Inhibition of prefrontal protein synthesis following recall does not disrupt memory for trace fear conditioning
© Blum et al; licensee BioMed Central Ltd. 2006
Received: 30 August 2006
Accepted: 06 October 2006
Published: 06 October 2006
The extent of similarity between consolidation and reconsolidation is not yet fully understood. One of the differences noted is that not every brain region involved in consolidation exhibits reconsolidation. In trace fear conditioning, the hippocampus and the medial prefrontal cortex (mPFC) are required for consolidation of long-term memory. We have previously demonstrated that trace fear memory is susceptible to infusion of the protein synthesis inhibitor anisomycin into the hippocampus following recall. In the present study, we examine whether protein synthesis inhibition in the mPFC following recall similarly results in the observation of reconsolidation of trace fear memory.
Targeted intra-mPFC infusions of anisomycin or vehicle were performed immediately following recall of trace fear memory at 24 hours, or at 30 days, following training in a one-day or a two-day protocol. The present study demonstrates three key findings: 1) trace fear memory does not undergo protein synthesis dependent reconsolidation in the PFC, regardless of the intensity of the training, and 2) regardless of whether the memory is recent or remote, and 3) intra-mPFC inhibition of protein synthesis immediately following training impaired remote (30 days) memory.
These results suggest that not all structures that participate in memory storage are involved in reconsolidation. Alternatively, certain types of memory-related information may reconsolidate, while other components of memory may not.
Protein synthesis dependency around the time of training is a hallmark of long-term memory consolidation . This has been repeatedly demonstrated across species and types of new information learned . Reactivation of information already committed to long-term memory may induce an additional protein synthesis dependent period, during which the original memory can be disturbed by administration of protein synthesis inhibitors, suggestive of a reconsolidation process , although alternative interpretations have been suggested . While protein synthesis dependencies have been observed for both consolidation and reconsolidation, evidence suggests that consolidation and reconsolidation differ in several key aspects (for review see ). For example, a protein synthesis-dependent period follows every training paradigm (consolidation), whereas not every reactivation of memory results in a re-dependency on protein synthesis (reconsolidation). Whether a reactivated memory undergoes reconsolidation appears to depend on several factors, including the age of the memory and training intensity [7, 8] For example, a conditioned taste aversion (CTA) memory resulting from a more intense, two day, training protocol is susceptible to reconsolidation, whereas a one day training paradigm results in a memory susceptible to extinction . An additional key difference is that not all brain regions involved in consolidation are involved in reconsolidation. For example, in inhibitory avoidance, protein synthesis in the dorsal hippocampus is required for consolidation but not reconsolidation of memory . Similarly, in CTA, consolidation depends on protein synthesis in the central nucleus of the amygdala, whereas there is no renewed protein synthesis dependency in this region following memory reactivation . In young chicks, following a reminder, expression of the transcription factor c-Fos is seen only in one of the two regions involved in consolidation , further suggesting that not all structures involved in consolidation participate in reconsolidation.
Trace fear conditioning is a form of learning that involves the association between a tone (conditioned stimulus, CS) and a footshock (unconditioned stimulus, US) that are separated by a time gap (trace period). The hippocampus and the medial prefrontal cortex (mPFC) are required for making the CS-US association, and both are required for consolidation of memory for trace fear conditioning [12–17] We have recently reported that, in addition to its role in consolidation of trace fear memory, the hippocampus is a site of renewed protein synthesis dependency following trace fear memory reactivation . Consistent with previous reports, only a more intense (two day) training protocol resulted in a memory susceptible to reconsolidation, whereas a one day training resulted in a memory susceptible to extinction. In the present study, we examine whether the mPFC is also a site of protein synthesis dependent reconsolidation following reactivation of trace fear memory for one- or two-day training paradigms.
Inhibition of prefrontal protein synthesis following recall of a one-day trace fear conditioning paradigm does not disrupt memory
During the reactivation trial, the animals demonstrated freezing behavior at a level comparable to the level acquired during training. Minimal freezing was observed in the novel context prior to CS presentation (pre-CS) and during CS presentation. A significant increase in freezing behavior during the trace period (Fig. 1B). Immediately following the reactivation trial, animals were divided into two comparable groups based on freezing behavior during the trace period of the reactivation trial, and one group was bilaterally infused with anisomycin (250 ug/2 ul per mPFC), while the other received vehicle (2 ul per mPFC) (trace period freezing: anisomycin n = 10: react = 82.5 ± 8.0 %; vehicle n = 9: react = 90.0 ± 6.0 %; n.s.). Retention was tested two days following reactivation by exposing the animals to four CS-alone trials and measuring trace period freezing. The two groups both remembered that the termination of the CS predicts the subsequent footshock as demonstrated by an increase in fear during the trace period as compared to that seen in either the pre-CS or the CS periods. However, the freezing percentage recorded for the anisomycin-infused animals during these time periods did not differ from that observed in the saline-treated controls (trace period freezing: anisomycin n = 10: retention = 65.8 ± 13.0 %; vehicle n = 9: retention = 77.8 ± 11.3 %; n.s.) (Fig. 1B–C), indicating no disruption in memory due to the protein synthesis inhibition. Contextual fear, assessed by placing the animal back into the original training chamber, also did not differ between the two groups (data not shown).
Inhibition of prefrontal protein synthesis following recall of a two-day trace conditioning paradigm does not disrupt memory
Inhibition of prefrontal protein synthesis following remote recall of a two-day trace fear conditioning paradigm does not disrupt memory
Reconsolidation refers to the experimental observations that following reactivation, a memory trace may once again become susceptible to protein synthesis inhibition [3, 9, 19] Although this is similar to the protein synthesis dependency of initial stabilization or consolidation of memory, one important distinction is that not every brain region involved in consolidation is involved in reconsolidation [9–11] In trace fear conditioning, memory for the trace relationship is stored in both the hippocampus and mPFC [16, 28] We have recently demonstrated that inhibition of hippocampal protein synthesis following reactivation of trace fear memory results in reconsolidation . In contrast, the present study demonstrates that memory for trace fear conditioning does not undergo protein synthesis-dependent reconsolidation in the mPFC, regardless of the intensity of the training, and regardless of whether the memory is recent or remote.
As we are reporting that we find no evidence of reconsolidation in the mPFC, we have to consider the possibility that this negative finding is due to an insufficient dose of the protein synthesis inhibitor. Effects in reconsolidation studies have previously been observed in structure-targeted infusions of as little as 62.5 μg of anisomycin . In the present study, we infused four times this amount (250 μg), a dose that has previously been shown to result in reconsolidation when infused into the hippocampus immediately following reactivation [18, 19]. Furthermore, a dose of 160 μg anisomycin, two-thirds that used in the reconsolidation experiments, was sufficient to impair long-term consolidation within the mPFC. It is therefore unlikely that insufficient protein synthesis inhibition underlies the absence of a reconsolidation effect. Several studies have suggested that specific reactivation conditions may be required to induce reconsolidation in some situations [29, 30] For example, in an object recognition task, reactivation of memory in the original context is required in order to observe zif268 dependent reconsolidation . Although the present study did not identify the specific conditions, if any, required for reconsolidation in the mPFC, it demonstrated that the paradigm that induces this phenomenon in the hippocampus is not capable of causing reconsolidation in the mPFC.
At present, it is still debated whether reconsolidation is analogous to a recapitulation of initial consolidation, if it reveals a late component of the initial consolidation process, or if it is related to memory retrieval (for review see )[5, 6]. Recently, it has been shown that impaired memory as a result of inhibition of hippocampal protein synthesis following memory recall can be rescued by a reminder).). This, along with studies demonstrating that memory impairments as a result of hippocampal reconsolidation reverse over time [4, 33]., suggests that the memory deficits may result from a dysfunction in memory retrieval. The mPFC has several characteristics which may explain the lack of reconsolidation in this structure, regardless of whether reconsolidation reflects memory storage or retrieval deficits. First, if we assume that reconsolidation is reflective of a memory storage deficit, the mPFC may serve a different function in memory storage than the hippocampus. Most models of long-term memory consolidation for hippocampus-dependent memories, such as trace fear conditioning, posit that the hippocampus plays a temporary role, and that remote, long-lasting memories are dependent on neocortical areas such as the mPFC. Although long-term plasticity is observed in both the mPFC and the hippocampus as a direct result of training, this does not imply that the memories stored in these structures are of equivalent strength. It is possible that plasticity within the mPFC is more stable, and not susceptible to reconsolidation, allowing for persistence of memory over months to years. Second, if reconsolidation reflects a retrieval deficit, the absence of reconsolidation in the mPFC may reflect the difference between the roles played by the hippocampus and the mPFC in this process. One possibility is that information storage within the hippocampus may be involved in the reactivation of various components of information stored in other structures and memory reactivation results in protein synthesis-dependent modifications within the hippocampus that are necessary for subsequent memory retrieval of updated memories . In contrast, in the trace fear conditioning paradigm, the mPFC may be involved in storing some aspect of the information that is retrieved and, therefore, may not undergo reconsolidation. Finally, we would like to point out that despite the fact that anisomycin has been the most widely used protein synthesis inhibitor, both in consolidation and reconsolidation experiments (for review see ), this inhibitor has a number of non-specific effects, including apoptosis [35, 36]. At present, it is not known if these non-specific influences may have contributed to the observation of reconsolidation in the hippocampus, and its lack in the mPFC. As more insight is gained into the processes underlying the observations of reconsolidation, it should become possible to determine the differences between the hippocampus and the mPFC in respect to their role in reconsolidation.
The present study demonstrates three key findings: 1) trace fear memory does not undergo protein synthesis dependent reconsolidation in the PFC, regardless of the intensity of the training, and 2) regardless of whether the memory is recent or remote, and 3) intra-mPFC inhibition of protein synthesis immediately following training impaired remote (30 days) memory.
Male Sprague Dawley rats (Charles River Laboratory, Wilmington, MA) weighing 250 to 300 gm were pair-housed under temperature-controlled conditions with a 12 hr light/dark cycle. All rats were given ad libitum access to water and food. Protocols regarding the training and surgery of the animal subjects were approved by the Institutional Animal Welfare Committee and were in compliance with NIH's Guide for Care and Use of Laboratory Animals.
Animals were anesthetized under 5% isoflurane with a 2:1 N2O/O2 mixture and then maintained under 2.5% isoflurane with a 2:1 N2O/O2 mixture via a face mask. Twenty-two gauge stainless-steel guide cannulae were implanted into the mPFC (bregma + 3.2 mm, lateral ± 0.75 mm, and depth -2.5 mm) using a stereotaxic device . Animals were given a ten-day rest period following surgery before behavioral testing. For the 30 day reconsolidation study, animals were trained in the trace fear conditioning task first, and the guide cannulae were implanted ten days prior to the reactivation/reconsolidation testing. This was done to prevent excessive scar tissue formation, and drift of the guide cannulae, which may occur over a one month period. Infusion cannulae that extended 1.5 mm past the end of the guide cannulae were used for drug infusions. After the completion of all behavioral experiments, representative animals were killed and the brains post-fixed in a 4% paraformaldehyde solution for histological analysis of cannulae location.
Anisomycin (Sigma, St. Louis, MO) was dissolved in 1 N hydrochloric acid, neutralized with NaOH, and then diluted in artificial cerebrospinal fluid (ACSF), pH 7.4. Anisomycin was bilaterally infused into the mPFC (250 μg/2 μl per side for reconsolidation; 160 μg/side for consolidation), at a rate of 0.25 μl per minute. The dose of 250 μg/2 μl has previously been shown to be effective in blocking reconsolidation [18, 19]. Anisomycin was infused immediately following a single CS-alone exposure in a novel context (reactivation trial). Vehicle control infusions consisted of the same volume ACSF with the same pH as the anisomycin solution. Following the infusion, the needle was left in place for two minutes to allow for diffusion of the drug. All infusions were performed using a motorized infusion pump (Stoelting, Wood Dale, IL).
Trace fear conditioning/recall
All behavioral tests were performed by an investigator who was blind to the treatment groups. Animals were placed in the training context (Habitest Unit, Coulbourn Instruments, Allentown, PA) and given a 120 second habituation period. Conditioning trials began with a 10 second tone (CS) followed by a 20 second trace period, after which the animal received a 0.8 mA foot-shock (US) for 0.7 seconds. Each CS-US paired training trial was separated by a pseudorandom inter-trial interval (ITI) that varied between 1–4 minutes. A pseudorandom ITI was used so that amount of time between foot-shocks could not be used as a cue for the US. For reactivation of memory, animals were placed in a novel context, given a 120 second habituation period followed by one presentation of the CS without the presentation of the US (CS-alone reactivation trial). Immediately following the reactivation trial, animals were divided into two comparable groups based on freezing behavior during the trace period of the reactivation trial. One group was bilaterally infused with anisomycin and the other with vehicle. For retention testing, each animal was placed in a novel context and given a 120 second habituation period. In the absence of foot-shock, four presentations of the CS were given separated by a varied ITI period. During the retention and reactivation trials, freezing behavior (defined as the absence of all movement excluding movement caused by respiration) was measured during the CS, trace, and ITI periods. Freezing behavior was recorded every two seconds during scoring periods. Freezing behavior during the four CS presentations was averaged for each animal. Following trace CS-US retention testing; contextual retention was measured by placing the animals back into the original training context for a 90 sec period during which freezing was scored, without exposure to the CS or US.
A two-way repeated measures ANOVA was used to compare the percentage of trace period freezing during retention testing between the anisomycin- and vehicle-infused groups as well as to compare trace period freezing between reactivation and retention trials, within and between the groups. An unpaired t-test was used to compare CS as well as contextual freezing between the anisomycin- and vehicle-infused groups. A p value of ≤ 0.05 was used as the criterion for statistical significance. Raw data were used for all statistical analyses.
conditioned taste aversion
medial prefrontal cortex
The authors would like to thank Anthony Moore for his insightful suggestions and comments. The authors are appreciative of the technical assistance offered by Melanie Moody. Grant sponsors: PK Dash NIH NS035457, MH072933, NS049160.
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