PHB-10546; No of Pages 7 Physiology & Behavior xxx (2014) xxx–xxx
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Relearning a context-shock association after forgetting is an NMDAr-independent process Diana Chan ⁎, Kathryn D. Baker, Rick Richardson School of Psychology, The University of New South Wales, Sydney 2052, Australia
H I G H L I G H T S • • • •
Infant animals forget context-shock associations rapidly. Forgotten context-shock associations still change the mechanisms of future context learning. Exposing animals to the shock only also causes these changes. Exposing animals to the context only caused a less robust effect.
a r t i c l e
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Article history: Received 30 August 2014 Received in revised form 31 October 2014 Accepted 3 November 2014 Available online xxxx Keywords: Fear Infantile amnesia NMDA receptor MK801
a b s t r a c t Infantile amnesia (i.e., the rapid rate of forgetting in young animals) is at least partially due to a memory retrieval, rather than a storage, failure as studies have shown that these engrams can continue to influence later behavior. For example, prior conditioning affects the neural mechanisms underlying future learning. In adult animals, the initial learning of a context-shock association depends upon N-Methyl-D-Aspartate (NMDA) receptors, but this conditioning renders subsequent learning to a similar context NMDAr-independent. In the present study, we examined whether this transition from NMDAr-dependent to NMDAr-independent context conditioning occurs even after infantile amnesia. Experiment 1 demonstrated that infant (i.e., postnatal day 17) rats acquire a context-shock association when trained with multiple shocks, as assessed by context freezing one day later. However, they exhibit significant forgetting of this association 10 days later. Experiments 2 and 3 showed that even when animals had forgotten the initial learning experience, future conditioning to the same context was NMDAr-independent. There was evidence of a transition to NMDAr-independent context fear learning in animals exposed only to the foot shock in infancy (Experiment 3) or only to the context in infancy (Experiment 3 but not Experiment 2). These latter results suggest that animals do not have to be exposed to the entire conditioning procedure at postnatal day 17 to show a transition to NMDAr-independent context learning. These experiments add to a growing body of evidence that forgotten infant memories can continue to affect later behavior by demonstrating that prior experience alters the mechanisms of future learning. © 2014 Elsevier Inc. All rights reserved.
1. Introduction Perhaps the most common view of forgetting is that it is an inability to behaviorally express a memory. Although memory loss occurs across the lifespan, memories that are formed early in life are particularly vulnerable to being forgotten. For example, most adults do not remember events that occurred before they were 4 years old [1]. This effect is known as infantile (or childhood) amnesia [2]. Importantly, this phenomenon has been demonstrated extensively across species, and is observed even when levels of learning are equated across younger and older animals [3]. ⁎ Corresponding author. Tel.: +61 2 9385 1048; fax: +61 2 93853641. E-mail addresses:
[email protected] (D. Chan),
[email protected] (K.D. Baker),
[email protected] (R. Richardson).
Even though our memories for early experiences are particularly fragile and easily forgotten it is widely accepted that aversive childhood experiences contribute to the development of adult psychopathology [4]. On the surface, this idea seems counter to the occurrence of infantile/childhood amnesia, but the work of Spear and his colleagues suggests a possible resolution to this apparent paradox. In particular, Spear has been instrumental in establishing that infantile amnesia is at least partially due to a memory retrieval, rather than a storage, failure. That is, an ‘engram’ of the memory persists even though it is not behaviorally expressed. In several classic studies, Spear and his colleagues demonstrated that apparently forgotten memories acquired early in life can be retrieved under certain conditions. This work built on research by Campbell and Jaynes [5] showing that periodic (i.e., weekly) ‘reminder’ treatments (in the form of abbreviated training sessions) markedly reduced forgetting in infant rats tested several
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Please cite this article as: Chan D, et al, Relearning a context-shock association after forgetting is an NMDAr-independent process, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.11.004
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weeks after being trained on an avoidance procedure. In several studies, Spear and his students showed that a single presentation of an unconditioned stimulus (US), by itself, was sufficient to reactivate a memory acquired early in life [e.g., 6]. This effect has been replicated numerous times, in rodents and humans [e.g., 7,8], and clearly shows that apparently forgotten memories formed early in life are not gone and that they can be recalled in certain test conditions. This foundational work by Spear has been extended by demonstrations that apparently forgotten infant memories can be assessed through implicit measures. For example, Newcombe and Fox [9] tested primary school aged children for recognition of their preschool classmates. Children exhibited a higher skin conductance response to pictures of their old classmates relative to pictures of unfamiliar children regardless of whether they explicitly recognized their former peers or not. Thus, even though early-life memories were not explicitly recalled these past memories continued to influence later behavior. Recently, Li and Richardson [10] used animal studies to explore this ‘implicit’ influence of forgotten memories at a molecular level. Specifically, they used a finding from the adult literature showing that prior experience can alter the neurobiology of learning. It is well established that the initial learning (i.e., acquisition) of a conditioned stimulus (CS)-US association requires activation of N-Methyl-DAspartate (NMDA) receptors, which trigger a molecular cascade leading to long-term potentiation (LTP). LTP is a long-lasting enhancement in synaptic strength and is thought to be a neurobiological substrate of learning and memory storage. In support of this view is a wealth of evidence that NMDA receptor (NMDAr) antagonists inhibit LTP and impair the acquisition of a CS-US association [e.g., 11–13]. While there is robust evidence that NMDA receptors are required for the initial learning of a CS-US association, recent studies have demonstrated that these receptors are not involved in learning an association for the second time. For example, Wiltgen et al. [14] sequentially conditioned two groups of adult mice to associate two different contexts with shock and tested the effect of blocking NMDA receptors during the first or second context conditioning episode. The animals were given systemic injections of either saline or an NMDAr antagonist, CPP, before conditioning to the first context (A) on day 1. The following day the mice received an injection of the alternative solution (i.e., those animals given saline on day 1 received CPP on day 2 and vice versa) before conditioning to the second context (context B). Both groups were then tested for their fear of each context on days 3 and 4. The results showed that CPP impaired conditioning to context A but not to context B, suggesting that while NMDA receptors are involved in learning the initial context-shock association they are not required for subsequent conditioning to a second context. This is a robust finding that has been replicated using systemic injections as well as infusions of a different NMDAr-antagonist into the hippocampus, and is not due to animals being unable to discriminate between the two contexts [e.g., 15]. Taken together, this work shows that there is a transition from an NMDAr-dependent learning process to an NMDAr-independent relearning process. All of the studies described above that examined the transition from NMDAr-dependent to NMDAr-independent learning used adult rodents, which are known to retain memories for context-shock associations for at least 16 months [16]. Thus, at the time of the second conditioning session animals had an intact memory of the first training experience. Li and Richardson [10] extended this work by examining whether the modifications that allow neurons to support NMDArindependent learning persist after infantile amnesia. At postnatal day (P) 17, rats were given pairings of a noise CS with a shock US. Although the noise CS elicited high levels of freezing 1 day after training it elicited negligible levels of freezing after 2 weeks, indicating that the fear memory had been forgotten (i.e., it was no longer behaviorally expressed). Nevertheless, re-acquisition of the noise CS-shock US association at this later age was unaffected by the NMDAr-antagonist MK801. These results suggest that infant memories that are no longer
behaviorally expressed nonetheless alter the cellular mechanisms involved in later learning about that stimulus. The transition from NMDAr-dependent to NMDAr-independent learning was stimulusspecific in that study. In addition, Li and Richardson [10] found that only associative learning at P17 caused the transition from NMDArdependence to NMDAr-independence. Merely exposing the infant animals to unpaired presentations of the noise CS and shock US did not lead to NMDAr-independent learning later in life. In the present study we examined whether a similar effect would occur following context conditioning early in life. While a similar NMDAr transition may be predicted, there are a number of differences between context and cued fear conditioning that could lead to different results. For example, these forms of learning differ in their dependence on particular physiological mechanisms [e.g., corticosterone is involved in long term memory for context but not cued conditioning; 17] and involvement of brain regions such as the hippocampus [e.g., lesions impair context but not cued conditioning in adult animals; 18,19]. The hippocampal formation is a relatively late developing structure, and therefore it has typically been found that infant rats (i.e., around P17) are impaired in retaining memory for conditioning to a context, but not an auditory CS, relative to older (e.g., juvenile P23) animals [e.g., 20]. Rudy and colleagues found that infant (i.e., P17) and juvenile (i.e., P23) animals exhibit equivalent context fear immediately after conditioning, but the infant rats had poorer memory at a 24 h retention interval. This effect occurred regardless of whether the foot shock was signaled by a discrete-cue (e.g., a tone) or not [20–22]. This impaired context learning in young rodents compared to older ones has been reported in a number of other studies [23–26] but there is evidence that infant rodents can acquire a context memory under certain conditions. For example, in contrast to the study by Rudy and Morledge [20], Brasser and Spear [27] found that CS-US conditioning potentiated fear learning to the background context relative to when the CS and US were presented in an unpaired manner. Other studies have also found that young animals are able to exhibit context learning when the context includes a novel olfactory element [e.g., 28,29]. Because of these several differences between context and cue conditioning, it is unclear whether the transition from NMDAr-dependent learning to NMDAr-independent relearning observed by Li and Richardson [10] with auditory cued fear conditioning will also be observed for a “forgotten” context fear memory. Therefore, in the present study we examined this issue. In Experiment 1 we demonstrated that giving infant rats (P17) multiple shocks at training led to conditioned fear at a 1-day test [see also 30,31], and that this memory was forgotten after a 10-day interval (infantile amnesia). Building upon this finding we investigated whether prior contextual fear conditioning induces the transition to NMDAr-independent relearning, even when the original memory is no longer behaviorally expressed (Experiment 2). Finally, we examined whether exposing animals to only part of the conditioning procedure (i.e., context or US only) similarly leads to such changes (Experiment 3). 2. Material and methods 2.1. Subjects The animals used in this study were male, experimentally naive, Sprague–Dawley rats that were born and raised in the breeding colony maintained in the School of Psychology at The University of New South Wales. Rats were housed with a 12-hr light-dark cycle (lights on at 0700) and all experiments were conducted during the light phase. Food and water were provided ad libitum. The date of birth was designated as P0. Litters were culled to 8 pups between P1 and P3, and were housed with their mother in plastic boxes (24.5 × 27 × 27 cm) covered with a wire lid until the end of the study. Only one rat per litter was used in each experimental group. Animals were cared for according to the principles of The Australian Code of Practice for the Care and Use of
Please cite this article as: Chan D, et al, Relearning a context-shock association after forgetting is an NMDAr-independent process, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.11.004
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Animals for Scientific Purposes (7th Ed.; 2004). All experimental procedures were approved by The University of New South Wales Animal Care and Ethics committee.
A second scorer who was blind to the experimental condition of each animal cross-scored a random 30% of the test data. The inter-rater reliability was high for all experiments (r = 0.94–0.99).
2.2. Apparatus
2.4. Drug administration
All experiments were conducted in rectangular chambers (13.5 × 9 × 9 cm) that were housed within wood boxes to attenuate external light and noise. The ceiling, front, and back walls of the chamber were constructed from clear Plexiglas, while the sidewalls and floor were built from steel rods (3 mm in diameter and 1 cm apart center-to-center). The chamber was cleaned with tap water after each experimental session. An infrared camera, with red light-emitting diodes (LEDs), was mounted on the back wall of the chamber to allow the animals' behavior to be recorded. Ventilation fans produced a steady, low level of background noise (48 dB).
In Experiments 2 and 3, rats were injected with MK801 (SigmaAldrich, Castle Hill, New South Wales) or saline 10 min prior to conditioning at P27. MK801 was dissolved in 0.9% sterile saline and was injected at a dose of 0.1 mg/kg. Control animals were given injections of saline. All injections were given subcutaneously (in the nape of the neck) at a volume of 1 mL/kg.
2.3. Procedure
The purpose of Experiment 1 was to demonstrate that, using the current parameters, infant rats could 1) acquire a context fear memory, and 2) that they forgot this memory over a 10 day interval. Animals in this experiment were conditioned to fear the context at P17 and then tested either 1 or 10 days later. Freezing during the 2-minute adaptation period prior to the delivery of shock was assessed as an index of the animals' pre-conditioning levels of fear, whereas freezing during the 2-minute test session was taken as an index of whether the infants learned a context-shock association (i.e., 1-day interval) and whether they forgot that association over time (i.e., 10-day interval). Prior to the onset of the shock (on P17), all animals in both groups exhibited 0% freezing, indicating that none were fearful of the context prior to conditioning. In contrast, when tested 1-day later, animals exhibited more freezing to the context. After the 10-day interval, however, the levels of freezing returned to what was observed before conditioning (see Fig. 1). A mixed-design ANOVA revealed a significant effect of phase (preconditioning vs test), such that rats froze more at test than at preconditioning, F1, 13 = 17.71, p = .001. The main effect of group was also significant due to animals tested 1 day after conditioning freezing more than those tested after 10 days, F1, 13 = 10.71, p = .006. Importantly, there was also a significant interaction F1,13 = 10.71, p = .006, with follow-up paired samples t-tests showing that the levels of freezing at preconditioning and test differed for the 1 day group, t7 = −4.13, p = .004, but not for the 10 day group, t6 = −1.87, p = 0.11. These results suggest that the rats formed a memory for the contextshock association that persisted for at least 24 h, and that this memory
2.3.1. Fear conditioning at P17 At P17, rats that were conditioned to fear the context received 6 foot shocks (0.6 mA, 1 s) after a 2 min adaptation period. The inter-shock interval ranged from 30 s to 60 s with an average of 45 s. Animals were returned to their home cage 10 s after the last shock. The delivery of the shock was controlled by computer software that was customdeveloped at The University of New South Wales. 2.3.2. Context exposure at P17 Animals that were merely exposed to the conditioning chamber (Experiments 2 and 3) were placed in the conditioning context for an equivalent period of time as those in the conditioned groups, but were not shocked. 2.3.3. Immediate shock at P17 Rats in the immediate shock group (Experiment 3) were placed into the conditioning chamber and a single foot shock was immediately administered; these animals were returned to their home cage immediately after the shock delivery. 2.3.4. Fear conditioning at P27 All animals in Experiments 2 and 3 were fear conditioned at P27. Similar to conditioning at P17, this consisted of an initial 2 min adaptation period prior to the onset of the first shock. However unlike at P17, animals were given 3 (0.6 mA, 1 s) rather than 6 shocks. A pilot study indicated that animals that were given 3 shocks or 6 shocks at P27 exhibited comparable levels of freezing. Therefore, the lower number was chosen to minimize the amount of discomfort that subjects experienced. The inter-shock interval at P27 ranged between 30 and 45 s.
3. Results 3.1. Experiment 1 — demonstration of Infantile Amnesia
Experiment 1 80
% Freezing
60 2.3.5. Test At test, animals were returned to the experimental chamber for 2 min but no shock was delivered. Freezing during this time was assessed as an index of the rats' fear, and thus their memory for the context-shock association. Freezing was considered as the absence of movement except that required for breathing [32], and was measured using a time sampling method where a judgment was made regarding whether the animal was freezing or not freezing every 3 s. The proportion of observations scored as freezing was converted to a percentage and used for all analyses. Animals were omitted from data analyses if their level of freezing at test was more than 3 standard deviations away from the group mean. This resulted in two exclusions in Experiment 1 (one from each group), one exclusion in Experiment 2 (from the Exposed-Saline group), and two exclusions in Experiment 3 (one each from the Conditioned-Mk801 and Exposed-Mk801 groups).
*
40
20
0 1-day test
10 day test
Group Fig. 1. Mean (±SEM) level of freezing at test for the groups tested at 1 day (n = 8) and 10 days (n = 7) after conditioning. * denotes a significant difference (p b .05).
Please cite this article as: Chan D, et al, Relearning a context-shock association after forgetting is an NMDAr-independent process, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.11.004
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was no longer behaviorally expressed after 10 days (i.e., forgetting had occurred). It should also be noted that the levels of freezing at the 1- and 10-day tests differed (independent samples t-test, t8.053 = 3.48, p = .0081). 3.2. Experiment 2 — does the transition to NMDAr-independent context learning occur even after the initial training experience has been forgotten? In adult rodents which retain context fear memories for extended periods, blockade of NMDA receptors impairs context conditioning to the initial context-shock association but not for subsequent conditioning to a second context [14], suggesting a transition to NMDAr-independent subsequent context learning. The purpose of Experiment 2 was to investigate whether subsequent learning is NMDAr-independent when the animal no longer behaviorally expresses a memory of the initial context-shock association. A summary of the procedure for Experiment 2 is shown in Table 1. Rats received conditioning or exposure to the context at P17, and then underwent context conditioning to the same environment at P27. Ten minutes prior to conditioning at P27, rats received injections of either MK801 or saline. Based on Li and Richardson's [10] results with discrete CSs, we expected that animals that were conditioned to the context at P17 would exhibit NMDAr-independent conditioning at P27 while animals that were merely exposed to the context would not show this transition. 3.2.1. Infantile amnesia of P17 context-shock association at P27 Freezing during the adaptation period of the P27 conditioning session was analyzed to examine whether animals that were conditioned at P17 had forgotten (i.e., no longer behaviorally expressed) the context-shock association. Animals that were injected with MK801 prior to conditioning at P27 were excluded from this analysis as some studies have reported that MK801 can affect motor behavior [33]. An independent samples t-test confirmed that there were no significant differences in levels of freezing for the conditioned-saline and exposed-saline groups during the 2 min adaptation period at P27, t15 = − 0.51, p = .61, (conditioned-saline: M = 0.28%, SEM = 0.28; exposed-saline; M = 0.63%, SEM = 0.55), demonstrating that rats that were previously conditioned to the context at P17 froze at comparable levels to the animals that were merely exposed to the context and not shocked. This indicates that animals that had been conditioned at P17 had forgotten the context-shock association by P27, replicating the findings of Experiment 1. 3.2.2. Test A 2 × 2 ANOVA with the factors of experience (at P17) and drug (at P27) revealed a near significant effect of drug, F1,30 = 3.89, p = .06, such that animals that were injected with saline prior to conditioning at P27 showed a trend towards more freezing at test than those that were given MK801. Groups that had been conditioned at P17 froze significantly more at the P28 test than those that were previously exposed to the context (main effect of experience at P17, F1,30 = 8.22, p = .01). Importantly, the interaction between experience (at P17) and drug (at P27) was also significant, F1,30 = 5.33, p = .03 (see Fig. 2). Subsequent post-hoc analyses were used to determine the source of this interaction. Independent samples t-tests revealed that in animals only exposed to the context at P17, MK801 given prior to conditioning at P27 significantly reduced the level of freezing exhibited the following day compared to saline-treated animals, t10.94 = −3.40, p = .01.2 This demonstrates that MK801 attenuated the acquisition of context
Table 1 Summary of the procedure for Experiment 2. Group
P17
P27
P28
Conditioned
6 shocks
Test
Exposed
Exposed to the context
Sal → 3 shocks MK801 → 3 shocks Sal → 3 shocks MK801 → 3 shocks
conditioning for the first time. However, there was no effect of MK801 on re-acquisition in animals previously conditioned to the context at P17, t16 = .22, p = .83. These results demonstrate that even though the context-shock association was completely forgotten through the process of infantile amnesia, as assessed by levels of freezing, the animals given this prior training transitioned to an NMDAr-independent form of context learning during re-acquisition. This transition was not observed in those rats merely exposed to the context at P17, as they exhibited impaired context learning when injected with MK801 prior to training on P27. 3.3. Experiment 3 — is associative conditioning at P17 required for the transition to NMDAr-independent context learning? The results of Experiment 2 show that prior context conditioning causes future context conditioning to be NMDAr-independent, even when the original context-shock association has been forgotten. In contrast, animals that were only exposed to part of the training procedure at P17 (i.e., context-exposure alone) did not make this transition. However, it is worth noting that the exposed-MK801 group in Experiment 2 did exhibit some learning about the context (i.e., they froze 28% at test) and this might be more than would have been observed in animals totally naive to the conditioning context given MK801 prior to conditioning at P27. That is, it may be the case that the context alone exposure led to a partial transition to NMDArindependent learning but this was not detected because there was no naive control group in Experiment 2. In addition, it should be noted that Experiment 2 did not test whether the stress and arousal associated with experience of the US foot shock alone are sufficient to cause a transition to NMDAr-independent context learning, although this was insufficient in our previous work with cued fear conditioning [10]. One way to administer a foot shock without inducing the learning of a context-shock association is to use the immediate shock protocol. In this procedure, animals are placed into the context and immediately given a foot shock. When subsequently tested, these animals exhibit very low levels of freezing in the context [34,35], which has been attributed to animals in this condition having insufficient time to form a context representation to associate with the shock US. To address these concerns, Experiment 3 investigated whether experiencing part of the conditioning procedure at P17 (i.e., exposure to the context or foot shock only) was sufficient to induce at least a partial switch from NMDAr-dependent to NMDAr-independent learning. The design involved 5 groups (see Table 2). Two groups were handled at P17 (i.e., these animals were naive), and another two groups received either conditioning or context exposure only (as in Experiment 2). The fifth group received only a foot shock immediately after placement in the context. At P27 animals received an injection of either saline or
Table 2 Summary of the procedure for Experiment 3. Group
P17
P27
P28
Naive-saline Naive-MK801 Conditioned-MK801 Exposed-MK801 ImmedShock-MK801
– – 6 shocks Exposure to the context 1 immediate shock
Sal → 3 shocks MK801 → 3 shocks MK801 → 3 shocks MK801 → 3 shocks MK801 → 3 shocks
Test Test Test Test Test
1
Levene's test indicated that the variances for the 1 day and 10 day groups were significantly different, F1,13 = 20.23, p = .001, and thus the adjusted degrees of freedom and p value have been reported. 2 Levene's test indicated that the variances of the exposed-MK801 and exposed-saline groups were significantly different (F1, 14 = 7.35, p = .02), and thus the adjusted degrees of freedom and p value have been reported for this comparison.
Test
Please cite this article as: Chan D, et al, Relearning a context-shock association after forgetting is an NMDAr-independent process, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.11.004
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Experiment 2 80
MK801
*
Saline
% Freezing
60
40
20
0 Exposed
Conditioned
Experience at P17 Fig. 2. Mean (±SEM) level of context-elicited freezing during test. Rats were either conditioned or merely exposed to the context at P17, and then received either injections of MK801 or saline prior to conditioning at P27 (n = 8–9 for each group). All animals were tested approximately 24 h later. * denotes a significant difference (p b .05).
MK801 10 min prior to conditioning to the context, and were tested for their levels of context-elicited freezing 24 h later (drug free). Animals in one of the naive groups were injected with MK801 prior to conditioning at P27 while those in the other naive group received saline (see Table 2). All three of the non-naive groups were injected with MK801 prior to training at P27. 3.3.1. Preconditioning freezing at P27 Freezing during the adaptation period at P27 was not scored because all of the non-naive animals in this experiment were administered MK801 prior to conditioning. As noted before, prior studies have shown that MK801 can affect motor behavior [33]. Therefore, scoring freezing behavior in these animals prior to conditioning would not have been a true reflection of context-elicited fear. However, the results from Experiment 1 and from the preconditioning period in Experiment 2 suggest that even when animals are conditioned to the context at P17 using our parameters, they forget this context-shock association by P27. 3.3.2. Test Fig. 3 depicts the mean level of context-elicited freezing exhibited at test by each of the five groups. Most groups showed a high level of freezing to the context after conditioning at P27, however, the naive-MK801 Experiment 3
% Freezing
80 60
*
40 20 0 Naive Saline
Naive MK801
Conditioned MK801
Exposed ImmedShock MK801 MK801
Group Fig. 3. Mean (±SEM) level of context-elicited freezing at test. At P17, animals were handled (naive), conditioned or exposed to the context, or immediately shocked after being placed into the experimental chamber. At P27, rats that were conditioned (n = 8), exposed (n = 7) or given immediate shock (n = 9) at P17, and one naive group (n = 9) were given injections of MK801 prior to conditioning. The other naive group (n = 7) was given saline. All animals were tested approximately 24 h later. * denotes a significant difference (p b .05).
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group showed a low level of freezing to the context at test. A one-way ANOVA confirmed that there was a significant difference in levels of contextual freezing between the groups, F4,39 = 4.97, p = .003. Post-hoc analyses, using Tukey's HSD test, demonstrated that the naive-MK801 group froze significantly less at test than the other groups (largest p = .04), which did not differ from each other (smallest p = .98). These findings demonstrate that as expected, naive animals showed evidence of successful context conditioning at P27 when tested the following day and that MK801 impaired the acquisition of this context conditioning. We also replicated our finding in Experiment 2 that MK801 did not affect re-acquisition at P27 in animals which were previously conditioned to the same context in infancy but had forgotten the original context-shock association. These findings again demonstrate that animals that were previously conditioned to fear a context make a transition to NMDAr-independent learning when later re-trained to fear that context, even though they would have forgotten the original context-shock association. Perhaps somewhat surprisingly, NMDAr-independent learning was also observed in rats that were administered the foot shock only at P17 (ImmedShock-MK801). This finding suggests that the transition to NMDAr-independent contextual fear learning does not require the previous experience to involve associative learning. This is in contrast to learning about discrete CSs, where exposure to the CS and US in an unpaired manner at P17 did not cause a transition to NMDAr-independent learning at P31 [10]. Finally, in contrast to the results reported in Experiment 2, in this experiment mere exposure to the context at P17 was sufficient to cause the transition to NMDAr-independent learning at P27. 4. Discussion In this study we examined the transition from NMDAr-dependent to NMDAr-independent learning. Studies using adult rodents have shown that while NMDA receptors are involved in learning an initial contextshock association, they are not required for subsequent conditioning to a second context [14,15]. As adult animals have an intact longlasting memory for the initial learning experience we investigated whether the same transition to NMDAr-independent learning for a second context-shock association occurred after infantile amnesia, when the initial has been forgotten. After demonstrating in Experiment 1 that contextual conditioning in infant (P17) animals led to learned fear at a 1-day test and that this memory was forgotten after a 10-day interval (infantile amnesia), we conducted two experiments testing whether prior contextual fear conditioning induces the transition to NMDAr-independent relearning when the original memory is no longer behaviorally expressed. In both of these experiments there was a transition to NMDAr-independent relearning in animals previously trained in infancy. In one of these two experiments there was a transition to NMDAr-independent contextual fear learning in animals exposed only to the context in infancy. The final experiment also demonstrated that a transition to NMDAr-independent context learning occurred in animals exposed to only the foot shock (i.e., the US only) in infancy. Importantly, the data suggests that our results are not due to state dependent learning. For example, in Experiment 2 the conditionedMK801 and exposed-MK801 groups did not differ in their experience of the drug as both groups were systemically injected with the same dose and volume of MK801 before training at P27. No injections were administered prior to test so therefore if MK-801 was producing statedependent learning then both groups would show low levels of freezing at test. The data clearly shows that this was not the case. The same argument can be made for the results reported in Experiment 3, where only one group injected with MK-801 prior to fear conditioning on P27 (i.e., the naïve-MK801 group) exhibited low levels of freezing at test while the other three groups given MK801 in exactly the same way had high levels of freezing at test. Our findings are consistent with reports using adult animals showing that there is a transition from NMDAr-dependent to NMDAr-
Please cite this article as: Chan D, et al, Relearning a context-shock association after forgetting is an NMDAr-independent process, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.11.004
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independent learning when similar contexts are used [14,15]. We extended these findings by demonstrating that the transition occurs even when the animals no longer behaviorally express a memory of that prior experience. This finding of a transition to NMDArindependent learning after infantile amnesia with contextual fear is similar to results reported by Li and Richardson [10] who examined fear learning for discrete CSs. In that study, re-acquisition of a noise CS-shock US association after infantile amnesia was unaffected by the NMDAr-antagonist MK801, and in the current study the same effect occurred for re-acquisition of a context-shock association. Taken together, these results provide strong evidence that relearning after forgetting is an NMDAr-independent process. In Experiment 2, animals only exposed to the context, but not the US, at P17 did not make a transition to NMDAr-independent learning as learning at P27 was impaired by injections of the NMDAr antagonist MK801. However, these animals did exhibit some learning about the context (i.e., mean freezing of 28%), indicating that the context alone exposure may have led to a partial transition to NMDAr-independent learning that was not detected because the experimental design did not include a naive control group. To address this issue we included a naive control condition in Experiment 3. Although the naive control group given MK801 in the latter experiment exhibited levels of contextual freezing very similar to that observed in the exposed-MK801 rats in Experiment 2 (indicating that there was not any transition), the performance of the exposed-MK801 rats in Experiment 3 was identical to that of the rats which were previously trained (indicating a complete transition). From this it can be concluded that while previous context-shock training reliably causes a transition to NMDAr-independent learning after forgetting, only exposing the animal to the context is not as reliable or robust of an experience for causing a transition to NMDArindependent learning. We also observed that animals exposed to an immediate foot shock at P17 showed NMDAr-independent learning at P27 (Experiment 3), suggesting that exposure to the US alone caused a transition to NMDAr-independent learning. In contrast, Li and Richardson [10] found that associative learning was required for animals to transition to NMDAr-independent cue conditioning because exposing the infant animals to unpaired presentations of the noise CS and shock US did not lead to NMDAr-independent learning later in life. The current results suggest that animals do not need to be exposed to the entire conditioning procedure to exhibit the transition to NMDArindependent learning for context conditioning. It is worth noting that a similar inconsistency has been found in the adult literature. Specifically Tayler et al. [15] and Wiltgen et al. [14] found that exposing a rat to Context A leads to NMDAr-independent learning in a physically similar context. However, Wang et al. [36] reported that they have unpublished data that indicates that context exposure alone in adult animals is not sufficient to cause this transition. It is clear across several studies now that prior training causes a transition to subsequent learning not requiring NMDA receptors and this can occur in adulthood or even after infantile amnesia, for both discrete CS and contextual fear conditioning. Some idea of potential molecular mechanisms involved in the transition from NMDAr-dependent to NMDAr-independent learning after forgetting can be gained from findings in the adult literature. Tayler et al. [15] suggested that in adult animals an initial context conditioning experience activates and induces plasticity in a specific subset of neurons. This leads to an enhancement in synaptic strength and the expression of novel receptor proteins (e.g., specific AMPA receptors), which can support subsequent learning without NMDA receptors. Thus, if these cells are involved in learning to fear a second context, they are able to support conditioning in an NMDAr-independent manner. From this perspective, if reacquisition after infantile amnesia activates neurons which were involved in encoding the initial learning experience, then these neurons may support the subsequent learning in an NMDAr-independent manner. However, if the initial experience and subsequent conditioning session activate different subsets of neurons then this transition would
not be observed. Another relevant finding which Tayler et al. [15] reported was that the transition from NMDAr-dependent to NMDArindependent learning was stronger if the two experiences were very similar (e.g., if subsequent conditioning occurred in a highly similar context) and the transition became weaker the more the experiences differed (e.g., moderately similar contexts). Therefore, the weaker transition to NMDAr-independent learning observed in animals only exposed to the context in infancy is likely to arise from activation of a partially overlapping population of cells supporting the two experiences, whereas prior conditioning would activate more overlapping cells to induce a full transition. In the present study, the two learning experiences may activate populations of neurons in the hippocampus and basolateral amygdala (BLA), as NMDA receptors in these regions are critical for context fear conditioning in naive animals (i.e., those that are learning for the first time). In particular, these receptors in the hippocampus are implicated in supporting the animal's formation of a configural representation of the context [37], while those in the BLA are required for associating this representation with the shock [13,38]. Therefore, if an overlapping population of cells is activated by the two learning experiences, the treatments administered at P17 must have modified, and thus activated, neurons in both the hippocampus and BLA. In adult animals, context exposure and context conditioning increase markers of neuronal activity (e.g., c-Fos mRNA) in the hippocampus and BLA [39–43] but the experience of foot shock only does not [42,44,45]. Taken together, it seems that the transition to NMDAr-independent learning observed in animals that were either conditioned or exposed to the context at P17 could be due to activation of specific sub-populations of neurons in the hippocampus and BLA during both conditioning or context exposure experiences, but it is not clear how the transition observed in those exposed to the US only is mediated. Future studies could address the cellular mechanisms that mediate learning in the absence of NMDAr activation. One likely candidate is calcium (Ca2 +)-permeable AMPA receptors. The insertion of Ca2 +permeable AMPA receptors into the postsynaptic membrane is experience-dependent and this process is upregulated by fear conditioning in regions such as the lateral amygdala [46]. Findings from Wiltgen et al. [14] provide evidence that these receptors are involved in NMDAr-independent learning. They demonstrated that blocking Ca2+-permeable AMPA receptors with systemic injections of the antagonist IEM-1460 impaired learning to a second context in adult mice (when an NMDAr antagonist did not impair acquisition), suggesting that Ca2 +-permeable AMPA receptors can support learning when plasticity is NMDAr-independent. Further studies by this group found that this form of NMDAr-independent learning involves the hippocampus [15]. It would be interesting for further work to investigate whether Ca2 +-permeable AMPA receptors are recruited during relearning a context-shock association after it has been forgotten and whether the hippocampus and/or amygdala are required for this learning. An important caveat that should be noted here is that the sensitivity of synaptic transmission to Ca2 +-permeable AMPA receptor antagonists in the amygdala of adult mice appears within hours after conditioning, peaks at 24 h, and disappears by 7 days after fear conditioning [46], suggesting that the involvement of these receptors in NMDAr-independent learning could be time-limited and may not last when infantile amnesia is observed (e.g., after 10 days in the current study). Therefore, it is possible that the finding of Wiltgen et al. [14] that Ca2 +-permeable AMPA receptors support NMDAr-independent learning is only applicable to learning in adult animals soon after the initial experience, where the second conditioning occurs when the animal has an intact and recent memory of the original training. The results of comparing the role of these AMPA receptors in infant and adult animals should reveal whether there are qualitative or quantitative differences in the mechanism(s) mediating the transition from NMDAr-dependent learning to NMDAr-independent relearning across development. In much of his work on memory development, Spear championed the
Please cite this article as: Chan D, et al, Relearning a context-shock association after forgetting is an NMDAr-independent process, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.11.004
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idea that the processes/mechanisms mediating memory and forgetting were qualitatively similar across development. The present findings, when taken in conjunction with the findings of Wiltgen and others with adult rodents, support such a view. However, novel insight into this issue will be gained by comparing the mechanisms that mediate NMDAr-independent learning in adults (e.g., Ca2+-permeable AMPA receptors) to the mechanisms that mediate this type of learning following infantile amnesia. In conclusion, we have demonstrated that similar to relearning about a discrete CS [10], relearning a context-shock association after forgetting is an NMDAr-independent process. However, unlike discrete CS conditioning, which required associative learning in order for the transition to occur, we found that exposure to the context only may induce a weaker transition to NMDAr-independent learning. These findings lead to other important directions for future research. Particularly, all of our experiments examined the effects of infantile amnesia on the transition to NMDAr-independent learning. However, forgetting is a phenomenon that occurs across the lifespan and therefore, it will also be important to examine whether similar results are obtained using adult (or aging) animals for types of learning susceptible to forgetting. This would allow us to examine whether studying infantile amnesia can inform our understanding of forgetting in adulthood or old age. Is amnesia in infancy an exaggerated form of the memory loss that occurs in mature animals, or is forgetting across the life span mediated by a different mechanism? Spear would, we believe, clearly favor the former possibility as illustrated by his statement “In my opinion there is still no substantial evidence that the causes of our ‘amnesia’ for the events of infancy are any different from what causes forgetting of the events of our adulthood” [47; p. 328]. In any case, the present findings build upon prior research suggesting that early experiences, even if forgotten, can impact on learning in late life and may provide insight into understanding how early experiences contribute to adult pathologies. Acknowledgments This research was reported as part of an Honours thesis by DC, who was supported by an Australian Postgraduate Award during preparation of the manuscript. The research was supported by grants from the Australian Research Council (DP120104925) and the National Health and Medical Research Council (APP1031688) to RR. KB is a National Health and Medical Research Council Peter Doherty Early Career Fellow (APP1054642). References [1] Howe ML. The nature of infantile amnesia. In: John HB, editor. Learning and memory: a comprehensive reference. Oxford: Academic Press; 1998. p. 287–97. [2] Jack F, Hayne H. Childhood amnesia: empirical evidence for a two-stage phenomenon. Memory 2010;18:831–44. [3] Campbell BA, Spear NE. Ontogeny of memory. Psychol Rev 1972;79:215–36. [4] Taylor SE, Way BM, Seeman TE. Early adversity and adult health outcomes. Dev Psychopathol 2011;23:939–54. [5] Campbell BA, Jaynes J. Reinstatement. Psychol Rev 1966;73:478–80. [6] Spear NE, Parsons PJ. Analysis of a reactivation treatment: ontogenetic determinants of alleviated forgetting. In: Medin DL, Roberts WA, Davis RT, editors. Processes of animal memory. Hillsdale, NJ: Erlbaum; 1976. p. 135–65. [7] Rovee-Collier C, Haye H. Reactivation of infant memory: implications for cognitive development. In: Hayne WR, editor. Advances in child development and behavior, vol. 20. JAI; 1987. p. 185–238. [8] Richardson R, Riccio DC, McKenney M. Stimulus attributes of reactivated memory: alleviation of ontogenetic forgetting in rats is context specific. Dev Psychobiol 1988;21:135–43. [9] Newcombe N, Fox NA. Infantile amnesia: through a glass darkly. Child Dev 1994;65: 31–40. [10] Li S, Richardson R. Traces of memory: reacquisition of fear following forgetting is NMDAr-independent. Learn Mem 2013;20:174–82. [11] Collingridge GL, Kehl SJ, McLennan H. Excitatory amino acids in synaptic transmission in the Schaffer collateral-commissural pathway of the rat hippocampus. J Physiol 1983;334:33–46. [12] Morris RGM, Anderson E, Lynch GS, Baudry M. Selective impairment of learning and blockade of long-term potentiation by an N-methyl-D-aspartate receptor antagonist, AP5. Nature 1986;319:774–6. http://dx.doi.org/10.1038/319774a0.
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Please cite this article as: Chan D, et al, Relearning a context-shock association after forgetting is an NMDAr-independent process, Physiol Behav (2014), http://dx.doi.org/10.1016/j.physbeh.2014.11.004