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Engram duplication in the day-old chick
BEHAVIORAL A ND N E U R A L BIOLOGY
31, 283-298 (1981)
Engram Duplication in the Day-Old Chick ROBYN J. HODGE, MARIE E .
GIaBS, AND
K . T. N G 1
Department of Psychology, La Trobe University, Bundoora, Victoria 3083, Australia Monocular training on a single-trial aversive discrimination task in day-old chickens resulted in memory being formed only in the hemisphere served by the eye used in training. The original memory could be duplicated in the other hemisphere when the other eye was used to retrieve the information from the trained hemisphere, but the success of this was dependent on the time after training when this retrieval was carried out. One retrieval within 10 min after training, when short-term memory was maximally active in the trained hemisphere, was successful in duplicating the memory in the naive hemisphere. However, if the trials were begun at 30 rain after training when the original short-term memory had decayed somewhat, five retrievals were necessary for optimum duplication of the memory. It was suggested that duplication of memory depended on information available in the short-term memory phase of the original engram. If the memory was retrieved through the untrained eye and memory formation was inhibited in the untrained hemisphere, the decay of memory in the trained hemisphere was delayed, the effect being greater the more times the information was retrieved. This effect was interpreted in terms of maintenance of labile memory beyond its normal active life by restimulation of memory formation processes in the trained hemisphere.
The complete decussation of the optic nerves in birds has stimulated interest in the issue of lateralization of memory storage for visual tasks (see Zeier, 1975, for review). Studies in which the different commissures that connect the chick forebrains have been severed demonstrated interhemispheric transfer of visual information (Cuenod, 1974). The possibility of interhemispheric "cross-talk" raises the issue of whether tasks learned with only one eye open result in bilateral or unilateral memory formation. Lesion studies using domestic chicks and singletrial passive avoidance learning have yielded results which suggest bilateral establishment of memory following learning (Benowitz, 1974; Greif, 1976). Cherkin (1970), Gaston (1978), and Bell and Gibbs (1977) have demonstrated interocular transfer of monocular learning in unoperated 1 The authors wish to express their gratitude to Dr. G. Coleman for a computerized system for recording and extracting data in these experiments.
283 0163 - 1047/81/030283-16502.00/0 Copyright© 1981 by AcademicPress, Inc. All rightsof reproductionin any formreserved.
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chicks. Bell and Gibbs (1977) interpreted their results as evidence for formation of a unilateral memory in the hemisphere receiving the information on training, with the information available to the untrained hemisphere. Successful transfer and duplication of a unilateral engram following operant learning in the rat have also been demonstrated by Russell and Ochs (1961). We have previously postulated a model for the development of memory for a single-trial discrimination task in young chickens (Gibbs & Ng, 1976, 1977). In this model, memory formation for binocular learning follows three behavioral sequentially dependent phases: a short-term phase (STM) decaying after 10 min following learning; a labile phase decaying after 30 rain following learning; and a long-term phase (LTM) developing after 30 min. These three phases are blocked, respectively, by intracranial injection of a depolarizing agent glutamate, the sodium pump inhibitor ouabain, or the protein synthesis inhibitor cycloheximide (CXM). Bell and Gibbs (1977) found that when the discrimination task was learned monocularly, intracranial administration of the labile memory inhibitor ouabain or the long-term memory inhibitor CXM to the trained hemisphere inhibited the formation of labile memory or LTM, respectively. Injection to the untrained side did not. Their conclusion was that memory for the task was established only in the hemisphere served by the eye used in training and the presence of memories in the trained side was not sufficient to establish memory in the naive side. Furthermore, Bell and Gibbs (1979) reported evidence suggesting that there could be duplication of memory in the untrained hemisphere when the chick performed the task using the untrained eye and therefore retrieved information by way of the untrained hemisphere. The efficiency of the duplication was a function of the time of accessing the information after monocular learning and the number of times the information was accessed or retrieved through the untrained hemisphere. When CXM was administered into the trained hemisphere just after learning, the information was available for retrieval for up to 30 min after learning, before it decayed in that hemisphere. If the information was retrieved once at 7½ min after learning using the untrained eye, the memory in the trained side was available, and when these chicks were tested again 24 hr later, there was no evidence of memory loss, irrespective of which eye was used for this later test. Chicks not receiving a first (access) trial at 7½ min did not show memory at 24 hr, nor did chicks which received bilateral injections of CXM. This latter finding weakens the possibility of a reminder effect as the reason for the good retention at 24 hr in chicks receiving one access trial. Under these same conditions, a single access trial at 30 min did not result in good retention 24 hr later, even though the memory was unaffected at 30 rain on the access trial. However, if five access trials were given, beginning at 30 min and spaced 5 min apart, memory was good at 24
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hr. These results can be interpreted within the model of memory formation mentioned above. The success of duplication of memory depends on which stage the memory in the trained hemisphere is in at the time of retrieving it through the untrained hemisphere. The results of Bell and Gibbs (1977, 1979) are consistent with the hypothesis that monocular learning in day-old chicks resulted in formation of a unilateral engram in the hemisphere contralateral to the eye used in training and this memory can be duplicated in the ipsilateral (untrained) hemisphere under appropriate conditions. A further finding of interest in the Bell and Gibbs (1979) study is the fact that in the presence of CXM in the trained hemisphere, retention levels remained high during all five access trials from 30 to 50 min after learning. In binocular learning, with bilateral injection of CXM, Gibbs and Ng (1977) report high retention levels at 30 rain but amnesia by 60 min after learning. If, during the access trials, information was being taken from the labile phase of memory in the trained hemisphere, some decrease in retention levels from 30 to 50 min might have been expected. Bell and Gibbs (1979) suggested that the access trials begun 30 min after learning may have retarded the decay of labile memory in the trained hemisphere. If this is so, it may be argued that the high retention levels that they subsequently obtained at 24 hr after learning with the untrained eye may have resulted from the access trials overcoming the effects of CXM in the trained hemisphere rather than from duplication of memory in the untrained hemisphere. However, this does not explain why, under these conditions of accessing, CXM administered to both the trained and the untrained hemisphere prevented high retention levels at 24 hr. The conclusion appears inescapable that duplication of the engram does in fact occur following accessing but this is independent of the effect of accessing on the original engram. The question remains as to why engram duplication becomes more difficult with increasing age of the unilateral engram (Bell & Gibbs, 1979). The possibility arises that engram duplication is dependent solely on information derived from the short-term memory phase of the original engram, and becomes progressively more difficult with an increasingly decaying STM after 10 min following learning. An equally interesting, if perhaps less important, question concerns under what conditions, if any, repeated accessing will lead to the inhibitory action of CXM in the trained hemisphere being overcome. In this paper we report a series of experiments directed at answering these two questions. METHOD Procedure. The basic experimental paradigm follows that described by Gibbs and Ng (1977). Figure 1 summarizes the experimental procedures
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by the exposed eye during training to the contralateral hemisphere and retrieval of the information by the untrained eye during access and retention trials. The trained hemisphere is marked with an (*) to represent initial engram formation. Only the hemispheres are represented in (B), with the symbols indicating which hemisphere received the learning trial (*), access trial(s) (dashed arrow) or retention trial (wide arrow). In (C), we show how the diagrammatic representation used in the subsequent figures was compiled from (A) and (B). Cross hatching in the subsequent figures indicates the hemisphere receiving the injection of the specified drug. specific to these experiments. Day-old white L e g h o r n - b l a c k Australorp cockerels were placed in pairs in wooden b o x e s shortly after arrival f r o m a local hatchery, and were kept in pairs throughout all subsequent manipulations. In all but one experiment, 20 different chicks were assigned for each data point. Pretraining. The chicks were trained to peck at a 4-mm red glass bead, dipped in water, presented for 10 sec. This was followed by a similar 10-sec trial with a 4-mm blue glass bead. Monocular learning. The right eye was occluded with an opaque Band Aid Spot. Ten minutes later, on a single aversive discrimination learning trial, chicks were presented a red bead similar to the one used in pretraining, but dipped in the chemical aversant methyl anthranilate, for 10 sec. The data for chicks failing to peck on this learning trial or to show the characteristic " d i s g u s t " responses of shaking their heads and wiping their beaks were discarded at the end of the experiment. The n u m b e r of chicks discarded was n e v e r m o r e than 15% of the original sample and the actual sample size for each data point is reported in the relevant figure legends. The eye patch was r e m o v e d immediately after the learning trial. Accessing trials. An accessing trial is one in which the eye occluded in the monocular learning trial is used to retrieve information about the
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aversive bead. Thus, where a chick is trained with the left eye (right eye occluded) accessing of memory is carried out by presenting a dry bead to the right eye. No blue bead is presented on an access trial. Five minutes prior to accessing, the left eye was occluded. On each access trial, a dry red bead was presented for 10 sec. The eye patch is removed immediately after the last access trial. Retention trials. A retention trial is one in which the trained eye is used to test for memory. Five minutes prior to the retention trial, the eye not used in training is occluded and the eye patch is removed immediately after the trial. On each retention trial a dry red bead is presented for 10 sec, followed by a dry blue bead. Retention is indexed by the proportion of chicks avoiding the red bead. Drugs and injections. All drugs were made up in sterile NaC1 (0.15 M; 0.9%). Ouabain (Sigma; 0.027 mM), cycloheximide (Upjohn, 3.5 raM), or saline (0.15 M) was administered 5 min before learning into the right hemisphere of each chick. Injections of 10-/A volumes for CXM and saline or 5- or 10-/zl volumes for ouabain were freehand into the center of the forebrain to a depth of 3.0 mm, in the neostriatal area. RESULTS
Formation of Unilateral Engram Ten microliters of ouabain or saline was administered into the right hemisphere of each chick, 5 rain prior to monocular training with the left eye, in order to prevent formation of labile and long-term memory in that hemisphere. Information was accessed through the right (naive) eye at 10, 30, 60, 120, 180, or 1440 min after training. Using the method of planned contrasts on proportions and a type I error rate of a = 0.05 (Rodger, 1969), significant differences were found between saline- and ouabain-treated chicks in the proportion avoiding the red bead at 30, 60, 120, 180, and 1440 min after training (F(1, oo = 13.55, 5.46, 11.30, 23.95, and 4.31 respectively). The difference at 10 rain after training was not significant. No differences were apparent between the drug and saline groups in proportion avoiding the blue bead (Fig. 2). The results suggest that, with ouabain inducing amnesia in the trained hemisphere after 10 min following learning (Gibbs & Ng, 1977), no memory is available from the untrained hemisphere alone.
Effect of Variation in Number of Access Trials on Engram Duplication Five minutes prior to monocular training with the left eye 10/zl of CXM was administered ~nto the right hemisphere of each chick, in order to prevent formation of LTM in that hemisphere. Commencing 30 rain after monocular training zero, one, two, three, four, or five access trials 5 min apart were performed with the right eye. A control group of chicks were
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given five access trials, spaced 5 min apart, through the right eye commencing 60 min after training. An injection of CXM was given to the trained hemisphere 5 min before training. Memory for the learned response was tested with the right eye 24 hr after training. The proportion of chicks avoiding the red bead during access trials at 30 min after learning did not alter from group to group and was comparable to retention levels reported for 30 min after learning following CXM pretreatment (Gibbs & Ng, 1977; Fig. 3). However, the proportion of chicks avoiding the red bead 24 hr after training was a linear function of the number of access trials. There was a significant linear (F(1, oo) = 23.96, p < .05) and a nonsignificant quadratic trend. With no access trials the proportion of chicks avoiding the red bead at 24 hr is comparable to that reported for CXM-pretreated chicks trained and tested binocularly (Gibbs & Ng, 1977). After 5 access trials, the proportion is comparable to that for untreated or saline-pretreated chicks (Gibbs & Ng, 1977). When five access trials were given 60 rain after training, the proportion of chicks avoiding the red bead during these trials was comparable to that obtained with CXM-pretreated chicks at 60-90 min after learning (Gibbs & Ng, 1977)..Retesting at 24 hr revealed no change in avoidance level (Fig. 4). The results suggest that five access trials, when begun 30 rain after learning, are necessary for complete engram duplication. However, the effect of repeated accessing appears to be additive, at least up to five
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access trials, provided the accessing is begun prior to the decay of the labile phase in the trained hemisphere.
Effect of Varying Time of Single-Access Trial on Engram Duplication Following learning with the left eye, a single access trial was performed through the right eye 10, 15, 20, 25, or 30 rain after learning. Ouabain or CXM was administered into the right (trained) hemisphere 5 min prior to monocular learning, to prevent formation of labile memory and LTM, respectively, in that hemisphere (Figs. 5A and B). Memory for the learned task was tested with the right eye at 24 hr. In the presence of ouabain in the trained hemisphere, the proportion of
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FIG. 4. Percentage of chicks in a group of 20 avoiding the red bead (A) on five access trials through the naive hemisphere commencing 60 min after monocular learning. The naive eye was tested again 24 hr after learning with the red bead and the blue bead (A). Monocular learning was given to the left eye (right hemisphere) with the right eye occluded 5 min after CXM was injected into the contralateral (right) hemisphere. All tests were carried out with the right eye, the left eye being occluded.
chicks avoiding the red bead during access trials follows the declining function observed with ouabain inhibition of labile memory in binocular learning (Gibbs & Ng, 1977), while in the presence of CXM, the proportions remain high and are comparable to those observed with CXM inhibition of LTM in binocular learning (Gibbs & Ng, 1977). The results suggest that performance during accessing reflects available memory in the original engram. In the absence of labile memory, information during accessing between 10 and 30 min after learning is retrieved from a decaying short-term memory. The proportion of ouabain-treated chicks avoiding the red bead at 180 min after learning is a declining function of the time of accessing, and the function is comparable to that observed during access trials (Fig. 5A). There is a significant linear trend in the function (F(1, oo) = 12.09, p < .05). However, the same declining function is observed at 24 hr after learning with CXM-treated chicks contrasting markedly with the function obtained during accessing (Fig. 5B). In fact, for each accessing time, there is no significant difference between proportion of ouabain-treated and CXM-treated chicks avoiding the red bead during retention tests. The two retention functions are indistinguishable from the retention functions obtained by Gibbs and Ng (1977) from retention tests between 10 and 30 min after learning with bilateral injections of ouabain or ethacrynic acid 5 min before binocular learning. Thus, while information during access trials is retrieved from whatever phase is active in the original engram at the time of accessing, information
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the right hemisphere on the percentage of chicks avoiding the dry red bead (open symbols) on the single access trial presented to the naive eye (in separate groups of chicks) at 10, 15, 20, 25, or 30 min after learning. On the right hand side is the percentage of chicks avoiding the dry red bead (open symbols) and the blue bead (filled symbols) on the 24-hr test, plotted according to the time of the access trial. Number of chicks in each group: A, 19-20; B, 18-20. for e n g r a m d u p l i c a t i o n f r o m o n e a c c e s s trial is o b t a i n e d f r o m t h e S T M p h a s e o f the o r i g i n a l e n g r a m .
Effect of Accessing Trials on Maintenance of Labile Memory in the Trained Hemisphere F i v e m i n u t e s p r i o r t o m o n o c u l a r l e a r n i n g w i t h t h e left e y e , C X M w a s a d m i n i s t e r e d into t h e right h e m i s p h e r e to inhibit f o r m a t i o n o f L T M in t h a t h e m i s p h e r e . T h i r t y m i n u t e s a f t e r l e a r n i n g z e r o , o n e , o r five a c c e s s trials w e r e c a r r i e d o u t w i t h t h e o t h e r e y e - - t h e right e y e . R e t e n t i o n t e s t s w e r e c a r r i e d o u t u s i n g t h e left ( t r a i n e d ) e y e at v a r i o u s t i m e s f r o m 10 t o 120 m i n after learning. C h i c k s r e c e i v i n g o n e o r five a c c e s s trials w e r e a d m i n i s t e r e d 1 0 / z l o f
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ouabain in the untrained hemisphere immediately after the first access trial to prevent formation of labile, and hence, also long-term memory in that hemisphere (Gibbs & Ng, 1977). In order to ensure that observed retention between 45 and 120 min after learning was not due to engram duplication in the untrained hemisphere, three groups of chickens given five access trials were administered 5 /~1 of ouabain in the untrained hemisphere at 30 min (immediately after the first access trial) and a further 5 /1.1 of ouabain at 45 min (immediately after the fourth access trial) following learning, and retention was tested at 60, 100, or 120 min after learning. With no access trials the time course of retention is comparable to that obtained with bilateral injections of CXM under binocular learning (Gibbs & Ng, 1977). Retention is high until 30 min following learning, and then declines rapidly, yielding the typical decay function of the labile phase. With five access trials, however, retention levels remain high for up to 100 min following learning (Fig. 6). At 70, 80, 90, and 100 min following learning the proportion of chicks avoiding the red bead after five access trials was significantly different from that observed with chicks given no access trials (F(1, ~) = 8.12, 5.76, 7.58, and 10.00, respectively; p < .05). At 60 or 120 min after learning, the difference is not significant. With one access trial, the results are equivocal. At 45 and 50 min after learning the retention levels remain high and are comparable to those observed during access trials. However, the proportions of chicks avoiding the red bead at these times were not significantly different from those observed with no access trials which still showed a little retention. The retention level drops markedly by 60 min after learning, and is maintained at that level up to 100 min following learning (Fig. 6). The proportion of chicks avoiding the bead at these times was not significantly different from those observed in chicks given no access trials. The above results suggest that after five access trials labile memory is maintained for at least 70 min after the normal time of decay. With one access trial it is maintained for up to 20 min after the normal time of decay. The absence of statistically significant differences here may be attributed to the power of the statistical test used and the fact that we are dealing with a declining retention function for the no access trial condition. The high retention levels observed between 60 and 180 min after learning cannot be attributed to duplication of the engram in the ipsilateral (naive) hemisphere. No significant differences in proportion of chicks avoiding the red bead were obtained at the 60-, 100-, and 120-min learning-retention intervals between chicks given five accesses and 10/xl ouabain 30 min after learning (67, 60, and 30%, respectively) and chicks given five accesses and 5/xl ouabain at 30 and 45 min after learning (67, 56, and 25%, respectively).
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Effect of Access Trials on CXM-Induced Amnesia in the Trained Hemisphere At 30 min after m o n o c u l a r training with the left eye, chicks w e r e p r e s e n t e d 5 or 10 a c c e s s trials t h r o u g h the right eye. T h e intertrial interval for five a c c e s s trials was 10 min, while that for 10 a c c e s s trials was 5 rain. R e t e n t i o n was tested at 24 hr after learning with the left (training) eye. T e n microliters o f C X M w a s administered to the trained h e m i s p h e r e 5 min prior to training; 5 /1,1 o f o u a b a i n was a d m i n i s t e r e d to the ipsilateral h e m i s p h e r e at 30 min (immediately after the first a c c e s s trial) and at 55 rain after learning to inhibit f o r m a t i o n o f labile and h e n c e long-term m e m o r y in that h e m i s p h e r e . T w o injections o f o u a b a i n were c o n s i d e r e d n e c e s s a r y since this drug is not maximally effective for 50 rain (Gibbs & Ng, 1977). A control g r o u p o f chicks was given five a c c e s s trials s p a c e d 5 min apart, with 10 t~l o f o u a b a i n a d m i n i s t e r e d into the ipsilateral h e m i s p h e r e 30 min after learning. D u r i n g a c c e s s trials the p r o p o r t i o n o f chicks a v o i d i n g the red bead r e m a i n e d high for all three g r o u p s (Fig. 7). At 24 hr after learning, the p r o p o r t i o n o f chicks avoiding the red bead in the g r o u p receiving 10 a c c e s s trials was not significantly different f r o m that in the g r o u p receiv-
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FIG. 7. Percentage of chicks avoiding the dry red bead (filled symbols) at 24 hr after monocular training when the trained side was tested. Chicks were trained using the left eye and the right hemisphere was injected with CXM 5 min before the learning trial. Chicks received different combinations o f access trials through the naive hemisphere, which was injected with ouabain to prevent duplication o f memory in that hemisphere (see text for details). The accessing combinations, commencing at 30 rain after learning were: 10 access trials with 5-min intervals between each trial (©), n = 20; 5 access trials with 10-rain intervals between each trial (rq), n = 19; 5 access trials with 5-min intervals between each trial (A), n = 20.
ing 5 access trials 10 min apart but these two groups, on the average, showed a greater proportion avoiding the red bead than the control group receiving 5 access trials 5 min apart (F(1, oo) = 9.99, p < .05). The level for the control group is comparable to that observed at 24 hr with CXMtreated chicks under binocular training (Gibbs & Ng, 1977), while those for the other two groups are comparable to saline-treated chicks. The results suggest that either 10 accesses 5 min apart or 5 accesses 10 min apart are sufficient to overcome CXM-induced amnesia. However, 5 accesses spaced 5 min apart were not sufficient to overcome CXMamnesia and reinstate LTM formation in the trained hemisphere.
DISCUSSION The present results are consistent with Bell and Gibbs' (1977) contention that monocular learning of a single-trial passive avoidance discrimination task in the day-old chick results in the formation of a unilateral engram in the hemisphere contralateral to the eye used in training. Ouabain administered to the trained hemisphere resulted in decreasing levels of retention during single accessing trials with the untrained hemisphere after 10 min following learning (Fig. 5A). Under the Gibbs and Ng (1977) model of memory formation, ouabain would have inhibited formation of labile
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memory in the trained hemisphere. If an engram were in fact also present in the untrained hemisphere following monocular learning, there should not have been a declining retention function during the single access trials after 10 min from learning. Thus, the declining function obtained during these access trials would appear to reflect a decaying short-term memory trace in the trained hemisphere and to argue against bilateral engram formation arising from monocular learning. The above results would not be consistent with the claims of Benowitz (1974) and Greif (1976) that in their experiments bilateral engrams were formed from monocular training, since lesions in their studies were administered prior to training. A significant difference between the lesion studies and the experiments reported here is that the lesions in the former were primarily of the hyperstriatal area whereas the drugs in the latter were administered in the neostriatal area. It is possible that the two sets of studies may have dealt with different brain sites. The present results also confirm the claim by Bell and Gibbs (1977) that the engram in the trained hemisphere can be duplicated in the untrained hemisphere by accessing through the untrained eye. Of greater significance, however, is the finding that engram duplication appears to depend solely on information available in the short-term memory phase of the original engram. With ouabain in the trained hemisphere, a single access trial 10 min after learning led to a high retention level 24 hr after learning, suggesting successful duplication of the engram. However, single access trials after 10 min following learning yielded decreasing retention levels 24 hr after learning (Fig. 5A). The retention function obtained at 24 hr after learning from single access trials between 10 and 30 min following learning correspond to the decaying STM trace obtained with binocular learning and bilateral injections of ouabain (Gibbs and Ng, 1977). As noted earlier, retention levels during the single access trials between 10 and 30 rain after learning show a similar decline (Fig. 5A). Cycloheximide administered to the trained hemisphere should prevent formation of LTM, but leave STM and labile memory intact in that hemisphere. We have evidence to show that CXM administered to one hemisphere resulted in significant inhibition of [14C]leucine incorporation in that hemisphere compared with saline-treated controls when the labeled amino acid was administered intracranially. No significant inhibition was observed in the untreated hemisphere (Gibbs, Richdale, & Ng, 1979). Thus memory in the trained hemisphere should remain high until after 30 min following learning when labile memory begins to decay (Gibbs & Ng, 1977). This is confirmed by the finding that single access trials between 10 and 30 min after learning yielded high retention levels (Fig. 5B). However, only the access trial at 10 rain after learning led to high retention levels at 24 hr. Single access trials after 10 min following
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learning led to decreasing retention levels at 24 hr comparable to those obtained with ouabain (Fig. 5). These results support the contention that duplication of the engram depends only on the status of STM in the trained hemisphere at the time of accessing, while retention levels during the access trials reflect whatever memory is available in the trained hemisphere at that time. The above would account for the finding that, with CXM in the trained hemisphere, access trials begun 30 min after learning and spaced 5 min apart, yielded retention levels at 24 hr which were linearly dependent on the number of access trials (Fig. 3). Furthermore, while five access trials begun 30 min after learning successfully duplicated the engram, five access trials begun 60 rain after learning were insufficient for engram duplication (Fig. 4). In the latter case, STM in the trained hemisphere may be expected to have decayed considerably. The results of the present experiments also confirm the Bell and Gibbs (1979) hypothesis that accessing information through the untrained hemisphere maintains labile memory in the trained hemisphere, despite the presence of the protein synthesis inhibitor, beyond the normal time of decay of this phase. Thus following one access trial at 30 min after learning, retention levels remain high until 50 min after learning when tested through the trained eye (Fig. 6) with ouabain administered to the untrained hemisphere to prevent engram duplication in that hemisphere. Similarly, five access trials begun 30 min after learning led to high retention levels when tested with the trained eye, until after 100 rain following learning. Furthermore, provided repeated accessing is carried out for a sufficient length of time, the labile phase can be maintained long enough to overcome CXM-induced amnesia (Fig. 7). Ten access trials spaced 5 min apart or five access trials spaced 10 min apart begun 30 rain after learning resulted in high retention levels, when tested with the trained eye, 24 hr after learning. Five access trials spaced 5 rain apart did not. Again, ouabain in the untrained hemisphere would have prevented engram duplication in that hemisphere. The precise mechanism underlying the maintaining action of repeated accessing is not clearly understood. We have demonstrated that CXMinduced amnesia can be overcome by amphetamine, norepinephrine, and diphenylhydantoin administered up to 30 min following learning (Gibbs, 1976a, 1976b; Gibbs & Ng, 1976). We suggested that the common action of these drugs in overcoming CXM-induced amnesia may be based on a reactivating of labile memory formation processes perhaps through stimulating Na,K ATPase activity (Gibbs & Ng, 1977; Jeffrey & Gibbs, 1976). This may also be the basis of action of multiple accessing in counteracting CXM-induced amnesia. Alternatively the possibility exists that repeated accessing may be extending maintenance mechanisms since
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the precise mechanism of normal labile memory maintenance following sodium pump dependent formation processes is not known. Either or both of the above actions of repeated accessing may lead to the keeping of relevant synapses identified or addressed for an increased period of time, until protein synthesis related to LTM formation recovers from CXM inhibition. In this sense, repeated accessing corresponds in some sense to Gibbs' (1976b) concept of physiological reinforcement, and may provide clues to the mechanisms supporting the effectiveness of rehearsal in strengthening memory traces. Whatever the nature of the mechanism underlying the action of accessing trials in maintaining the active life of the labile phase of memory of the original engram in the trained hemisphere, it is clear that this action of the accessing trials is quite independent of their action in duplicating the engram in the untrained hemisphere (cf. Bell & Gibbs, 1979). Thus, five access trials begun 30 min after learning and spaced 5 min apart did not overcome CXM-induced amnesia in the trained hemisphere (Fig. 7) but successfully duplicated the engram in the untrained hemisphere (Fig. 3). Finally, as shown in Fig. 3, while during the one to five access trials begun 30 min after learning, retention levels measured with the untrained eye remained constant and high, retention levels at 24 hr, measured with the same eye, were an increasing function of the number of access trials. The results from these experiments do not rule out the possibility that, under normal conditions, spontaneous duplication of memory takes place in the untrained hemisphere only after formation of the engram in the trained hemisphere has passed beyond the CXM-sensitive protein synthesis-dependent phase (see Gibbs & Ng, 1977). This possibility cannot be tested pharmacologically but could be tested by commisurotomy at relevant times following learning, although the fact that in these experiments the success in duplication appears to depend only on the status of STM in the original engram would tend to cast some doubts on it. In any event, this hypothesis would not be relevant to an explanation of duplication processes under conditions where LTM formation in the trained hemisphere has been inhibited by pharmacological intervention. In summary, the present experiments confirm Bell and Gibbs' (1977) hypothesis that monocular learning of a single-trial aversive task in dayold chicks leads, at least initially, to formation of a unilateral engram which is accessible to retrieval by the untrained hemisphere. Accessing information through the untrained hemisphere at appropriate times after learning and with an appropriate number of access trials can lead to engram duplication in the untrained hemisphere. Success in duplication appears to be dependent on the status of the original STM at the time of accessing. Independently of the above, accessing will also serve to maintain memory traces in the trained hemisphere which would otherwise
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decay in the presence of protein-synthesis inhibitors. If maintained long enough, amnesia induced by protein synthesis inhibition can be overcome. REFERENCES Bell, G. A., & Gibbs, M. E. (1977). Unilateral storage of monocular engram in day old chicks. Brain Research, 124, 263-270. Bell, G. A., & Gibbs, M. E. (1979). Interhemispheric engram transfer in chick. Neuroscience Letters, 13, 163-168. Benowitz, L. (1974). Conditions for the bilateral transfer of monocular learning in chicks. Brain Research, 65, 203-213. Cherkin, A. (1970). Eye to eye transfer of an early response modification in chicks. Nature (London) 227, 1153. Cuenod, M. (1974). Commissural pathways in the visual information in the pigeon. In F. O. Schmitt & F. G. Worden (Eds.), The Neurosciences, Third Study Program. Cambridge, Mass: MIT Press. Gaston, K. E. (1978). Interocular transfer of a visually mediated conditioned food aversion in chicks. Behavioral Biology, 24, 272-278. Gibbs, M. E. (1976). Effects of amphetamine on short-term protein independent memory in day-old chickens. Pharmacology, Biochemistry and Behavior, 4, 305-309. Gibbs, M. E. (1976). Modulation of cycloheximide-resistant memory by sympathomimetic agents. Pharmacology, Biochemistry and Behavior, 4, 703-707. (b) Gibbs, M. E., Gibbs, C. L., & Ng. K. T. (1978). A possible physiological mechanism for short-term memory. Physiology and Behavior, 20, 619-627. Gibbs, M. E., & Ng, K. T. (1976). Memory formation: A new three-phase model. Neuroscience Letters, 2, 165-169. Gibbs, M. E., & Ng, K. T. (1977). Psychobiology of memory: Towards a model of memory formation. Biobehavioral Reviews, 1, 113-136. Gibbs, M. E., Richdale, A. L., & Ng, K. T. (1979). Biochemical aspects of protein synthesis inhibition by cycloheximide in one or both hemispheres of the chick brain. Pharmacology, Biochemistry and Behavior, 10, 929-931. Greif, K. F. (1976). Bilateral memory for monocular one-trial passive avoidance in chicks. Behavioral Biology, 16, 453-462. Jeffrey, P. L., & Gibbs, M. E. (!976). Biochemical actions of sympathomimetic drugs which overcome cycloheximide-induced amnesia. Pharmacology, Biochemistry and Behavior, 5, 571-575. Rodger, R. S. (1969). Linear hypothesis in 2 x a frequency tables. British Journal of Mathematical and Statistical Psychology, 22, 29-48. Russell, I. S., & Ochs, S. (1961). One-trial interhemispheric transfer of a learning engram. Science, 153, 1077-1078. Zeier, H. (1975). Interhemispheric interactions. In P. Wright, P. G. Caryl, & D. M. Vowles (Eds.), Neural and Endocrine Aspects of Behavior in Birds, pp. 163-180. Amsterdam: Elsevier.
31, 283-298 (1981)
Engram Duplication in the Day-Old Chick ROBYN J. HODGE, MARIE E .
GIaBS, AND
K . T. N G 1
Department of Psychology, La Trobe University, Bundoora, Victoria 3083, Australia Monocular training on a single-trial aversive discrimination task in day-old chickens resulted in memory being formed only in the hemisphere served by the eye used in training. The original memory could be duplicated in the other hemisphere when the other eye was used to retrieve the information from the trained hemisphere, but the success of this was dependent on the time after training when this retrieval was carried out. One retrieval within 10 min after training, when short-term memory was maximally active in the trained hemisphere, was successful in duplicating the memory in the naive hemisphere. However, if the trials were begun at 30 rain after training when the original short-term memory had decayed somewhat, five retrievals were necessary for optimum duplication of the memory. It was suggested that duplication of memory depended on information available in the short-term memory phase of the original engram. If the memory was retrieved through the untrained eye and memory formation was inhibited in the untrained hemisphere, the decay of memory in the trained hemisphere was delayed, the effect being greater the more times the information was retrieved. This effect was interpreted in terms of maintenance of labile memory beyond its normal active life by restimulation of memory formation processes in the trained hemisphere.
The complete decussation of the optic nerves in birds has stimulated interest in the issue of lateralization of memory storage for visual tasks (see Zeier, 1975, for review). Studies in which the different commissures that connect the chick forebrains have been severed demonstrated interhemispheric transfer of visual information (Cuenod, 1974). The possibility of interhemispheric "cross-talk" raises the issue of whether tasks learned with only one eye open result in bilateral or unilateral memory formation. Lesion studies using domestic chicks and singletrial passive avoidance learning have yielded results which suggest bilateral establishment of memory following learning (Benowitz, 1974; Greif, 1976). Cherkin (1970), Gaston (1978), and Bell and Gibbs (1977) have demonstrated interocular transfer of monocular learning in unoperated 1 The authors wish to express their gratitude to Dr. G. Coleman for a computerized system for recording and extracting data in these experiments.
283 0163 - 1047/81/030283-16502.00/0 Copyright© 1981 by AcademicPress, Inc. All rightsof reproductionin any formreserved.
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chicks. Bell and Gibbs (1977) interpreted their results as evidence for formation of a unilateral memory in the hemisphere receiving the information on training, with the information available to the untrained hemisphere. Successful transfer and duplication of a unilateral engram following operant learning in the rat have also been demonstrated by Russell and Ochs (1961). We have previously postulated a model for the development of memory for a single-trial discrimination task in young chickens (Gibbs & Ng, 1976, 1977). In this model, memory formation for binocular learning follows three behavioral sequentially dependent phases: a short-term phase (STM) decaying after 10 min following learning; a labile phase decaying after 30 rain following learning; and a long-term phase (LTM) developing after 30 min. These three phases are blocked, respectively, by intracranial injection of a depolarizing agent glutamate, the sodium pump inhibitor ouabain, or the protein synthesis inhibitor cycloheximide (CXM). Bell and Gibbs (1977) found that when the discrimination task was learned monocularly, intracranial administration of the labile memory inhibitor ouabain or the long-term memory inhibitor CXM to the trained hemisphere inhibited the formation of labile memory or LTM, respectively. Injection to the untrained side did not. Their conclusion was that memory for the task was established only in the hemisphere served by the eye used in training and the presence of memories in the trained side was not sufficient to establish memory in the naive side. Furthermore, Bell and Gibbs (1979) reported evidence suggesting that there could be duplication of memory in the untrained hemisphere when the chick performed the task using the untrained eye and therefore retrieved information by way of the untrained hemisphere. The efficiency of the duplication was a function of the time of accessing the information after monocular learning and the number of times the information was accessed or retrieved through the untrained hemisphere. When CXM was administered into the trained hemisphere just after learning, the information was available for retrieval for up to 30 min after learning, before it decayed in that hemisphere. If the information was retrieved once at 7½ min after learning using the untrained eye, the memory in the trained side was available, and when these chicks were tested again 24 hr later, there was no evidence of memory loss, irrespective of which eye was used for this later test. Chicks not receiving a first (access) trial at 7½ min did not show memory at 24 hr, nor did chicks which received bilateral injections of CXM. This latter finding weakens the possibility of a reminder effect as the reason for the good retention at 24 hr in chicks receiving one access trial. Under these same conditions, a single access trial at 30 min did not result in good retention 24 hr later, even though the memory was unaffected at 30 rain on the access trial. However, if five access trials were given, beginning at 30 min and spaced 5 min apart, memory was good at 24
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hr. These results can be interpreted within the model of memory formation mentioned above. The success of duplication of memory depends on which stage the memory in the trained hemisphere is in at the time of retrieving it through the untrained hemisphere. The results of Bell and Gibbs (1977, 1979) are consistent with the hypothesis that monocular learning in day-old chicks resulted in formation of a unilateral engram in the hemisphere contralateral to the eye used in training and this memory can be duplicated in the ipsilateral (untrained) hemisphere under appropriate conditions. A further finding of interest in the Bell and Gibbs (1979) study is the fact that in the presence of CXM in the trained hemisphere, retention levels remained high during all five access trials from 30 to 50 min after learning. In binocular learning, with bilateral injection of CXM, Gibbs and Ng (1977) report high retention levels at 30 rain but amnesia by 60 min after learning. If, during the access trials, information was being taken from the labile phase of memory in the trained hemisphere, some decrease in retention levels from 30 to 50 min might have been expected. Bell and Gibbs (1979) suggested that the access trials begun 30 min after learning may have retarded the decay of labile memory in the trained hemisphere. If this is so, it may be argued that the high retention levels that they subsequently obtained at 24 hr after learning with the untrained eye may have resulted from the access trials overcoming the effects of CXM in the trained hemisphere rather than from duplication of memory in the untrained hemisphere. However, this does not explain why, under these conditions of accessing, CXM administered to both the trained and the untrained hemisphere prevented high retention levels at 24 hr. The conclusion appears inescapable that duplication of the engram does in fact occur following accessing but this is independent of the effect of accessing on the original engram. The question remains as to why engram duplication becomes more difficult with increasing age of the unilateral engram (Bell & Gibbs, 1979). The possibility arises that engram duplication is dependent solely on information derived from the short-term memory phase of the original engram, and becomes progressively more difficult with an increasingly decaying STM after 10 min following learning. An equally interesting, if perhaps less important, question concerns under what conditions, if any, repeated accessing will lead to the inhibitory action of CXM in the trained hemisphere being overcome. In this paper we report a series of experiments directed at answering these two questions. METHOD Procedure. The basic experimental paradigm follows that described by Gibbs and Ng (1977). Figure 1 summarizes the experimental procedures
286
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by the exposed eye during training to the contralateral hemisphere and retrieval of the information by the untrained eye during access and retention trials. The trained hemisphere is marked with an (*) to represent initial engram formation. Only the hemispheres are represented in (B), with the symbols indicating which hemisphere received the learning trial (*), access trial(s) (dashed arrow) or retention trial (wide arrow). In (C), we show how the diagrammatic representation used in the subsequent figures was compiled from (A) and (B). Cross hatching in the subsequent figures indicates the hemisphere receiving the injection of the specified drug. specific to these experiments. Day-old white L e g h o r n - b l a c k Australorp cockerels were placed in pairs in wooden b o x e s shortly after arrival f r o m a local hatchery, and were kept in pairs throughout all subsequent manipulations. In all but one experiment, 20 different chicks were assigned for each data point. Pretraining. The chicks were trained to peck at a 4-mm red glass bead, dipped in water, presented for 10 sec. This was followed by a similar 10-sec trial with a 4-mm blue glass bead. Monocular learning. The right eye was occluded with an opaque Band Aid Spot. Ten minutes later, on a single aversive discrimination learning trial, chicks were presented a red bead similar to the one used in pretraining, but dipped in the chemical aversant methyl anthranilate, for 10 sec. The data for chicks failing to peck on this learning trial or to show the characteristic " d i s g u s t " responses of shaking their heads and wiping their beaks were discarded at the end of the experiment. The n u m b e r of chicks discarded was n e v e r m o r e than 15% of the original sample and the actual sample size for each data point is reported in the relevant figure legends. The eye patch was r e m o v e d immediately after the learning trial. Accessing trials. An accessing trial is one in which the eye occluded in the monocular learning trial is used to retrieve information about the
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aversive bead. Thus, where a chick is trained with the left eye (right eye occluded) accessing of memory is carried out by presenting a dry bead to the right eye. No blue bead is presented on an access trial. Five minutes prior to accessing, the left eye was occluded. On each access trial, a dry red bead was presented for 10 sec. The eye patch is removed immediately after the last access trial. Retention trials. A retention trial is one in which the trained eye is used to test for memory. Five minutes prior to the retention trial, the eye not used in training is occluded and the eye patch is removed immediately after the trial. On each retention trial a dry red bead is presented for 10 sec, followed by a dry blue bead. Retention is indexed by the proportion of chicks avoiding the red bead. Drugs and injections. All drugs were made up in sterile NaC1 (0.15 M; 0.9%). Ouabain (Sigma; 0.027 mM), cycloheximide (Upjohn, 3.5 raM), or saline (0.15 M) was administered 5 min before learning into the right hemisphere of each chick. Injections of 10-/A volumes for CXM and saline or 5- or 10-/zl volumes for ouabain were freehand into the center of the forebrain to a depth of 3.0 mm, in the neostriatal area. RESULTS
Formation of Unilateral Engram Ten microliters of ouabain or saline was administered into the right hemisphere of each chick, 5 rain prior to monocular training with the left eye, in order to prevent formation of labile and long-term memory in that hemisphere. Information was accessed through the right (naive) eye at 10, 30, 60, 120, 180, or 1440 min after training. Using the method of planned contrasts on proportions and a type I error rate of a = 0.05 (Rodger, 1969), significant differences were found between saline- and ouabain-treated chicks in the proportion avoiding the red bead at 30, 60, 120, 180, and 1440 min after training (F(1, oo = 13.55, 5.46, 11.30, 23.95, and 4.31 respectively). The difference at 10 rain after training was not significant. No differences were apparent between the drug and saline groups in proportion avoiding the blue bead (Fig. 2). The results suggest that, with ouabain inducing amnesia in the trained hemisphere after 10 min following learning (Gibbs & Ng, 1977), no memory is available from the untrained hemisphere alone.
Effect of Variation in Number of Access Trials on Engram Duplication Five minutes prior to monocular training with the left eye 10/zl of CXM was administered ~nto the right hemisphere of each chick, in order to prevent formation of LTM in that hemisphere. Commencing 30 rain after monocular training zero, one, two, three, four, or five access trials 5 min apart were performed with the right eye. A control group of chicks were
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given five access trials, spaced 5 min apart, through the right eye commencing 60 min after training. An injection of CXM was given to the trained hemisphere 5 min before training. Memory for the learned response was tested with the right eye 24 hr after training. The proportion of chicks avoiding the red bead during access trials at 30 min after learning did not alter from group to group and was comparable to retention levels reported for 30 min after learning following CXM pretreatment (Gibbs & Ng, 1977; Fig. 3). However, the proportion of chicks avoiding the red bead 24 hr after training was a linear function of the number of access trials. There was a significant linear (F(1, oo) = 23.96, p < .05) and a nonsignificant quadratic trend. With no access trials the proportion of chicks avoiding the red bead at 24 hr is comparable to that reported for CXM-pretreated chicks trained and tested binocularly (Gibbs & Ng, 1977). After 5 access trials, the proportion is comparable to that for untreated or saline-pretreated chicks (Gibbs & Ng, 1977). When five access trials were given 60 rain after training, the proportion of chicks avoiding the red bead during these trials was comparable to that obtained with CXM-pretreated chicks at 60-90 min after learning (Gibbs & Ng, 1977)..Retesting at 24 hr revealed no change in avoidance level (Fig. 4). The results suggest that five access trials, when begun 30 rain after learning, are necessary for complete engram duplication. However, the effect of repeated accessing appears to be additive, at least up to five
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access trials, provided the accessing is begun prior to the decay of the labile phase in the trained hemisphere.
Effect of Varying Time of Single-Access Trial on Engram Duplication Following learning with the left eye, a single access trial was performed through the right eye 10, 15, 20, 25, or 30 rain after learning. Ouabain or CXM was administered into the right (trained) hemisphere 5 min prior to monocular learning, to prevent formation of labile memory and LTM, respectively, in that hemisphere (Figs. 5A and B). Memory for the learned task was tested with the right eye at 24 hr. In the presence of ouabain in the trained hemisphere, the proportion of
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chicks avoiding the red bead during access trials follows the declining function observed with ouabain inhibition of labile memory in binocular learning (Gibbs & Ng, 1977), while in the presence of CXM, the proportions remain high and are comparable to those observed with CXM inhibition of LTM in binocular learning (Gibbs & Ng, 1977). The results suggest that performance during accessing reflects available memory in the original engram. In the absence of labile memory, information during accessing between 10 and 30 min after learning is retrieved from a decaying short-term memory. The proportion of ouabain-treated chicks avoiding the red bead at 180 min after learning is a declining function of the time of accessing, and the function is comparable to that observed during access trials (Fig. 5A). There is a significant linear trend in the function (F(1, oo) = 12.09, p < .05). However, the same declining function is observed at 24 hr after learning with CXM-treated chicks contrasting markedly with the function obtained during accessing (Fig. 5B). In fact, for each accessing time, there is no significant difference between proportion of ouabain-treated and CXM-treated chicks avoiding the red bead during retention tests. The two retention functions are indistinguishable from the retention functions obtained by Gibbs and Ng (1977) from retention tests between 10 and 30 min after learning with bilateral injections of ouabain or ethacrynic acid 5 min before binocular learning. Thus, while information during access trials is retrieved from whatever phase is active in the original engram at the time of accessing, information
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Effect of Accessing Trials on Maintenance of Labile Memory in the Trained Hemisphere F i v e m i n u t e s p r i o r t o m o n o c u l a r l e a r n i n g w i t h t h e left e y e , C X M w a s a d m i n i s t e r e d into t h e right h e m i s p h e r e to inhibit f o r m a t i o n o f L T M in t h a t h e m i s p h e r e . T h i r t y m i n u t e s a f t e r l e a r n i n g z e r o , o n e , o r five a c c e s s trials w e r e c a r r i e d o u t w i t h t h e o t h e r e y e - - t h e right e y e . R e t e n t i o n t e s t s w e r e c a r r i e d o u t u s i n g t h e left ( t r a i n e d ) e y e at v a r i o u s t i m e s f r o m 10 t o 120 m i n after learning. C h i c k s r e c e i v i n g o n e o r five a c c e s s trials w e r e a d m i n i s t e r e d 1 0 / z l o f
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HODGE, GIBBS, AND NG
ouabain in the untrained hemisphere immediately after the first access trial to prevent formation of labile, and hence, also long-term memory in that hemisphere (Gibbs & Ng, 1977). In order to ensure that observed retention between 45 and 120 min after learning was not due to engram duplication in the untrained hemisphere, three groups of chickens given five access trials were administered 5 /~1 of ouabain in the untrained hemisphere at 30 min (immediately after the first access trial) and a further 5 /1.1 of ouabain at 45 min (immediately after the fourth access trial) following learning, and retention was tested at 60, 100, or 120 min after learning. With no access trials the time course of retention is comparable to that obtained with bilateral injections of CXM under binocular learning (Gibbs & Ng, 1977). Retention is high until 30 min following learning, and then declines rapidly, yielding the typical decay function of the labile phase. With five access trials, however, retention levels remain high for up to 100 min following learning (Fig. 6). At 70, 80, 90, and 100 min following learning the proportion of chicks avoiding the red bead after five access trials was significantly different from that observed with chicks given no access trials (F(1, ~) = 8.12, 5.76, 7.58, and 10.00, respectively; p < .05). At 60 or 120 min after learning, the difference is not significant. With one access trial, the results are equivocal. At 45 and 50 min after learning the retention levels remain high and are comparable to those observed during access trials. However, the proportions of chicks avoiding the red bead at these times were not significantly different from those observed with no access trials which still showed a little retention. The retention level drops markedly by 60 min after learning, and is maintained at that level up to 100 min following learning (Fig. 6). The proportion of chicks avoiding the bead at these times was not significantly different from those observed in chicks given no access trials. The above results suggest that after five access trials labile memory is maintained for at least 70 min after the normal time of decay. With one access trial it is maintained for up to 20 min after the normal time of decay. The absence of statistically significant differences here may be attributed to the power of the statistical test used and the fact that we are dealing with a declining retention function for the no access trial condition. The high retention levels observed between 60 and 180 min after learning cannot be attributed to duplication of the engram in the ipsilateral (naive) hemisphere. No significant differences in proportion of chicks avoiding the red bead were obtained at the 60-, 100-, and 120-min learning-retention intervals between chicks given five accesses and 10/xl ouabain 30 min after learning (67, 60, and 30%, respectively) and chicks given five accesses and 5/xl ouabain at 30 and 45 min after learning (67, 56, and 25%, respectively).
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F[o. 6. The effect of varying the number of access trials through the naive hemisphere on retention from the trained hemisphere. The right hemisphere was injected with CXM 5 rain before the learning trial. One (A) or five (D) access trials were given through the left hemisphere commencing at 30 min after learning, with this hemisphere receiving an injection of ouabain immediately after the first access trial at 30 min to inhibit duplication of memory in this side. The right hemisphere (A, I ) was tested in separate groups of chicks at various times after the completion of the access trial(s). Separate groups of chicks receiving no access trials were tested at various times after learning (O) commencing at 10 min after learning. Number of chicks per group: 18-20.
Effect of Access Trials on CXM-Induced Amnesia in the Trained Hemisphere At 30 min after m o n o c u l a r training with the left eye, chicks w e r e p r e s e n t e d 5 or 10 a c c e s s trials t h r o u g h the right eye. T h e intertrial interval for five a c c e s s trials was 10 min, while that for 10 a c c e s s trials was 5 rain. R e t e n t i o n was tested at 24 hr after learning with the left (training) eye. T e n microliters o f C X M w a s administered to the trained h e m i s p h e r e 5 min prior to training; 5 /1,1 o f o u a b a i n was a d m i n i s t e r e d to the ipsilateral h e m i s p h e r e at 30 min (immediately after the first a c c e s s trial) and at 55 rain after learning to inhibit f o r m a t i o n o f labile and h e n c e long-term m e m o r y in that h e m i s p h e r e . T w o injections o f o u a b a i n were c o n s i d e r e d n e c e s s a r y since this drug is not maximally effective for 50 rain (Gibbs & Ng, 1977). A control g r o u p o f chicks was given five a c c e s s trials s p a c e d 5 min apart, with 10 t~l o f o u a b a i n a d m i n i s t e r e d into the ipsilateral h e m i s p h e r e 30 min after learning. D u r i n g a c c e s s trials the p r o p o r t i o n o f chicks a v o i d i n g the red bead r e m a i n e d high for all three g r o u p s (Fig. 7). At 24 hr after learning, the p r o p o r t i o n o f chicks avoiding the red bead in the g r o u p receiving 10 a c c e s s trials was not significantly different f r o m that in the g r o u p receiv-
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HODGE, GIBBS, AND N G OUAB CXM
7o
100-
80-
1Ol5mrn ouabain *30,* 55
g6e-
5/lOrnln ouet:min -30,+ 55
N 20-
5/5mln ouobain *30
3'0
~'
~'0
go
~0
1440
LEARNING- ACCESS. TEST INTERVAL (rain)
FIG. 7. Percentage of chicks avoiding the dry red bead (filled symbols) at 24 hr after monocular training when the trained side was tested. Chicks were trained using the left eye and the right hemisphere was injected with CXM 5 min before the learning trial. Chicks received different combinations o f access trials through the naive hemisphere, which was injected with ouabain to prevent duplication o f memory in that hemisphere (see text for details). The accessing combinations, commencing at 30 rain after learning were: 10 access trials with 5-min intervals between each trial (©), n = 20; 5 access trials with 10-rain intervals between each trial (rq), n = 19; 5 access trials with 5-min intervals between each trial (A), n = 20.
ing 5 access trials 10 min apart but these two groups, on the average, showed a greater proportion avoiding the red bead than the control group receiving 5 access trials 5 min apart (F(1, oo) = 9.99, p < .05). The level for the control group is comparable to that observed at 24 hr with CXMtreated chicks under binocular training (Gibbs & Ng, 1977), while those for the other two groups are comparable to saline-treated chicks. The results suggest that either 10 accesses 5 min apart or 5 accesses 10 min apart are sufficient to overcome CXM-induced amnesia. However, 5 accesses spaced 5 min apart were not sufficient to overcome CXMamnesia and reinstate LTM formation in the trained hemisphere.
DISCUSSION The present results are consistent with Bell and Gibbs' (1977) contention that monocular learning of a single-trial passive avoidance discrimination task in the day-old chick results in the formation of a unilateral engram in the hemisphere contralateral to the eye used in training. Ouabain administered to the trained hemisphere resulted in decreasing levels of retention during single accessing trials with the untrained hemisphere after 10 min following learning (Fig. 5A). Under the Gibbs and Ng (1977) model of memory formation, ouabain would have inhibited formation of labile
ENGRAM DUPLICATION
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memory in the trained hemisphere. If an engram were in fact also present in the untrained hemisphere following monocular learning, there should not have been a declining retention function during the single access trials after 10 min from learning. Thus, the declining function obtained during these access trials would appear to reflect a decaying short-term memory trace in the trained hemisphere and to argue against bilateral engram formation arising from monocular learning. The above results would not be consistent with the claims of Benowitz (1974) and Greif (1976) that in their experiments bilateral engrams were formed from monocular training, since lesions in their studies were administered prior to training. A significant difference between the lesion studies and the experiments reported here is that the lesions in the former were primarily of the hyperstriatal area whereas the drugs in the latter were administered in the neostriatal area. It is possible that the two sets of studies may have dealt with different brain sites. The present results also confirm the claim by Bell and Gibbs (1977) that the engram in the trained hemisphere can be duplicated in the untrained hemisphere by accessing through the untrained eye. Of greater significance, however, is the finding that engram duplication appears to depend solely on information available in the short-term memory phase of the original engram. With ouabain in the trained hemisphere, a single access trial 10 min after learning led to a high retention level 24 hr after learning, suggesting successful duplication of the engram. However, single access trials after 10 min following learning yielded decreasing retention levels 24 hr after learning (Fig. 5A). The retention function obtained at 24 hr after learning from single access trials between 10 and 30 min following learning correspond to the decaying STM trace obtained with binocular learning and bilateral injections of ouabain (Gibbs and Ng, 1977). As noted earlier, retention levels during the single access trials between 10 and 30 rain after learning show a similar decline (Fig. 5A). Cycloheximide administered to the trained hemisphere should prevent formation of LTM, but leave STM and labile memory intact in that hemisphere. We have evidence to show that CXM administered to one hemisphere resulted in significant inhibition of [14C]leucine incorporation in that hemisphere compared with saline-treated controls when the labeled amino acid was administered intracranially. No significant inhibition was observed in the untreated hemisphere (Gibbs, Richdale, & Ng, 1979). Thus memory in the trained hemisphere should remain high until after 30 min following learning when labile memory begins to decay (Gibbs & Ng, 1977). This is confirmed by the finding that single access trials between 10 and 30 min after learning yielded high retention levels (Fig. 5B). However, only the access trial at 10 rain after learning led to high retention levels at 24 hr. Single access trials after 10 min following
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learning led to decreasing retention levels at 24 hr comparable to those obtained with ouabain (Fig. 5). These results support the contention that duplication of the engram depends only on the status of STM in the trained hemisphere at the time of accessing, while retention levels during the access trials reflect whatever memory is available in the trained hemisphere at that time. The above would account for the finding that, with CXM in the trained hemisphere, access trials begun 30 min after learning and spaced 5 min apart, yielded retention levels at 24 hr which were linearly dependent on the number of access trials (Fig. 3). Furthermore, while five access trials begun 30 min after learning successfully duplicated the engram, five access trials begun 60 rain after learning were insufficient for engram duplication (Fig. 4). In the latter case, STM in the trained hemisphere may be expected to have decayed considerably. The results of the present experiments also confirm the Bell and Gibbs (1979) hypothesis that accessing information through the untrained hemisphere maintains labile memory in the trained hemisphere, despite the presence of the protein synthesis inhibitor, beyond the normal time of decay of this phase. Thus following one access trial at 30 min after learning, retention levels remain high until 50 min after learning when tested through the trained eye (Fig. 6) with ouabain administered to the untrained hemisphere to prevent engram duplication in that hemisphere. Similarly, five access trials begun 30 min after learning led to high retention levels when tested with the trained eye, until after 100 rain following learning. Furthermore, provided repeated accessing is carried out for a sufficient length of time, the labile phase can be maintained long enough to overcome CXM-induced amnesia (Fig. 7). Ten access trials spaced 5 min apart or five access trials spaced 10 min apart begun 30 rain after learning resulted in high retention levels, when tested with the trained eye, 24 hr after learning. Five access trials spaced 5 rain apart did not. Again, ouabain in the untrained hemisphere would have prevented engram duplication in that hemisphere. The precise mechanism underlying the maintaining action of repeated accessing is not clearly understood. We have demonstrated that CXMinduced amnesia can be overcome by amphetamine, norepinephrine, and diphenylhydantoin administered up to 30 min following learning (Gibbs, 1976a, 1976b; Gibbs & Ng, 1976). We suggested that the common action of these drugs in overcoming CXM-induced amnesia may be based on a reactivating of labile memory formation processes perhaps through stimulating Na,K ATPase activity (Gibbs & Ng, 1977; Jeffrey & Gibbs, 1976). This may also be the basis of action of multiple accessing in counteracting CXM-induced amnesia. Alternatively the possibility exists that repeated accessing may be extending maintenance mechanisms since
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the precise mechanism of normal labile memory maintenance following sodium pump dependent formation processes is not known. Either or both of the above actions of repeated accessing may lead to the keeping of relevant synapses identified or addressed for an increased period of time, until protein synthesis related to LTM formation recovers from CXM inhibition. In this sense, repeated accessing corresponds in some sense to Gibbs' (1976b) concept of physiological reinforcement, and may provide clues to the mechanisms supporting the effectiveness of rehearsal in strengthening memory traces. Whatever the nature of the mechanism underlying the action of accessing trials in maintaining the active life of the labile phase of memory of the original engram in the trained hemisphere, it is clear that this action of the accessing trials is quite independent of their action in duplicating the engram in the untrained hemisphere (cf. Bell & Gibbs, 1979). Thus, five access trials begun 30 min after learning and spaced 5 min apart did not overcome CXM-induced amnesia in the trained hemisphere (Fig. 7) but successfully duplicated the engram in the untrained hemisphere (Fig. 3). Finally, as shown in Fig. 3, while during the one to five access trials begun 30 min after learning, retention levels measured with the untrained eye remained constant and high, retention levels at 24 hr, measured with the same eye, were an increasing function of the number of access trials. The results from these experiments do not rule out the possibility that, under normal conditions, spontaneous duplication of memory takes place in the untrained hemisphere only after formation of the engram in the trained hemisphere has passed beyond the CXM-sensitive protein synthesis-dependent phase (see Gibbs & Ng, 1977). This possibility cannot be tested pharmacologically but could be tested by commisurotomy at relevant times following learning, although the fact that in these experiments the success in duplication appears to depend only on the status of STM in the original engram would tend to cast some doubts on it. In any event, this hypothesis would not be relevant to an explanation of duplication processes under conditions where LTM formation in the trained hemisphere has been inhibited by pharmacological intervention. In summary, the present experiments confirm Bell and Gibbs' (1977) hypothesis that monocular learning of a single-trial aversive task in dayold chicks leads, at least initially, to formation of a unilateral engram which is accessible to retrieval by the untrained hemisphere. Accessing information through the untrained hemisphere at appropriate times after learning and with an appropriate number of access trials can lead to engram duplication in the untrained hemisphere. Success in duplication appears to be dependent on the status of the original STM at the time of accessing. Independently of the above, accessing will also serve to maintain memory traces in the trained hemisphere which would otherwise
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decay in the presence of protein-synthesis inhibitors. If maintained long enough, amnesia induced by protein synthesis inhibition can be overcome. REFERENCES Bell, G. A., & Gibbs, M. E. (1977). Unilateral storage of monocular engram in day old chicks. Brain Research, 124, 263-270. Bell, G. A., & Gibbs, M. E. (1979). Interhemispheric engram transfer in chick. Neuroscience Letters, 13, 163-168. Benowitz, L. (1974). Conditions for the bilateral transfer of monocular learning in chicks. Brain Research, 65, 203-213. Cherkin, A. (1970). Eye to eye transfer of an early response modification in chicks. Nature (London) 227, 1153. Cuenod, M. (1974). Commissural pathways in the visual information in the pigeon. In F. O. Schmitt & F. G. Worden (Eds.), The Neurosciences, Third Study Program. Cambridge, Mass: MIT Press. Gaston, K. E. (1978). Interocular transfer of a visually mediated conditioned food aversion in chicks. Behavioral Biology, 24, 272-278. Gibbs, M. E. (1976). Effects of amphetamine on short-term protein independent memory in day-old chickens. Pharmacology, Biochemistry and Behavior, 4, 305-309. Gibbs, M. E. (1976). Modulation of cycloheximide-resistant memory by sympathomimetic agents. Pharmacology, Biochemistry and Behavior, 4, 703-707. (b) Gibbs, M. E., Gibbs, C. L., & Ng. K. T. (1978). A possible physiological mechanism for short-term memory. Physiology and Behavior, 20, 619-627. Gibbs, M. E., & Ng, K. T. (1976). Memory formation: A new three-phase model. Neuroscience Letters, 2, 165-169. Gibbs, M. E., & Ng, K. T. (1977). Psychobiology of memory: Towards a model of memory formation. Biobehavioral Reviews, 1, 113-136. Gibbs, M. E., Richdale, A. L., & Ng, K. T. (1979). Biochemical aspects of protein synthesis inhibition by cycloheximide in one or both hemispheres of the chick brain. Pharmacology, Biochemistry and Behavior, 10, 929-931. Greif, K. F. (1976). Bilateral memory for monocular one-trial passive avoidance in chicks. Behavioral Biology, 16, 453-462. Jeffrey, P. L., & Gibbs, M. E. (!976). Biochemical actions of sympathomimetic drugs which overcome cycloheximide-induced amnesia. Pharmacology, Biochemistry and Behavior, 5, 571-575. Rodger, R. S. (1969). Linear hypothesis in 2 x a frequency tables. British Journal of Mathematical and Statistical Psychology, 22, 29-48. Russell, I. S., & Ochs, S. (1961). One-trial interhemispheric transfer of a learning engram. Science, 153, 1077-1078. Zeier, H. (1975). Interhemispheric interactions. In P. Wright, P. G. Caryl, & D. M. Vowles (Eds.), Neural and Endocrine Aspects of Behavior in Birds, pp. 163-180. Amsterdam: Elsevier.