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Recovery from hypoxia and ECS-induced amnesia after a single exposure to training environment
Physiology and Behavior, Vol. 10, pp. 85-89, Brain Research Publications Inc., 1973. Printed in U.S.A.
Recovery from Hypoxia and ECS-Induced Amnesia After a Single Exposure to Training Environment S. J. S A R A l
Center for Experimental and Comparative Psychology University o f Louvain, Pellenberg, Belgium
(Received 1 August 1972)
SARA, S. J. Recovery from hypoxia and ECS-induced amnesia after a single exposure to training environment. PHYSIOL. BEHAV. 10(1) 85-89, 1973.A single exposure to the training environment for 3 rain was sufficient to produce recovery from hypoxia-induced amnesia when tested 30 rain later. Recovery from ECS-induced amnesia was evidenced 5 rain after exposure to the training environment. The results make a state dependent learning explanation for memory loss after ECS or hypoxia unfeasible, since the recovery is seen immediately after the memory loss is evidenced. The author proposes that amnestie treatments prevent the integration of the training event into existing functional memory systems and consequently the training has no significance for the animal. Reexposure to the training environment precipitates a continuation of these integrative processes and the memory becomes organized in such a way that it has a behavioral consequence (avoidance) on the subsequent trial. Memory
Retrograde amnesia
Hypoxia
ECS
Avoidance learning
SARA, S. J. La r~cuperation de l'amn~sie induite par hypoxie aprbs exposition une seule lois ~ l'environment d'apprentissage. PHYSIOL. BEHAV. 10(1) 85-89,1973.- La r6cuperation de l'amn6sie induite par hypoxie peut 6tre obtenue en exposant les rats une seule fois ~ l'environment d'apprentissage ce qui est confirm6 par un test effectu~ 30 min apr~s exposition. La r6cuperation de l'amndsie induite par ECS fur raise en 6vidence d~s les 5 min qui suivaient l'exposition des sujets h l'environment de l'apprentissage. D'apr~s ces rdsultats, une interpretation de type "stated dependent learning" ne parait pas adequate pour expliquer l'anm~sie apr6s un ECS ou une hypoxie, vu que ron observe une rdcuperation presque immediate apr~.s avoir 6tabli l'amn6sie. Scion l'auteur, les interventions amn~siques emp~cheraient l'int6gration des traitments qui caract~rissent ia situation d'apprentissage, dans les syst6mes fonetionels de la m6moire. C'est pourquoi la situation d'apprentissage perd sa signification pour l'animal. Pourtant, lorsque l'animal est r6expose ~ l'environment d'apprentissage, ces proeesSus d'int6gration sont de nouveau d~clench6s et la m~moire s'organise de telle sorte qu'elle peut avoir une consequence observable lors de l'essai suivant. M6moire
Amn6sie induite
Hypoxie
ECS
IT HAS been well established that recovery f r o m experimentally induced amnesia is possible. The c o n d i t i o n s for recovery of m e m o r y vary according t o the e x p e r i m e n t a l conditions and in s o m e studies an a t t e m p t at recovery was not successful. Kohlenberg and Tarbasso [5] have shown that there can be spontaneous recovery f r o m retrograde amnesia, as have Young and GaUuscio [ I l ]. Galluscio [ 3 ] found recovery from amnesia after giving a reminder footshock. Quartermain, McEwen and Azmitia [8] report results which showed recovery f r o m amnesia p r o d u c e d by b o t h ECS and cycloheximide. These results are particularly impressive because t h e y include t w o amnestic agents whose m o d e of action is presumably different. The authors point
out that the conditions for producing this recovery are rather limited. T h e y tried unsuccessfully to restore m e m o r y for f o o t s h o c k by giving a reminder shock in an environment different f r o m that of the training, o n the day after the training and before the test. Further studies revealed that a testing session before the reminder shock was necessary for the reminder to be effective in restoring memory. On this testing session an amnesia for the previous f o o t s h o c k was, of course, evidenced. The authors indicate that, although this first test had not been considered by others to be a crucial factor in reactivating m e m o r y , it was, in fact, included in the experimental procedures of two o t h e r reports of m e m o r y recovery after a reminder [6 ].
1The author would like to thank Dr. C. Giurgea for critical comment and helpful guidance. 85
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SARA
Z i n k i n a n d Miller [ 1 2 ] f o u n d a slight t e n d e n c y for m e m o r y t o r e a p p e a r a f t e r r e p e a t e d testing, b u t this s t u d y lacks t h e p r o p e r c o n t r o l s to i n d i c a t e if t h e recovery was t h e result of t h e r e p e a t e d testing or t h e mere passage o f time. Herz and Peeke [4] in a similar e x p e r i m e n t , s h o w e d t h a t it was t h e r e p e a t e d testing w h i c h a c c o u n t e d f o r t h e r e c o v e r y and n o t t h e passage o f time. This was i n t e r p r e t e d as d u e to a "gradual adaptation to the experimental situation". But these results m i g h t just as well b e e n i n t e r p r e t e d as evidence of a r e m i n d e r effect of the s i t u a t i o n itself. P e r h a p s t h e amnesia evidenced during t h e first test was only partial, a n d not due to t h e failure of t h e m e m o r y to c o n s o l i d a t e , b u t to o t h e r factors, such as t h e lack of a n a d e q u a t e i n t e g r a t i o n of the s h o c k e x p e r i e n c e with t h e e n v i r o n m e n t a l cues. T h e p r e s e n t e x p e r i m e n t investigates t h e possibility t h a t m e m o r y can be recovered w i t h a single test acting as a r e m i n d e r , t h e r e c o v e r y being evidenced o n a s u b s e q u e n t test. EXPERIMENT I. HYPOXIA-INDUCED AMNESIA AND RECOVERY
Method Animals. F i f t y - f o u r male rats of t h e Wistar strain were o b t a i n e d f r o m U n i o n C h i m i q u e Beige (UCB). T h e y weighed 1 5 0 - 2 0 0 g at t h e t i m e o f t h e e x p e r i m e n t and were h o u s e d 8 - 10 to a cage w i t h free access t o f o o d a n d water. Apparatus. B o t h t h e e x p e r i m e n t a l t r a i n i n g cage a n d t h e e q u i p m e n t for i n d u c i n g h y p o x i a were t h e same as was used by Sara a n d Lefevre [ 9 ] . T h e t r a i n i n g cage was a large c o m p a r t m e n t m a d e of a l u m i n u m a n d p a i n t e d white, w i t h a gravel floor, measuring 3 0 x 3 0 x 3 0 cm a n d was c o n n e c t e d b y a 6x6 c m guillotine d o o r t o a small c o m p a r t m e n t , p a i n t e d black, m e a s u r i n g 1 0 x l 0 x l 0 cm. T h e f l o o r o f t h e small c o m p a r t m e n t was m a d e of m e t a l rods o f 2.5 m m w i d t h and 12 m m apart. T h e s h o c k was delivered t h r o u g h a F o r i n g e r s h o c k scrambler. T h e shock source was a 7 0 0 V, 50 Hz t r a n s f o r m e r w h o s e o u t p u t c u r r e n t is limited b y a variable series resistence. A n a m p e r e m e t e r in place o f t h e rat s h o w e d a c u r r e n t of ~ 500 m i c r o a m p s . T h e cage for i n d u c i n g h y p o x i a consisted of an airtight Plexiglas b o x measuring 5 0 x 3 0 x 3 0 cm. Pure n i t r o g e n was i n t r o d u c e d into t h e cage and was mixed w i t h t h e a t m o sphere b y a v e n t i l a t o r a t t a c h e d to o n e o f t h e walls. A B e c k m a n 777 a n a l y z e r c o n t i n u o u s l y m e a s u r e d t h e p e r c e n t age o f o x y g e n in t h e a t m o s p h e r e of t h e cage. Experimental plan and procedure. T h e animals were divided r a n d o m l y i n t o f o u r groups. T h e t r a i n i n g p r o c e d u r e was as follows. T h e a n i m a l was placed in t h e large c o m p a r t m e n t of t h e a p p a r a t u s , facing away f r o m t h e d o o r to the small c o m p a r t m e n t . It was allowed to explore t h e t w o c o m p a r t m e n t s freely for 3 min. T h e l a t e n c y to e n t e r and t h e t o t a l t i m e s p e n t in t h e small c o m p a r t m e n t was n o t e d for each animal. A f t e r t h e 3 m i n f a m i l i a r i z a t i o n period t h e d o o r was lowered and t h e a n i m a l received 20 sec of f o o t s h o c k and was t h e n r e m o v e d f r o m t h e small c o m p a r t m e n t . T h e FS-NHYP g r o u p were c o n f i n e d for l 0 m i n in a Plexiglas cage, a f t e r w h i c h t h e y were r e m o v e d t o the h o m e cage. T h e FS-HYP g r o u p was i n t r o d u c e d immediately a f t e r t r a i n i n g i n t o t h e h y p o x i a cage at 31A% o x y g e n level and was m a i n t a i n e d at this level f o r 10 min. A f t e r r e c u p e r a t i o n t h e a n i m a l was r e t u r n e d to t h e h o m e cage. T w e n t y - f o u r hr later t h e rat was placed in the large c o m p a r t m e n t in the same m a n n e r as o n Day 1 and t h e
latency a n d t o t a l t i m e spent in the small c o m p a r t m e n t was noted. T h e rat was r e m o v e d from t h e a p p a r a t u s and r e t u r n e d t o t h e g r o u p cage. T h i r t y min later t h e rat was replaced in the t r a i n i n g cage a n d t h e latency and t o t a l time in the small c o m p a r t m e n t were again recorded. Later a f i f t h g r o u p was a d d e d to t h e e x p e r i m e n t to d e t e r m i n e if t h e effect o f t h e a m n e s i a p r o d u c e d b y t w o successive h y p o x i a t r e a t m e n t s w o u l d be resistant t o t h e r e m i n d e r effect. T h e p r o c e d u r e was t h e same as f o r t h e group F S - H Y P e x c e p t t h a t 15 m i n a f t e r removal f r o m t h e h y p o x i a cage t h e rat was r e i n t r o d u c e d i n t o t h e cage at 31A% and again m a i n t a i n e d for 10 rain ( G r o u p F S - H Y P - H Y P ) .
Results T h e measure of r e t e n t i o n was t h e t o t a l t i m e spent in t h e small c o m p a r t m e n t . O n Day 1, b e f o r e training, t h e animals spent a b o u t 80% of t h e t o t a l time in t h e dark. A Kruskal-Wallis one way analysis of variance s h o w e d t h a t t h e r e was no d i f f e r e n c e a m o n g g r o u p s o n Day 1 as t o t o t a l time s p e n t in t h e dark. ( H = 4 . 8 6 , dr=4, p > 0 . 3 0 ) As can be seen f r o m Fig. 1, t h e r e was a m a r k e d d i f f e r e n c e in t h e time s p e n t in the dark b e t w e e n T, and T2 for t h e g r o u p F S - N H Y P , indicating t h a t t h e t r a i n i n g is effective. T h e fact t h a t t h e r e is no difference b e t w e e n T~ and T3 for this g r o u p indicates t h a t no e x t i n c t i o n is seen o n t h e second test. T h a t the p e r f o r m a n c e of t h e c o n t r o l g r o u p N F S - N H Y P was stable over t h e t h r e e t i m e s indicates t h a t t h e r e is no s p o n t a n e o u s decrease in t h e a t t r a c t i o n o f the small dark c o m p a r t m e n t . T h a t h y p o x i a is n o t aversive is shown by the performance of the group NFS-HYP, which does n o t avoid t h e d a r k over t h e t h r e e times. T h e g r o u p F S - H Y P , significantly d i f f e r e n t f r o m t h e F S - H Y P group at T~ ( M a n n - W h i t n e y U test, two-tailed, U = 3 9 , p < 0 . 0 2 ) has c o m p l e t e l y r e c u p e r a t e d at T3. A W i l c o x o n m a t c h e d pairs signed r a n k s test shows a highly significant d i f f e r e n c e b e t w e e n T2 a n d T3 for this group. T h e r e is no d i f f e r e n c e b e t w e e n G r o u p s F S - H Y P and F S - N H Y P at T3, i n d i c a t i n g recovery f r o m the amnesia seen at T , . T h e g r o u p F S - - H Y P - H Y P was n o t d i f f e r e n t f r o m t h e group F S - H Y P at a n y t i m e a n d can, t h e r e f o r e , be c o n s i d e r e d as a c h e c k o n t h e reliability o f t h e findings.
Discussion T h e results of E x p e r i m e n t 1 verify t h e findings of Sara and Lefevre [9] t h a t h y p o x i a does, u n d e r certain conditions, p r o d u c e r e t r o g r a d e amnesia. However, this m e m o r y loss is n o t p e r m a n e n t and a r e m i n d e r serves t o reactivate the m e m o r y . T h e results agree w i t h t h o s e o f Q u a r t e r m a i n et aL [ 8 ] , b u t also i n d i c a t e t h a t a n i n t e r m e d i a r y test is n o t only a necessary c o n d i t i o n , b u t a sufficient c o n d i t i o n as well, for the r e c o v e r y p h e n o m e n o n . EXPERIMENT 2 ECS INDUCED AMNESIA AND RECOVERY This e x p e r i m e n t a t t e m p t s to generalise t h e results seen w i t h h y p o x i a by e m p l o y i n g a m o r e widely studied a m n e s t i c agent, ECS.
Method Animals. T h e animals were 24 male Wistar rats, 1 6 0 - - 2 0 0 g, o b t a i n e d f r o m t h e same source a n d h o u s e d u n d e r the same c o n d i t i o n s as E x p e r i m e n t I. Apparatus. T h e t r a i n i n g cage was t h e same as used in
RECOVERY FROM HYPOXIA AND E C S - I N D U C E D AMNESIA
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20
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T1
12
T3
FIG. 1. Median time in sec spent in the small dark compartment before training (T,), 24 hr after training (T 2 ) and 24 hr and 30 rain after training (Ta). Note the performance decrement of both hypoxia groups at the first test (T2) and the total recuperation at T3,30 min later. Duration of each trial: 180 sec. Experiment 1. ECS was effected by means of a transtemporal shock from a 1500 V transformer from which a 50 mA shock of 0.25 sec was delivered via a 10 Ks2 series resistance. Experimental plan and procedure. The animals were divided into three groups. The training was the same as in Experiment 1. Group F S - E C S received training followed immediately by ECS outside of the training cage. Group F S - N E C S had training and sham ECS; Group N F S - E C S was permitted to explore the apparatus for 3 rain and then was removed and convulsed. A slight variation in the testing procedure on Day 2 was that the time between T2 and T3 was reduced to 5 rain.
Results Figure 2 indicates the median time spent in the dark on T I , T2 and T3. The results are similar to those of Experiment 1. A Kruskal-Wallis one-way analysis of variance indicates no difference among groups at T~ before training. (H-l, dr=2, p>0.50). Group F S - E C S was significantly different from Group F S - N E C S at T , , indicating a memory loss (Mann-Whitney U test one-tailed U = I 4 , p=0.025). A Wilcoxon Matched Pairs Signed Ranks test shows that the difference between T~ and Ts for Group F S - E C S is significant (T=4, p<0.02, two-tailed), thus indicating recovery from amnesia. No other group showed a difference between T2 and T3. That the ECS treatment itself does not cause avoidance of the small compartment at T2 is indicated by the fact that Group N F S - E C S spends about the same amount of t i m e in the dark on each of the three occasions.
Discussion The results of Experiment 2 indicate that ECS induced
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amnesia can be reversed by merely placing the animal in the training environment for 3 rain, 24 hr after footshock ECS treatment. A reminder shock in a neutral environment is not necessary to recovery. This experiment shows that the reminder can precede the recovery of performance by as little as 5 min. GENER AL DISCUSSION Quartermain et al. [8] showed that reexposure to the training environment is a necessary condition for the restitution of memory; the present experiments indicate that this reexposure is a sufficient condition. The results do not support the interpretation of a similar phenomenon by Young and Galluscio [ 12] that it is due to the fact that the original loss was a result of suppression of the conditioned emotional response (CER). According to these authors, it was the CER which later reappeared as a function of time, manifesting itself as recovery from amnesia. In the present experiments the time between the first test, which serves as the reminder, and the second test is only 30 rain in Experiment l and 5 rain in Experiment 2, which seems to rule out any interpretation due solely to time factors. Some authors have interpreted the amnesic results seen with ECS as due to a state dependent learning phenomenon. (DeVietti and Larson [ 2 ] , Chorover and DeLuca [1]). The performance decrement seen on Day 2 is attributed to the fact that the physiological changes introduced by the synergistic interaction of the ECS and the footshock have not completely attenuated 24 hr later. The animal is in a different brain state during the test than during the acquisition of the response. Recovery occurs with later testing (48 or 72 hr, according to Zinkin and Miller [13]) because it is then that the rat has fully recovered from the postictal effects of the ECS and is in the same brain state as during the training. Therefore the
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140 I 120 100 80 \
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FIG. 2. Median time in sec spent in the small dark compartment before training (TI), 24 hr after training (T2) and 24 hr and 5 min after training (T~). Note the performance decrement of the FS-ECS group at T2 and the total recuperation at T3,5 min later. Duration of each trial: 180 sec.
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SARA
m e m o r y is retrievable. However, T h o m p s o n and Neely [ 11 ] have shown that rats convulsed 5 sec after training do not show amnesia 24 hr later, if they received another convulsion 20 min before the test. The effect of the second ECS was to introduce the same physiological state as during the storage phase, i m m e d i a t e l y after training. ECS-induced amnesia they have attributed to dissociated learning which presumably is not unlike the state d e p e n d e n t learning, except in one case the dissociated state is dissociated from the condition immediately after training and in the other case it is the training condition which is dissociated. There is also disagreement between the two as to the c o n d i t i o n of the brain at the time of test. T h o m p s o n and Neely must give ECS before training in order to arrive at the same physiological conditions as during storage processes; DeVietti and Larson attribute m e m o r y loss 24 hr later to the fact that the animal is still under the effects of the ECS. That there is a state d e p e n d e n t factor in learning has been well d o c u m e n t e d [7]. However, the use o f such a p h e n o m e n o n to explain results in experimental amnesia should be done with caution. To do so puts limitations on m e m o r y mechanisms which are not supported by observation or experience. F u r t h e r m o r e such a limitation on m e m o r y processes is not biologically adaptive. Memories for experiences lived under one physiological c o n d i t i o n are certainly available for use in decision making when the organism is in a dissimilar physiological state, although reinstatement of the original c o n d i t i o n might facilitate recall of the event. The results of the present experiments show that the state d e p e n d e n t learning hypothesis is not adequate to explain the recovery from amnesia p h e n o m e n o n because recovery was seen one half hr or 5 rain after the amnesia had been d e m o n s t r a t e d (at the first test, T2 ). It would be difficult to explain this recovery in terms of brain state change, unless the recovery was only partial or only seen in a few animals, because this change in brain state must, presumably, take place gradually. As the results indicate, the difference in avoidance scores between T2 and T3 for the F S - H Y P in E x p e r i m e n t 1 and the F S - E C S in E x p e r i m e n t 2 was quite dramatic. There is another possible explanation for this recovery p h e n o m e n o n . Perhaps the elements of the learning are acquired during the training session; the intervention of the amnestic agent prevents a subsequent organization and integration into existing functional systems. During the first
testing session, T2, the rat has the o p p o r t u n i t y to retrieve the elements, i.e. the footshock, the environmental cues of the cage, the discrimination between the dark and the light c o m p a r t m e n t , and to reorganize these elements into a significant ensemble. Since the stimulus array during the first test did not change the natural t e n d e n c y o f the rat to enter the dark c o m p a r t m e n t , it can be said that it did not have a significance for the animal. The activity of reorganization and integration of the elements, including the m e m o r y of the footshock, is required. It is proposed that this is the activity which is interrupted by the amnestic treatment, and it is resumed again during T~. At T3 it can be inferred from the abrupt change in the behavior that the stimulus array has b e c o m e significant for the animal. This hypothesis accounts for Quartermain's findings that a reminder shock given in a neutral e n v i r o n m e n t was not sufficient to produce recovery of memory. Perhaps it is not the reminder shock at all that is important, but the effect of being placed in the experimental environment which produces recovery in his experiment as well as in the present one. CONCLUSION The results of these experiments are not compatible with the consolidation hypothesis because recovery from retrograde amnesia was seen. If the amnesia produced by hypoxia and ECS was due to failure o f m e m o r y to consolidate, there would be no possibility for a recovery, because there would be nothing to recover. The results are also incompatible with the interpretation of Suboski et al. [10] or Young and Galluscio [121, that the performance d e c r e m e n t is due to the blockage of the incubation of the CER, because of the short time in which recovery was seen. F u r t h e r m o r e , the results are not due to a state dependent learning factor as suggested by DeVietti and Larson and others, because the time difference b e t w e e n T2 and T3 is not great enough to produce such a dramatic change in behavior as was seen at T3, based on a change of physiological state. A possible explanation could be that the experience at T: provides the animal with the o p p o r t u n i t y to organize the previously learned elements into a significant stimulus array. This kind of interpretation requires a dynamic long-term m e m o r y , a m e m o r y which is constantly undergoing a reorganization as a function of experience.
REFERENCES
1. Chorover, S. and P. DeLuca. Transient change in electrocortiographic reaction to ECS in the rat following footshock. Z comp. physiol. PsychoL 59: 141-149, 1969. 2. DeVietti, T. and R. Larson. ECS effects: evidence supporting state-dependent learning in rats. J. cornp, physiol. Psychol. 74: 407-415, 1971. 3. Galluscio, E. Retrograde amnesia induced by electroconvulsive shock and carbon dioxide anesthesia in rats: an attempt to stimulate recovery. J. comp. physiol. Psychol. 75: 136--140, 1971. 4. Herz, and H. Peeke. Permanence of retrograde amnesia p r o d u c e d by electroconvulsive shock. Science 163: 1396-1397, 1967.
5. Kohlenberg, R. and T. Trabasso. Recovery of a conditioned emotional response after one or two electroconvulsive shocks. .L comp. physioL PsychoL 65: 270-273, 1968. 6. Koppenaal, R., E. Jogada and J. Cruce. Recovery from ECS produced amnesia following a reminder. Psychonom. ScL 9: 293-294, 1967. 7. Overton, D. State dependent or dissociated learning produced with pentobarbital. J. comp. physiol. Psychol. 57: 3-12, 1964. 8. Quartermain, D. B. McEwen and E. Azmitia. Amnesia produced by electroconvulsive shock or cycloheximide: conditions for recovery. Science 169: 683-686, 1970. 9. Sara, S. and D. Lefevre. Hypoxia induced amnesia and pharmacological protection by Piracetam. PsychopharmacoIogia 25: 32-40, 1972.
RECOVERY FROM HYPOXIA AND ECS-INDUCED AMNESIA 10. Suboski, M. D., A. A. Spevak, J. Litner and E. Beaumaster. Effects of ECS following one-trial discriminated avoidance conditioning. Neuropsychologia 7: 67-78, 1969. 1 I. Thompson, C, and J. Neely. Dissociated learning in rats produced by electroconvulsive shock. Physiol. Behav. 5: 783-786, 1970.
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12. Young, A. and E. Galluscio. Recovery from ECS produced amnesia. Psych ono m. S cL 22:149-151,1971. 13. Zinkin, S. and A. Miller. Recovery of memory after amnesia induced by electroconvulsive shock. Science 155:102 104.
Recovery from Hypoxia and ECS-Induced Amnesia After a Single Exposure to Training Environment S. J. S A R A l
Center for Experimental and Comparative Psychology University o f Louvain, Pellenberg, Belgium
(Received 1 August 1972)
SARA, S. J. Recovery from hypoxia and ECS-induced amnesia after a single exposure to training environment. PHYSIOL. BEHAV. 10(1) 85-89, 1973.A single exposure to the training environment for 3 rain was sufficient to produce recovery from hypoxia-induced amnesia when tested 30 rain later. Recovery from ECS-induced amnesia was evidenced 5 rain after exposure to the training environment. The results make a state dependent learning explanation for memory loss after ECS or hypoxia unfeasible, since the recovery is seen immediately after the memory loss is evidenced. The author proposes that amnestie treatments prevent the integration of the training event into existing functional memory systems and consequently the training has no significance for the animal. Reexposure to the training environment precipitates a continuation of these integrative processes and the memory becomes organized in such a way that it has a behavioral consequence (avoidance) on the subsequent trial. Memory
Retrograde amnesia
Hypoxia
ECS
Avoidance learning
SARA, S. J. La r~cuperation de l'amn~sie induite par hypoxie aprbs exposition une seule lois ~ l'environment d'apprentissage. PHYSIOL. BEHAV. 10(1) 85-89,1973.- La r6cuperation de l'amn6sie induite par hypoxie peut 6tre obtenue en exposant les rats une seule fois ~ l'environment d'apprentissage ce qui est confirm6 par un test effectu~ 30 min apr~s exposition. La r6cuperation de l'amndsie induite par ECS fur raise en 6vidence d~s les 5 min qui suivaient l'exposition des sujets h l'environment de l'apprentissage. D'apr~s ces rdsultats, une interpretation de type "stated dependent learning" ne parait pas adequate pour expliquer l'anm~sie apr6s un ECS ou une hypoxie, vu que ron observe une rdcuperation presque immediate apr~.s avoir 6tabli l'amn6sie. Scion l'auteur, les interventions amn~siques emp~cheraient l'int6gration des traitments qui caract~rissent ia situation d'apprentissage, dans les syst6mes fonetionels de la m6moire. C'est pourquoi la situation d'apprentissage perd sa signification pour l'animal. Pourtant, lorsque l'animal est r6expose ~ l'environment d'apprentissage, ces proeesSus d'int6gration sont de nouveau d~clench6s et la m~moire s'organise de telle sorte qu'elle peut avoir une consequence observable lors de l'essai suivant. M6moire
Amn6sie induite
Hypoxie
ECS
IT HAS been well established that recovery f r o m experimentally induced amnesia is possible. The c o n d i t i o n s for recovery of m e m o r y vary according t o the e x p e r i m e n t a l conditions and in s o m e studies an a t t e m p t at recovery was not successful. Kohlenberg and Tarbasso [5] have shown that there can be spontaneous recovery f r o m retrograde amnesia, as have Young and GaUuscio [ I l ]. Galluscio [ 3 ] found recovery from amnesia after giving a reminder footshock. Quartermain, McEwen and Azmitia [8] report results which showed recovery f r o m amnesia p r o d u c e d by b o t h ECS and cycloheximide. These results are particularly impressive because t h e y include t w o amnestic agents whose m o d e of action is presumably different. The authors point
out that the conditions for producing this recovery are rather limited. T h e y tried unsuccessfully to restore m e m o r y for f o o t s h o c k by giving a reminder shock in an environment different f r o m that of the training, o n the day after the training and before the test. Further studies revealed that a testing session before the reminder shock was necessary for the reminder to be effective in restoring memory. On this testing session an amnesia for the previous f o o t s h o c k was, of course, evidenced. The authors indicate that, although this first test had not been considered by others to be a crucial factor in reactivating m e m o r y , it was, in fact, included in the experimental procedures of two o t h e r reports of m e m o r y recovery after a reminder [6 ].
1The author would like to thank Dr. C. Giurgea for critical comment and helpful guidance. 85
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SARA
Z i n k i n a n d Miller [ 1 2 ] f o u n d a slight t e n d e n c y for m e m o r y t o r e a p p e a r a f t e r r e p e a t e d testing, b u t this s t u d y lacks t h e p r o p e r c o n t r o l s to i n d i c a t e if t h e recovery was t h e result of t h e r e p e a t e d testing or t h e mere passage o f time. Herz and Peeke [4] in a similar e x p e r i m e n t , s h o w e d t h a t it was t h e r e p e a t e d testing w h i c h a c c o u n t e d f o r t h e r e c o v e r y and n o t t h e passage o f time. This was i n t e r p r e t e d as d u e to a "gradual adaptation to the experimental situation". But these results m i g h t just as well b e e n i n t e r p r e t e d as evidence of a r e m i n d e r effect of the s i t u a t i o n itself. P e r h a p s t h e amnesia evidenced during t h e first test was only partial, a n d not due to t h e failure of t h e m e m o r y to c o n s o l i d a t e , b u t to o t h e r factors, such as t h e lack of a n a d e q u a t e i n t e g r a t i o n of the s h o c k e x p e r i e n c e with t h e e n v i r o n m e n t a l cues. T h e p r e s e n t e x p e r i m e n t investigates t h e possibility t h a t m e m o r y can be recovered w i t h a single test acting as a r e m i n d e r , t h e r e c o v e r y being evidenced o n a s u b s e q u e n t test. EXPERIMENT I. HYPOXIA-INDUCED AMNESIA AND RECOVERY
Method Animals. F i f t y - f o u r male rats of t h e Wistar strain were o b t a i n e d f r o m U n i o n C h i m i q u e Beige (UCB). T h e y weighed 1 5 0 - 2 0 0 g at t h e t i m e o f t h e e x p e r i m e n t and were h o u s e d 8 - 10 to a cage w i t h free access t o f o o d a n d water. Apparatus. B o t h t h e e x p e r i m e n t a l t r a i n i n g cage a n d t h e e q u i p m e n t for i n d u c i n g h y p o x i a were t h e same as was used by Sara a n d Lefevre [ 9 ] . T h e t r a i n i n g cage was a large c o m p a r t m e n t m a d e of a l u m i n u m a n d p a i n t e d white, w i t h a gravel floor, measuring 3 0 x 3 0 x 3 0 cm a n d was c o n n e c t e d b y a 6x6 c m guillotine d o o r t o a small c o m p a r t m e n t , p a i n t e d black, m e a s u r i n g 1 0 x l 0 x l 0 cm. T h e f l o o r o f t h e small c o m p a r t m e n t was m a d e of m e t a l rods o f 2.5 m m w i d t h and 12 m m apart. T h e s h o c k was delivered t h r o u g h a F o r i n g e r s h o c k scrambler. T h e shock source was a 7 0 0 V, 50 Hz t r a n s f o r m e r w h o s e o u t p u t c u r r e n t is limited b y a variable series resistence. A n a m p e r e m e t e r in place o f t h e rat s h o w e d a c u r r e n t of ~ 500 m i c r o a m p s . T h e cage for i n d u c i n g h y p o x i a consisted of an airtight Plexiglas b o x measuring 5 0 x 3 0 x 3 0 cm. Pure n i t r o g e n was i n t r o d u c e d into t h e cage and was mixed w i t h t h e a t m o sphere b y a v e n t i l a t o r a t t a c h e d to o n e o f t h e walls. A B e c k m a n 777 a n a l y z e r c o n t i n u o u s l y m e a s u r e d t h e p e r c e n t age o f o x y g e n in t h e a t m o s p h e r e of t h e cage. Experimental plan and procedure. T h e animals were divided r a n d o m l y i n t o f o u r groups. T h e t r a i n i n g p r o c e d u r e was as follows. T h e a n i m a l was placed in t h e large c o m p a r t m e n t of t h e a p p a r a t u s , facing away f r o m t h e d o o r to the small c o m p a r t m e n t . It was allowed to explore t h e t w o c o m p a r t m e n t s freely for 3 min. T h e l a t e n c y to e n t e r and t h e t o t a l t i m e s p e n t in t h e small c o m p a r t m e n t was n o t e d for each animal. A f t e r t h e 3 m i n f a m i l i a r i z a t i o n period t h e d o o r was lowered and t h e a n i m a l received 20 sec of f o o t s h o c k and was t h e n r e m o v e d f r o m t h e small c o m p a r t m e n t . T h e FS-NHYP g r o u p were c o n f i n e d for l 0 m i n in a Plexiglas cage, a f t e r w h i c h t h e y were r e m o v e d t o the h o m e cage. T h e FS-HYP g r o u p was i n t r o d u c e d immediately a f t e r t r a i n i n g i n t o t h e h y p o x i a cage at 31A% o x y g e n level and was m a i n t a i n e d at this level f o r 10 min. A f t e r r e c u p e r a t i o n t h e a n i m a l was r e t u r n e d to t h e h o m e cage. T w e n t y - f o u r hr later t h e rat was placed in the large c o m p a r t m e n t in the same m a n n e r as o n Day 1 and t h e
latency a n d t o t a l t i m e spent in the small c o m p a r t m e n t was noted. T h e rat was r e m o v e d from t h e a p p a r a t u s and r e t u r n e d t o t h e g r o u p cage. T h i r t y min later t h e rat was replaced in the t r a i n i n g cage a n d t h e latency and t o t a l time in the small c o m p a r t m e n t were again recorded. Later a f i f t h g r o u p was a d d e d to t h e e x p e r i m e n t to d e t e r m i n e if t h e effect o f t h e a m n e s i a p r o d u c e d b y t w o successive h y p o x i a t r e a t m e n t s w o u l d be resistant t o t h e r e m i n d e r effect. T h e p r o c e d u r e was t h e same as f o r t h e group F S - H Y P e x c e p t t h a t 15 m i n a f t e r removal f r o m t h e h y p o x i a cage t h e rat was r e i n t r o d u c e d i n t o t h e cage at 31A% and again m a i n t a i n e d for 10 rain ( G r o u p F S - H Y P - H Y P ) .
Results T h e measure of r e t e n t i o n was t h e t o t a l t i m e spent in t h e small c o m p a r t m e n t . O n Day 1, b e f o r e training, t h e animals spent a b o u t 80% of t h e t o t a l time in t h e dark. A Kruskal-Wallis one way analysis of variance s h o w e d t h a t t h e r e was no d i f f e r e n c e a m o n g g r o u p s o n Day 1 as t o t o t a l time s p e n t in t h e dark. ( H = 4 . 8 6 , dr=4, p > 0 . 3 0 ) As can be seen f r o m Fig. 1, t h e r e was a m a r k e d d i f f e r e n c e in t h e time s p e n t in the dark b e t w e e n T, and T2 for t h e g r o u p F S - N H Y P , indicating t h a t t h e t r a i n i n g is effective. T h e fact t h a t t h e r e is no difference b e t w e e n T~ and T3 for this g r o u p indicates t h a t no e x t i n c t i o n is seen o n t h e second test. T h a t the p e r f o r m a n c e of t h e c o n t r o l g r o u p N F S - N H Y P was stable over t h e t h r e e t i m e s indicates t h a t t h e r e is no s p o n t a n e o u s decrease in t h e a t t r a c t i o n o f the small dark c o m p a r t m e n t . T h a t h y p o x i a is n o t aversive is shown by the performance of the group NFS-HYP, which does n o t avoid t h e d a r k over t h e t h r e e times. T h e g r o u p F S - H Y P , significantly d i f f e r e n t f r o m t h e F S - H Y P group at T~ ( M a n n - W h i t n e y U test, two-tailed, U = 3 9 , p < 0 . 0 2 ) has c o m p l e t e l y r e c u p e r a t e d at T3. A W i l c o x o n m a t c h e d pairs signed r a n k s test shows a highly significant d i f f e r e n c e b e t w e e n T2 a n d T3 for this group. T h e r e is no d i f f e r e n c e b e t w e e n G r o u p s F S - H Y P and F S - N H Y P at T3, i n d i c a t i n g recovery f r o m the amnesia seen at T , . T h e g r o u p F S - - H Y P - H Y P was n o t d i f f e r e n t f r o m t h e group F S - H Y P at a n y t i m e a n d can, t h e r e f o r e , be c o n s i d e r e d as a c h e c k o n t h e reliability o f t h e findings.
Discussion T h e results of E x p e r i m e n t 1 verify t h e findings of Sara and Lefevre [9] t h a t h y p o x i a does, u n d e r certain conditions, p r o d u c e r e t r o g r a d e amnesia. However, this m e m o r y loss is n o t p e r m a n e n t and a r e m i n d e r serves t o reactivate the m e m o r y . T h e results agree w i t h t h o s e o f Q u a r t e r m a i n et aL [ 8 ] , b u t also i n d i c a t e t h a t a n i n t e r m e d i a r y test is n o t only a necessary c o n d i t i o n , b u t a sufficient c o n d i t i o n as well, for the r e c o v e r y p h e n o m e n o n . EXPERIMENT 2 ECS INDUCED AMNESIA AND RECOVERY This e x p e r i m e n t a t t e m p t s to generalise t h e results seen w i t h h y p o x i a by e m p l o y i n g a m o r e widely studied a m n e s t i c agent, ECS.
Method Animals. T h e animals were 24 male Wistar rats, 1 6 0 - - 2 0 0 g, o b t a i n e d f r o m t h e same source a n d h o u s e d u n d e r the same c o n d i t i o n s as E x p e r i m e n t I. Apparatus. T h e t r a i n i n g cage was t h e same as used in
RECOVERY FROM HYPOXIA AND E C S - I N D U C E D AMNESIA
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20
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T1
12
T3
FIG. 1. Median time in sec spent in the small dark compartment before training (T,), 24 hr after training (T 2 ) and 24 hr and 30 rain after training (Ta). Note the performance decrement of both hypoxia groups at the first test (T2) and the total recuperation at T3,30 min later. Duration of each trial: 180 sec. Experiment 1. ECS was effected by means of a transtemporal shock from a 1500 V transformer from which a 50 mA shock of 0.25 sec was delivered via a 10 Ks2 series resistance. Experimental plan and procedure. The animals were divided into three groups. The training was the same as in Experiment 1. Group F S - E C S received training followed immediately by ECS outside of the training cage. Group F S - N E C S had training and sham ECS; Group N F S - E C S was permitted to explore the apparatus for 3 rain and then was removed and convulsed. A slight variation in the testing procedure on Day 2 was that the time between T2 and T3 was reduced to 5 rain.
Results Figure 2 indicates the median time spent in the dark on T I , T2 and T3. The results are similar to those of Experiment 1. A Kruskal-Wallis one-way analysis of variance indicates no difference among groups at T~ before training. (H-l, dr=2, p>0.50). Group F S - E C S was significantly different from Group F S - N E C S at T , , indicating a memory loss (Mann-Whitney U test one-tailed U = I 4 , p=0.025). A Wilcoxon Matched Pairs Signed Ranks test shows that the difference between T~ and Ts for Group F S - E C S is significant (T=4, p<0.02, two-tailed), thus indicating recovery from amnesia. No other group showed a difference between T2 and T3. That the ECS treatment itself does not cause avoidance of the small compartment at T2 is indicated by the fact that Group N F S - E C S spends about the same amount of t i m e in the dark on each of the three occasions.
Discussion The results of Experiment 2 indicate that ECS induced
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amnesia can be reversed by merely placing the animal in the training environment for 3 rain, 24 hr after footshock ECS treatment. A reminder shock in a neutral environment is not necessary to recovery. This experiment shows that the reminder can precede the recovery of performance by as little as 5 min. GENER AL DISCUSSION Quartermain et al. [8] showed that reexposure to the training environment is a necessary condition for the restitution of memory; the present experiments indicate that this reexposure is a sufficient condition. The results do not support the interpretation of a similar phenomenon by Young and Galluscio [ 12] that it is due to the fact that the original loss was a result of suppression of the conditioned emotional response (CER). According to these authors, it was the CER which later reappeared as a function of time, manifesting itself as recovery from amnesia. In the present experiments the time between the first test, which serves as the reminder, and the second test is only 30 rain in Experiment l and 5 rain in Experiment 2, which seems to rule out any interpretation due solely to time factors. Some authors have interpreted the amnesic results seen with ECS as due to a state dependent learning phenomenon. (DeVietti and Larson [ 2 ] , Chorover and DeLuca [1]). The performance decrement seen on Day 2 is attributed to the fact that the physiological changes introduced by the synergistic interaction of the ECS and the footshock have not completely attenuated 24 hr later. The animal is in a different brain state during the test than during the acquisition of the response. Recovery occurs with later testing (48 or 72 hr, according to Zinkin and Miller [13]) because it is then that the rat has fully recovered from the postictal effects of the ECS and is in the same brain state as during the training. Therefore the
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140 I 120 100 80 \
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FIG. 2. Median time in sec spent in the small dark compartment before training (TI), 24 hr after training (T2) and 24 hr and 5 min after training (T~). Note the performance decrement of the FS-ECS group at T2 and the total recuperation at T3,5 min later. Duration of each trial: 180 sec.
88
SARA
m e m o r y is retrievable. However, T h o m p s o n and Neely [ 11 ] have shown that rats convulsed 5 sec after training do not show amnesia 24 hr later, if they received another convulsion 20 min before the test. The effect of the second ECS was to introduce the same physiological state as during the storage phase, i m m e d i a t e l y after training. ECS-induced amnesia they have attributed to dissociated learning which presumably is not unlike the state d e p e n d e n t learning, except in one case the dissociated state is dissociated from the condition immediately after training and in the other case it is the training condition which is dissociated. There is also disagreement between the two as to the c o n d i t i o n of the brain at the time of test. T h o m p s o n and Neely must give ECS before training in order to arrive at the same physiological conditions as during storage processes; DeVietti and Larson attribute m e m o r y loss 24 hr later to the fact that the animal is still under the effects of the ECS. That there is a state d e p e n d e n t factor in learning has been well d o c u m e n t e d [7]. However, the use o f such a p h e n o m e n o n to explain results in experimental amnesia should be done with caution. To do so puts limitations on m e m o r y mechanisms which are not supported by observation or experience. F u r t h e r m o r e such a limitation on m e m o r y processes is not biologically adaptive. Memories for experiences lived under one physiological c o n d i t i o n are certainly available for use in decision making when the organism is in a dissimilar physiological state, although reinstatement of the original c o n d i t i o n might facilitate recall of the event. The results of the present experiments show that the state d e p e n d e n t learning hypothesis is not adequate to explain the recovery from amnesia p h e n o m e n o n because recovery was seen one half hr or 5 rain after the amnesia had been d e m o n s t r a t e d (at the first test, T2 ). It would be difficult to explain this recovery in terms of brain state change, unless the recovery was only partial or only seen in a few animals, because this change in brain state must, presumably, take place gradually. As the results indicate, the difference in avoidance scores between T2 and T3 for the F S - H Y P in E x p e r i m e n t 1 and the F S - E C S in E x p e r i m e n t 2 was quite dramatic. There is another possible explanation for this recovery p h e n o m e n o n . Perhaps the elements of the learning are acquired during the training session; the intervention of the amnestic agent prevents a subsequent organization and integration into existing functional systems. During the first
testing session, T2, the rat has the o p p o r t u n i t y to retrieve the elements, i.e. the footshock, the environmental cues of the cage, the discrimination between the dark and the light c o m p a r t m e n t , and to reorganize these elements into a significant ensemble. Since the stimulus array during the first test did not change the natural t e n d e n c y o f the rat to enter the dark c o m p a r t m e n t , it can be said that it did not have a significance for the animal. The activity of reorganization and integration of the elements, including the m e m o r y of the footshock, is required. It is proposed that this is the activity which is interrupted by the amnestic treatment, and it is resumed again during T~. At T3 it can be inferred from the abrupt change in the behavior that the stimulus array has b e c o m e significant for the animal. This hypothesis accounts for Quartermain's findings that a reminder shock given in a neutral e n v i r o n m e n t was not sufficient to produce recovery of memory. Perhaps it is not the reminder shock at all that is important, but the effect of being placed in the experimental environment which produces recovery in his experiment as well as in the present one. CONCLUSION The results of these experiments are not compatible with the consolidation hypothesis because recovery from retrograde amnesia was seen. If the amnesia produced by hypoxia and ECS was due to failure o f m e m o r y to consolidate, there would be no possibility for a recovery, because there would be nothing to recover. The results are also incompatible with the interpretation of Suboski et al. [10] or Young and Galluscio [121, that the performance d e c r e m e n t is due to the blockage of the incubation of the CER, because of the short time in which recovery was seen. F u r t h e r m o r e , the results are not due to a state dependent learning factor as suggested by DeVietti and Larson and others, because the time difference b e t w e e n T2 and T3 is not great enough to produce such a dramatic change in behavior as was seen at T3, based on a change of physiological state. A possible explanation could be that the experience at T: provides the animal with the o p p o r t u n i t y to organize the previously learned elements into a significant stimulus array. This kind of interpretation requires a dynamic long-term m e m o r y , a m e m o r y which is constantly undergoing a reorganization as a function of experience.
REFERENCES
1. Chorover, S. and P. DeLuca. Transient change in electrocortiographic reaction to ECS in the rat following footshock. Z comp. physiol. PsychoL 59: 141-149, 1969. 2. DeVietti, T. and R. Larson. ECS effects: evidence supporting state-dependent learning in rats. J. cornp, physiol. Psychol. 74: 407-415, 1971. 3. Galluscio, E. Retrograde amnesia induced by electroconvulsive shock and carbon dioxide anesthesia in rats: an attempt to stimulate recovery. J. comp. physiol. Psychol. 75: 136--140, 1971. 4. Herz, and H. Peeke. Permanence of retrograde amnesia p r o d u c e d by electroconvulsive shock. Science 163: 1396-1397, 1967.
5. Kohlenberg, R. and T. Trabasso. Recovery of a conditioned emotional response after one or two electroconvulsive shocks. .L comp. physioL PsychoL 65: 270-273, 1968. 6. Koppenaal, R., E. Jogada and J. Cruce. Recovery from ECS produced amnesia following a reminder. Psychonom. ScL 9: 293-294, 1967. 7. Overton, D. State dependent or dissociated learning produced with pentobarbital. J. comp. physiol. Psychol. 57: 3-12, 1964. 8. Quartermain, D. B. McEwen and E. Azmitia. Amnesia produced by electroconvulsive shock or cycloheximide: conditions for recovery. Science 169: 683-686, 1970. 9. Sara, S. and D. Lefevre. Hypoxia induced amnesia and pharmacological protection by Piracetam. PsychopharmacoIogia 25: 32-40, 1972.
RECOVERY FROM HYPOXIA AND ECS-INDUCED AMNESIA 10. Suboski, M. D., A. A. Spevak, J. Litner and E. Beaumaster. Effects of ECS following one-trial discriminated avoidance conditioning. Neuropsychologia 7: 67-78, 1969. 1 I. Thompson, C, and J. Neely. Dissociated learning in rats produced by electroconvulsive shock. Physiol. Behav. 5: 783-786, 1970.
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12. Young, A. and E. Galluscio. Recovery from ECS produced amnesia. Psych ono m. S cL 22:149-151,1971. 13. Zinkin, S. and A. Miller. Recovery of memory after amnesia induced by electroconvulsive shock. Science 155:102 104.