last (Barbizet, 1970; Benson & Geschwind, 1967; Russell & Nathan, 1946). The present results, however, cannot strongly support or reject these ideas about recovery from retrograde amnesia. Comparisons across the three main conditions of the study (ECS-retest intervals of 7, 14, and 28 days) are complicated by the high variability of the scores in the various groups. In addition, some of the improvement in retention scores may reflect a diminution of the proactive effects of ECS on performance, rather than spontaneous recovery from retrograde amnesia. In particular, differences between the scores obtained 7 and 14 days after ECS could have been due in part to the fact that, in tests of the proactive effects of ECS, performance was marginally affected at 7 days, but not at 14 days, after treatment. Whether or not any recovery actually occurred within 28 days, the present study provides strong confirmation for the idea that retrograde amnesia can be temporally graded across a long time period. In a large number of previous studies, which typically involved a single ECS, retrograde amnesia covered a period of only seconds or minutes (for reviews, see Mah & Albert, 1973; McGaugh & Herz, 1972). In contrast, in the present study and in a previous one (Squire & Spanis, 1984) four ECS treatments produced retrograde amnesia covering a period up to a week or more. Thus, a long temporal gradient of retrograde amnesia is a robust and reproducible phenomenon in mice. This finding strengthens the continuity between results from experimental animals and previous results from patients receiving ECT (Squire & Cohen, 1979; Squire et al., 1975). In three different experimental conditions, from 7 to 28 days after ECS, the temporal gradient of retrograde amnesia had a characteristic shape. Specifically, retention after ECS was always significantly poorer for recent memories than for more remote memories. As discussed previously (Squire & Cohen, 1979; Squire, Cohen, & Nadel, 1984), it is this critical feature of the data that requires a construct like memory consolidation, whereby the structure of memory is presumed to change with the passage of time after learning (Glickman, 1961; McGaugh & Herz, 1972; Muller & Pilzecker, 1900). In addition, the length of the retrograde amnesia gradient in both mice and humans requires that these changes after training must occur in the neural substrates of long-term memory. Such changes could occur in concert with normal forgetting and result in a gradual reorganization of the original representations of stored information. As consolidation proceeds, representations acquire stability and become resistant to disruption by treatments like ECS. These same ideas are also consistent with the possibility that some recovery can occur for information that has partially consolidated at the time that memory is disrupted. In the extreme case of memories formed
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j u s t p r i o r to d i s r u p t i o n , w h i c h h a v e o n l y b e g u n to c o n s o l i d a t e , r e t r o g r a d e amnesia can be permanent. But for memories that have partially consolidated, storage processes may continue (and retrieval may succeed) a f t e r t h e e f f e c t s o f t h e d i s r u p t i v e a g e n t h a v e d i s s i p a t e d . B y this v i e w both the phenomenon of temporally graded retrograde amnesia and the p h e n o m e n o n o f r e c o v e r y ( s p e c i f i c a l l y , t h e s u g g e s t i o n that o l d e r m e m o r i e s r e c o v e r , n o t m e m o r i e s f o r m e d j u s t p r i o r to d i s r u p t i o n ) a r e c o n s i s t e n t with t h e c o n s o l i d a t i o n h y p o t h e s i s . T h u s t e m p o r a l l y g r a d e d r e t r o g r a d e a m n e s i a is c o n s i d e r e d to reflect a d i s r u p t i o n o f m e m o r y storage p r o c e s s e s , w h o s e s e v e r i t y d e p e n d s o n t h e age o f m e m o r y at t h e t i m e o f E C S . C o r r e s p o n d i n g l y , the e x t e n t o f r e c o v e r y is a l s o d e p e n d e n t o n t h e age o f m e m o r y at t h e t i m e o f E C S ( S q u i r e , 1987).
REFERENCES Barbizet, J. (1970). Human memory and its pathology. San Francisco: Freeman. Benson, F., & Geschwind, N. (1967). Shrinking retrograde amnesia. Journal of Neurology, Neurosurgery and Psychiatry, 30, 539-544. Chevalier, J. A. (1965). Permanence of amnesia after a single posttrial electroconvulsive seizure. Journal of Comparative and Physiological Psychology. 59, 125-127. Glickman, S. E. (1961). Perseverative neural processes and consolidation of the memory trace. Psychological Bulletin, 58, 218-233. Jarvik, M. E., & Kopp, R. (1967). An improved one-trial passive avoidance learning situation. Psychological Reports, 21, 221-224. King, R. A., & Glasser, R. L. (1970). Duration of electroconvulsive shock-reduced retrograde amnesia in rats. Physiology and Behavior, 5, 335-339. Luttges, M. W., & McGaugh, J. L. (1967). Permanence of retrograde amnesia produced by electroconvulsive shock. Science, 156, 408-410. Mah, C. J., & Albert, D. J. (1973). Electroconvulsive shock-induced retrograde amnesia: An analysis of the variation in the length of the amnesia gradient. Behavioral Biology, 9, 517-540. McGaugh, J. L., & Herz, M. M. (1972). Memory consolidation. San Francisco: Albion. Muller, G. E., & Pilzecker, A. (1900). Experimentelle Beitrage zur Lehre vom Gedachtniss [Experimental contributions to the theory of memory]. Zeitschrift fur Psychologie, 1, 1-288.
Russell, W. R., & Nathan, P. W. (1946). Traumatic amnesia. Brain, 69, 280-300. Squire, L. R. (1986). Mechanisms of memory. Science, 232, 1612-1619. Squire, L. R. (1987). Memory and brain. New York: Oxford Univ. Press. Squire, L. R., & Cohen, N. J. (1979). Memory and amnesia: Resistance to disruption develops for years after learning. Behavior and Neural Biology, 25, 115-125. Squire, L. R., Cohen, N. J., & Nadel, L. (1984). The medial temporal region and memory consolidation: A new hypothesis. In H. Weingartner & E. Parker (Eds.), Memory consolidation. Hillsdale, NJ: Erlbaum. Squire, L. R., Slater, P. C., & Chace, P. M. (1975). Retrograde amnesia: Temporal gradient in very long-term memory following electroconvulslve therapy. Science, 187, 77-79. Squire, L. R., Slater, P. C., & Miller, P. L. (1981). Retrograde amnesia and bilateral electroconvulsive therapy: Long-term follow-up. Archives of General Psychiatry, 38, 89-95. Squire, L. R., & Spanis, C. W. (1984). Long gradient of retrograde amnesia in mice: Continuity with the findings in humans. Behavioral Neuroscience, 98, 345-348. Zornetzer, S. F., Abraham, W. D., & Appleton, R. A. (1978). Locus coeruleus and labile memory. Pharmacology, Biochemistry, and Behavior, 9, 227-234.
48, 237-245 (1987)
Stability of Long Temporal Gradients of Retrograde Amnesia in Mice 1 CURT W.
SPANIS* AND LARRY R.
S Q U I R E t "2
*Department of Biology, University of San Diego, San Diego, California 92110; and kVeterans Administration Medical Center, San Diego, and Department of Psychiatry, School of Medicine, University of California, San Diego, San Diego, California 92161 Mice were given a single training trial and then received a series of four electroconvulsive shocks (ECS), 1 h apart, at one of several times after training (1-180 days). Retention was then tested at one of three times after ECS: 7, 14, or 28 days. Control animals that received sham treatment exhibited gradual forgetting with increasing training-retention intervals. Mice given ECS exhibited temporally graded retrograde amnesia, which affected memories acquired up to about 14 days before treatment. The retrograde amnesia was relatively stable. maintaining its temporally graded appearance for at least 28 days after ECS. Some recovery may have occun-ed in the case of memories acquired 7 days or longer before ECS, but memories acquired only 1 or 5 days before ECS did not recover. These findings extend the parallel between experimental amnesia in laboratory animals and human amnesia. © 1987AcademicPress,Inc.
Memory is not fixed at the moment of learning. It appears to change, or consolidate, as time passes. The best support for this idea comes from experimental studies of amnesia, which show that memory remains susceptible to disruption by convulsive stimulation (or other agents) for a period of time after learning. Eventually memory becomes resistant to disruption (Glickman, 1961 ; McGaugh & Herz, 1972). Nearly all previous studies of experimental amnesia following electroconvulsive shock (ECS) in mice and rats have involved a single treatment, which typically produces brief retrograde amnesia, That is, memory has been found to be susceptible to disruption from a few seconds up to several minutes after learning. This retrograde amnesia appears to be long lasting, if not permanent; it does not seem to recover spontaneously (Chevalier, 1965; King & Glasser, This work was supported by the Medical Research Service of the Veterans Administration, by Grant MH24600 from the National Institute of Mental Health, and by the Office of Naval Research. 2 We thank Arthur Shimamura for his comments on the manuscript. Correspondence and reprint requests should be sent to Larry R. Squire, Veterans Admimstration Medical Center, 3350 La Jolla Village Dr. (V116A). San Diego, CA 92161. 237 0163-1047/87 $3.00 Copyright © 1987 by Academic Press, lnc All rights of reproduction in any form reserved
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1970; Luttges & McGaugh, 1967; for review, see McGaugh & Herz, 1972). Studies of patients receiving a prescribed course of electroconvulsive therapy (ECT) showed that retrograde amnesia can be extensive (Squire & Cohen, 1979; Squire, Slater, & Chace, 1975). Patients sometimes forgot events that occurred a few years prior to the onset of amnesia without forgetting more remote events. Much of this retrograde amnesia recovered during the months following ECT (Squire, Slater & Miller, 1981). Recently, this finding that retrograde amnesia following convulsive stimulation is sometimes extensive rather than brief was confirmed in experimental animals. Four ECS treatments, spaced 1 h apart, disrupted memories acquired from 1 day to 3 weeks previously (Squire and Spanis, 1984). The retrograde amnesia was temporally graded across this time period. Temporally graded retrograde amnesia covering several days was also found in rats following a combination of locus coeruleus damage and ECS (Zornetzer, Abraham, & Appleton, 1978). There have been no studies in experimental animals of the stability of long temporal gradients of retrograde amnesia. The present study assessed the stability of retrograde amnesia in mice during 28 days following ECS.
METHOD Male Swiss-Webster albino mice (n = 1410, 35-50 g; Simonsen Laboratories, Gilroy, CA) were trained in a one-trial, step-through passive avoidance task, a standardized and much used method for the study of memory in rodents (Jarvik & Kopp, 1967). The apparatus was a troughshaped, two-compartment box, consisting of an outer lighted area (3 cm wide x 7 cm long x 12.5 cm high) and an inner dark area (3 cm x 23 cm x 12.5 cm high). Each mouse was trained and tested only once. For training, a mouse was placed in the start compartment, facing an opaque door to the dark inner compartment. After 10 s, the door to the inner compartment was raised. If the mouse turned to face away from the door to the inner compartment, either before or (more rarely) after the door was raised, the mouse was gently turned to face the dark compartment. When the mouse entered the inner compartment and touched the rear footplates, it received footshock (0.65 mA for 5 s) and then was allowed to escape back to the start compartment. Later, memory was tested in a second trial in which no footshock was delivered. This retest trial was otherwise identical to the training trial, except that the mouse was not turned around once the door was raised. After the door was raised, the time taken to enter the inner compartment and touch the rear plates was recorded automatically (step-through latency). Mice not entering the dark compartment within 600 s were given the maximum score of 600. All training and testing were scheduled between 1100 and 1800 hs. Mice were trained, given ECS at one of several intervals after training
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(ranging from 1 day to 180 days), and then retested either 7, 14, or 28 days after treatment. The results at 14 days after ECS were reported previously (Squire & Spanis, 1984) but are also included here. ECS was always administered four times at 1-h intervals. ECS was delivered transcorneally (60 Hz constant current, sinusoidal waveform, 35 mA for 200 ms), using small metal electrodes covered with saline-soaked cotton pledgets. Clonic-tonic seizures occurred in each case. Control mice, housed together with each ECS-treated group, were handled in an identical way except that no current was passed through the electrodes. Each ECS group consisted of 26-59 mice (mean = 37.9). Each control group consisted of 18-48 mice (mean = 29.2). In summary, there were two treatment groups (mice given ECS and mice given sham treatment), and there were three treatment-retest intervals (7, 14, and 28 days). Within each of these three conditions, there were six to eight different training-treatment intervals (ranging from 1 day to 180 days). The three treatment-retest conditions were employed in three separate experiments conducted at different times. However, within each condition, all training-treatment intervals and both groups (ECS and sham treatment) were tested concurrently.
RESULTS In previous studies involving this task, the response latencies at retest were often not normally distributed, and nonparametric statistics were typically employed. The distribution was examined by subtracting each of the 1410 raw scores from the mean of the group to which each score belonged. This made it possible to inspect the distribution of all 1410 scores around a single mean. The distribution had a single peak and was approximately symmetrical with a slight leftward skew. Only 7.2% of the animals obtained the maximum raw score of 600 s. Accordingly, parametric tests (all two-tailed) were used to analyze the data. However, in order to describe the findings fully, and to facilitate comparisons with previous studies of this task, the data have been presented separately as both mean scores and median scores. Moreover, all comparisons involving two groups were also evaluated with nonparametric tests. With two minor exceptions, noted below, nonparametric tests always yielded the same results as parametric tests. The mean step-through latency during original training was 7.4 s (median = 6 s). Figure 1 shows the results obtained in the retention tests for all ECS and control mice. The upper three panels show mean step-through latencies on the retention test for each of the three conditions (treatmentretest intervals of 7. 14, and 28 days), and for all training-treatment intervals. The standard error of the mean for the control groups ranged from 23 to 49 s (mean = 35 s). The standard error of the mean for the ECS groups ranged from 12 to 39 s (mean = 29 s). The lower three
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FIG. 1. Retention scores of mice given electroconvulsive shock (ECS, four treatments at hourly intervals) or sham treatment (Control) at one of several intervals after training on a one-trial passive avoidance task. Retention was always tested either 7 days (left), 14 days (middle), or 28 days (right) after ECS. The data are expressed both as mean stepthrough latencies on the retention test (upper panels) and as median step-through latencies (lower panels).
panels of Fig. 1 show the same data expressed as median step-through latencies. In all cases a high step-through latency indicates good retention of the training footshock, and a low step-through latency indicates poor retention. Control mice exhibited a forgetting curve, avoiding the inner compartment most strongly at short training-retest intervals. Mice given ECS exhibited marked retrograde amnesia, which diminished in severity as the trainingECS interval increased. Overall, the scores of the control mice and the ECS mice were different when the training-ECS interval was short, and the scores tended to converge when the training-ECS interval was long. Three separate analyses of variance were first done, using the data from each ECS-retest interval (in Figs. 1A-IC). Each analysis thus involved two factors: group (ECS and control) and training-ECS interval. In all three cases, the effect of group was highly significant (Fs > 10.0, ps < .002), showing that mice performed differently depending on whether they had received ECS or sham treatment. The effect of training-ECS
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interval was significant for the 7- and 14-day ECS-retest conditions (Fs > 2.2, ps < .05), but not for the 28-day ECS-retest condition (p > 0.1, Fig. 1C). This finding shows that retention was poorer overall when the training-ECS interval (and correspondingly, the training-retention interval) was long than when the training-ECS interval was short. (Mice in the 28-day condition were not tested at the longer intervals.) Finally, the interaction of group x training-ECS interval was highly significant (Fs > 3.4, ps < .003). This finding shows that ECS affected performance on the retention test differently depending on how long after training ECS was given. Memories formed from 1 day to about 1 or 2 weeks before ECS were disrupted. Memories acquired more than 14 days before ECS were not affected. The characteristics of the retrograde amnesia produced by ECS were explored further by individual comparisons within each ECS-retest condition. In Fig. 1A, comparisons involving corresponding ECS and control groups showed that memories formed 7 days or less before ECS were significantly disrupted (ts > 2.9, ps < .01). Memories formed 14 days or more before treatment were not affected (ts < 1.0, ps > 0.10). A single exception to this finding was that mice given ECS 28 days after training, and then tested 7 days later, performed more poorly than the corresponding control group (t(52) = 2.26, p < .05). However, this difference was not confirmed by a nonparametric test (Mann-Whitney test, p = .19, see Fig. 1D). Within the ECS group, retention was better for mice given ECS 14 days after training than for mice given ECS only 1 day after training (t(84) --- 3.7, p < .01). The results were similar when the interval between treatment and retest was extended to 14 days (Fig. 1B). Memories acquired 1 or 7 days before ECS were disrupted (ts > 2.8, ps < .01), but memories acquired 14 days or more before ECS were not affected (ts < 1.0, ps > 0.10). In addition, within the ECS group, retention was better for mice given ECS 14 days after training than for mice given ECS only 1 day after training (t(101) = 2.63, p < .01) (the nonparametric test for this comparison (Fig. 1E) yielded p = .06). When the interval between ECS and retest was extended to 28 days (Fig. 1C), temporally graded retrograde amnesia was also observed. However, unlike the findings for ECS-retest intervals of 7 and 14 days, in this condition memories acquired 7 days before ECS were not affected. Specifically, memories acquired 1 or 5 days before ECS were disrupted (ts > 2.16, ps < .05), but memories acquired 7 days or more before treatment were not affected (ts < 1.6, ps > 0.10). Within the ECS group, retention was better for mice given ECS 7 days after training than for mice given ECS only 1 day after training (t(62) = 2.01, p < .05). Taken together, these results show that ECS produced temporally graded retrograde amnesia and that amnesia persisted for at least 28 days
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after treatment. This conclusion seems valid, regardless whether the data are expressed as means or medians, and regardless whether the data are analyzed with parametric or nonparametric statistics. Comparisons across the three ECS-retest conditions (7, 14, and 28 days; Figs. 1A-1C) must be made with caution because the mice in these conditions were tested at different times. One way to begin to evaluate the effect of ECS-retest interval is to compare the scores of mice given ECS at the five training-ECS intervals (1, 7, 14, 21, and 28 days) that appeared in all three ECS-retest conditions. Accordingly, the data for the ECS mice were submitted to a two-way analysis of variance (3 E C S retest intervals x 5 training-ECS intervals). This analysis yielded the expected effect of training-ECS interval (F(4, 575) = 4.1, p < .003), confirming that ECS affected retention differently depending on how long after training ECS was given. In addition, there was a significant effect of ECS-retest interval (F(2, 575) = 3.6, p < .03) and a significant interaction between ECS-retest interval and training-ECS interval (F(8, 575) = 3.3, p < .002). The overall means for the ECS mice in the three ECS-retest conditions were 144 s (7 days), 198 s (14 days), and 202 s (28 days). The effect of ECS-retest interval shows that, despite the stability of temporally graded retrograde amnesia, some improvement in retention scores did occur as the interval between ECS and retest was extended. The significant interaction term suggests that the improvement in retention score was affected by the age of the memory at the time of ECS. If retention scores can improve as the ECS-retest interval is extended, one should not expect such improvement to occur equally across all training-ECS intervals. Indeed, it is difficult to see how improvement could occur, except at training-ECS intervals where memory was initially affected by ECS, i.e., training-ECS intervals of 1 to 14 days. The results confirm this expectation. Averaging across training-ECS intervals of 1, 7, and 14 days, the mean retention score improved from 120.2 s to 190.4 s to 213.6 s as the ECS-retest interval was extended from 7 to 14 to 28 days, respectively. For training-ECS intervals of 21 and 28 days, the corresponding retention scores were 204.8, 211.3, and 187.1 s for ECSretest intervals of 7, 14, and 28 days. Thus, to the extent that performance improved with increasing ECS-retest interval, the improvement seemed limited to mice given ECS from 1 to 14 days, but not longer, after training. One clear example of improvement occurred for mice given ECS 7 days after passive avoidance training. In tests done 7 and 14 days after the ECS (Figs. 1A and 1B), marked retrograde amnesia was present. In contrast, in the test done 28 days after ECS (Fig. 1C), memory for passive avoidance training was spared. To assess possible proactive effects of ECS on step-through latency, two additional groups of mice were given four ECS (n = 25) or four
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sham treatments (n = 19) 1 week prior to initial training and were retested 1 day after training. Two other groups were given four ECS (n = 24) or four sham treatments (n = 23) 2 weeks prior to training and were retested 1 day after training. The mean step-through latencies for the groups trained 1 week after treatment were 193 s (ECS) and 298 s (control) t(42) = 1.85, p = .07). For the groups trained 2 weeks after treatment, the scores were 262 s (ECS) and 250 s (control) t(45) = 0.2, p > 0.10). Thus at 1 week after ECS there was a marginal effect of ECS on stepthrough latencies, but by 2 weeks the effects of ECS had subsided.
DISCUSSION A series of four ECS, spaced 1 h apart, produced marked retrograde amnesia, which persisted for at least 28 days. The retrograde amnesia was temporally graded and diminished monotonically as the trainingECS interval increased. On average, memories acquired only a few days prior to treatment were severely disrupted by ECS, memories acquired 21 days or longer prior to ECS were not affected, and memories acquired 7 to 14 days prior to treatment were affected to an intermediate extent. These observations confirm the finding that retrograde amnesia in mice can be temporally graded across long time periods (Squire & Spanis, 1984). In addition, the results show that the phenomenon of temporally graded retrograde amnesia is relatively stable. It can be observed 7 days after ECS, and it is still present after 28 days. At the same time, the results raised the possibility that some recovery from retrograde amnesia may have occurred as time passed after ECS. For example, at 7 and 14 days after ECS, the retrograde amnesia gradient clearly extended to and included memories acquired 7 days before treatment. Yet at 28 days after ECS, only memories acquired 1 and 5 days before treatment were impaired; memories that were 7 days or older at the time of treatment were not significantly affected. Therefore, it is possible that 7-day-old memories were initially affected by ECS but that they spontaneously recovered within 28 days. According to this interpretation, 1-day-old memories were lost for at least 28 days following ECS, perhaps permanently; 7-day-old memories were lost but recovered by 28 days after ECS; memories older than 14 days were not affected, even at 7 days after ECS. This idea is consistent with the finding that retrograde amnesia in psychiatric patients following ECT is recoverable to a considerable extent (Squire, et al., 1981). It is also consistent with the finding that recovery occurs most readily for information acquired months and years before ECT, and less readily or not at all for information acquired only days or weeks before treatment. Finally, this idea also provides support for a widely held view of human retrograde amnesia (albeit one that is still incompletely documented with objective tests), namely, that recovery
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from retrograde amnesia follows a temporal sequence whereby oldest memories recover first and most recent memories recover last (Barbizet, 1970; Benson & Geschwind, 1967; Russell & Nathan, 1946). The present results, however, cannot strongly support or reject these ideas about recovery from retrograde amnesia. Comparisons across the three main conditions of the study (ECS-retest intervals of 7, 14, and 28 days) are complicated by the high variability of the scores in the various groups. In addition, some of the improvement in retention scores may reflect a diminution of the proactive effects of ECS on performance, rather than spontaneous recovery from retrograde amnesia. In particular, differences between the scores obtained 7 and 14 days after ECS could have been due in part to the fact that, in tests of the proactive effects of ECS, performance was marginally affected at 7 days, but not at 14 days, after treatment. Whether or not any recovery actually occurred within 28 days, the present study provides strong confirmation for the idea that retrograde amnesia can be temporally graded across a long time period. In a large number of previous studies, which typically involved a single ECS, retrograde amnesia covered a period of only seconds or minutes (for reviews, see Mah & Albert, 1973; McGaugh & Herz, 1972). In contrast, in the present study and in a previous one (Squire & Spanis, 1984) four ECS treatments produced retrograde amnesia covering a period up to a week or more. Thus, a long temporal gradient of retrograde amnesia is a robust and reproducible phenomenon in mice. This finding strengthens the continuity between results from experimental animals and previous results from patients receiving ECT (Squire & Cohen, 1979; Squire et al., 1975). In three different experimental conditions, from 7 to 28 days after ECS, the temporal gradient of retrograde amnesia had a characteristic shape. Specifically, retention after ECS was always significantly poorer for recent memories than for more remote memories. As discussed previously (Squire & Cohen, 1979; Squire, Cohen, & Nadel, 1984), it is this critical feature of the data that requires a construct like memory consolidation, whereby the structure of memory is presumed to change with the passage of time after learning (Glickman, 1961; McGaugh & Herz, 1972; Muller & Pilzecker, 1900). In addition, the length of the retrograde amnesia gradient in both mice and humans requires that these changes after training must occur in the neural substrates of long-term memory. Such changes could occur in concert with normal forgetting and result in a gradual reorganization of the original representations of stored information. As consolidation proceeds, representations acquire stability and become resistant to disruption by treatments like ECS. These same ideas are also consistent with the possibility that some recovery can occur for information that has partially consolidated at the time that memory is disrupted. In the extreme case of memories formed
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j u s t p r i o r to d i s r u p t i o n , w h i c h h a v e o n l y b e g u n to c o n s o l i d a t e , r e t r o g r a d e amnesia can be permanent. But for memories that have partially consolidated, storage processes may continue (and retrieval may succeed) a f t e r t h e e f f e c t s o f t h e d i s r u p t i v e a g e n t h a v e d i s s i p a t e d . B y this v i e w both the phenomenon of temporally graded retrograde amnesia and the p h e n o m e n o n o f r e c o v e r y ( s p e c i f i c a l l y , t h e s u g g e s t i o n that o l d e r m e m o r i e s r e c o v e r , n o t m e m o r i e s f o r m e d j u s t p r i o r to d i s r u p t i o n ) a r e c o n s i s t e n t with t h e c o n s o l i d a t i o n h y p o t h e s i s . T h u s t e m p o r a l l y g r a d e d r e t r o g r a d e a m n e s i a is c o n s i d e r e d to reflect a d i s r u p t i o n o f m e m o r y storage p r o c e s s e s , w h o s e s e v e r i t y d e p e n d s o n t h e age o f m e m o r y at t h e t i m e o f E C S . C o r r e s p o n d i n g l y , the e x t e n t o f r e c o v e r y is a l s o d e p e n d e n t o n t h e age o f m e m o r y at t h e t i m e o f E C S ( S q u i r e , 1987).
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Russell, W. R., & Nathan, P. W. (1946). Traumatic amnesia. Brain, 69, 280-300. Squire, L. R. (1986). Mechanisms of memory. Science, 232, 1612-1619. Squire, L. R. (1987). Memory and brain. New York: Oxford Univ. Press. Squire, L. R., & Cohen, N. J. (1979). Memory and amnesia: Resistance to disruption develops for years after learning. Behavior and Neural Biology, 25, 115-125. Squire, L. R., Cohen, N. J., & Nadel, L. (1984). The medial temporal region and memory consolidation: A new hypothesis. In H. Weingartner & E. Parker (Eds.), Memory consolidation. Hillsdale, NJ: Erlbaum. Squire, L. R., Slater, P. C., & Chace, P. M. (1975). Retrograde amnesia: Temporal gradient in very long-term memory following electroconvulslve therapy. Science, 187, 77-79. Squire, L. R., Slater, P. C., & Miller, P. L. (1981). Retrograde amnesia and bilateral electroconvulsive therapy: Long-term follow-up. Archives of General Psychiatry, 38, 89-95. Squire, L. R., & Spanis, C. W. (1984). Long gradient of retrograde amnesia in mice: Continuity with the findings in humans. Behavioral Neuroscience, 98, 345-348. Zornetzer, S. F., Abraham, W. D., & Appleton, R. A. (1978). Locus coeruleus and labile memory. Pharmacology, Biochemistry, and Behavior, 9, 227-234.