Retrieving a recently formed context fear memory reactivates neurons in the hippocampus, amygdala, and cortex. Several weeks after learning, the degree of reactivation is altered in hippocampal and amygdala networks but remains stable in the cortex.

H2B-GFP expression in transgenic mice was increased by learning and could be regulated by doxycycline (DOX). Using this system, we found a large network of neurons in the hippocampus, amygdala, and neocortex that were active during context fear conditioning and subsequent memory retrieval 2 days later. Reactivation was contingent on memory retrieval and was not observed when animals were trained and tested in different environments. When memory was retrieved several weeks after learning, reactivation was altered in the hippocampus and amygdala but remained unchanged in the cortex.

Episodic memories are encoded within hippocampal and neocortical circuits. Retrieving these memories is assumed to involve reactivation of neural ensembles that were established during learning. Although it has been possible to follow the activity of individual neurons shortly after learning, it has not been possible to examine their activity weeks later during retrieval. We addressed this issue by using a stable form of GFP (H2B-GFP) to permanently tag neurons that are active during contextual fear conditioning.

In the current experiments, we examined memory retrieval using a context fear conditioning task that is dependent on the hippocampus, amygdala, and neocortex []. Neurons were labeled with H2B-GFP during learning and then re-examined 2 days or 2 weeks later during memory retrieval. We found a large network of tagged neurons in the hippocampus, amygdala, and neocortex that were reactivated 2 days after learning. This result suggests that memory retrieval involves reactivation of individual neurons that were engaged during learning. These same networks were not reactivated when animals were trained and tested in different environments. Two weeks after learning, the pattern of reactivation was altered in the hippocampus and amygdala but remained largely unchanged in the cortex. This finding suggests that hippocampal and amygdala circuits are modified after learning, whereas cortical networks remain stable over time.

Fluorescence in situ hybridization (FISH) studies using the immediate early genes Arc and Homer indicate that hippocampal and neocortical neurons are reactivated when animals explore the same spatial environment twice within a 30 min period []. Longer intervals cannot be examined with this technique because mRNA for these genes rapidly decays. To overcome this issue, we used newly engineered transgenic mice that express a long-lasting, activity-dependent form of green fluorescent protein (GFP). In these animals, activation of the c-fos promoter during learning leads to the expression of human histone H2B-GFP, a fusion protein that takes several weeks to degrade []. As a result, the activity of neurons labeled with H2B-GFP can be examined days and weeks after learning. A similar strategy was used to demonstrate reactivation of amygdala neurons 3 days after learning in tau-LacZ reporter mice [].

Episodic and contextual memories can be retrieved months and years after they are formed. Retrieving these memories is assumed to involve reactivation of hippocampal and neocortical networks that were established during learning []. Consistent with this idea, many brain regions that are active during learning are also engaged during testing []. However, it has not been possible to determine whether the same neurons that encode memory in these regions are later reactivated during retrieval.

Cellular imaging of zif268 expression in the hippocampus and amygdala during contextual and cued fear memory retrieval: selective activation of hippocampal CA1 neurons during the recall of contextual memories.

The current data suggest that H2B-GFP-positive neurons are reactivated during testing when animals retrieve a memory for the training context. However, it is possible that reactivation is driven by other stimuli that are present during training and testing (e.g., transport cues, experimenter, and removal from the homecage). To examine this issue, we quantified double labeling in mice that were trained in context A and tested in a different environment (context B) 2 days later. These animals were exposed to the same background cues during training and testing but should not retrieve a memory for context A. Consistent with this idea, freezing levels in context Bwere significantly lower than those observed in our previous experiment, when mice were trained and tested in context A(main effect of group F (1, 8) = 5.85, p < 0.05). Figures 5 A and 5B show the percentage of neurons expressing H2B-GFP and c-fos in the hippocampus, amygdala, and cortex. Figures 5 C and 5D illustrate the percentage of reactivated neurons relative to chance (chance = percent GFP × percent c-fos). Analysis of double labeling for H2B-GFP and c-fos revealed that reactivation did not exceed chance in any of the brain regions examined (Fisher’s PLSD, pairwise comparisons, all p values > 0.05). These data suggest that reactivation of neural networks is only observed when memory is retrieved. However, there was a trend for reactivation in the BLA and CA1 suggesting that neurons in these regions may be sensitive to other stimuli that are present during training and testing. Nonetheless, our experiments demonstrate that the main determinant of reactivation is exposure to the context in which footshock was previously administered.

(D) Mice trained and tested in context B did not show greater than chance reactivation in any of the cortical regions examined. Error bars represent ± SEM. ∗ p < 0.05.

(C) The percentage of double-labeled neurons (H2B-GFP + c-fos) is shown relative to chance (percent H2B-GFP × percent c-fos). Mice trained in context A and tested in context B did not show greater than chance reactivation in the hippocampus or BLA.

Reactivation in RSPv did not exceed chance 14 days after learning because of an increase in c-fos expression (illustrated in Figure 2 D), not because of a decline in the percentage of reactivated neurons. This result is consistent with a previous study that found increased expression of c-fos in the retrosplenial cortex during the retrieval of remote spatial memories []. As shown in Figure 2 D, we did not observe a change in c-fos expression over time in any of the other cortical regions examined. Therefore, our data suggest that reactivation of cortical networks remains relatively stable after learning.

Finally, we examined reactivation of cortical regions when memory was retrieved 2 weeks after learning. Figure 4 B shows the percentage of reactivated neurons in each region relative to chance. We found that reactivation was significantly greater than chance in the ENTl and PTLp (Fisher’s PLSD, pairwise comparisons, p values < 0.05) but not in the RSPv or MOs (p values > 0.05). To determine whether there were changes in the number of reactivated neurons over time, we compared the percentage of H2B-GFP-positive cells colabeled with c-fosduring the recent and remote memory tests ( Figure 4 C). This analysis revealed that the percentage of reactivated neurons did not change over time in any of the cortical regions examined (all p values > 0.05).

We next examined reactivation of cortical areas that are involved in spatial and contextual learning. Figure 4 A shows the percentage of reactivated neurons in each region relative to chance (chance = percent GFP × percent c-fos). Reactivation was significantly greater than chance in the ENTl, RSPv, and PTLp (Fisher’s PLSD, pairwise comparisons, all p values < 0.05). As expected, reactivation did not exceed chance in MOs (p > 0.05). These results indicate that retrieval of a recently formed context fear memory involves widespread reactivation of cortical neurons that were engaged during learning.

(C) The percentage of H2B-GFP-positive neurons that were reactivated (i.e., colabeled with c-fos) is shown for mice tested 2 days (recent, n = 5) or 14 days (remote, n = 4) after training. The percentage of reactivated neurons did not change over time in any of the cortical regions examined. Error bars represent ± SEM. ∗ p < 0.05.

(B) Two weeks after learning, reactivation exceeded chance in ENTl and PTLp but not RSPv or MOs.

(A) TetTag mice were trained off DOX in context A and tested in the same environment 2 days later (n = 5). The percentage of double-labeled neurons is shown relative to chance (percent H2B-GFP × percent c-fos). Significant reactivation was observed in ENTl, RSPv, and PTLp. Reactivation was not observed in MOs.

These results deviated from our predictions about remote memory reactivation in two ways. First, we did not expect to find reactivation of hippocampal neurons given that this region was not required for memory retrieval 14 days after learning. The fact that CA3 and CA1 were reactivated suggests that the hippocampus normally contributes to memory retrieval but that other structures can compensate when this region is inactivated. Recent studies are consistent with this idea []. Second, we did not observe reactivation of the BLA even though the amygdala is required for the retrieval of fear memories months and years after learning []. Examination of our c-fos expression data ( Figure 2 D) indicates that activity in the amygdala at 2 weeks (9.9%) did not differ from that observed during memory retrieval at 2 days (7.6%) (no effect of time F (1, 7) = 1.31, p > 0.05). Therefore, it is possible that amygdala activity is required for remote memory retrieval even though reactivation of the same neurons that were engaged during learning is not. Alternatively, a smaller population of reactivated neurons (which did not exceed chance in the current experiment) may be sufficient to support memory retrieval at remote time points. To test the latter possibility, we examined the relationship between BLA reactivation and freezing. There was a strong linear relationship between the percentage of reactivated neurons and the amount of freezing at 2 days (r= 0.81) and 2 weeks (r= 0.62) that did not differ (no effect of time, F (1, 5) = 3.14, p > 0.05). This suggests that reactivation of BLA neurons is related to the amount of freezing during recent and remote memory retrieval. This finding is consistent with a recently published paper that used a similar genetic system to examine reactivation of amygdala neurons after fear conditioning [].

Role of the basolateral amygdala in the storage of fear memories across the adult lifetime of rats.

We next determined whether the same brain regions were reactivated when memory was retrieved 2 weeks after learning. Mice were trained as described above and then tested 14 days later. Analysis of double labeling revealed that neurons in CA3 and CA1 were significantly reactivated during remote memory retrieval (Fisher’s PLSD, pairwise comparisons, p values < 0.05). In contrast, reactivation in the BLA and DG did not exceed chance (p values > 0.05) ( Figure 3 C). To determine whether there were changes in the number of reactivated neurons over time, we compared the percentage of H2B-GFP-positive cells colabeled with c-fosduring the recent and remote memory tests ( Figure 3 D). Planned comparisons (Fisher’s PLSD) found a significant reduction in the percentage of reactivated neurons in the BLA and DG during the remote memory test (p values < 0.05), an increase in CA3 (p < 0.05), and no change in CA1 (p > 0.05).

Given that the hippocampus is required to retrieve recent context fear memories, we expected to see reactivation of neurons in this region 2 days after learning. Figure 3 B shows the percentage of reactivated neurons in TetTag mice relative to chance (chance = percent GFP × percent c-fos). Reactivation was significantly greater than chance in the DG, CA1, and BLA (Fisher’s PLSD, pairwise comparisons, all p values < 0.05). Reactivation did not exceed chance in CA3 (p > 0.05). These results suggest that memory retrieval involves reactivation of neurons in the hippocampus and amygdala that were previously activated during learning.

Previous work indicates that context fear gradually becomes independent of the hippocampus after learning []. Therefore, we next determined whether the hippocampus is required for memory retrieval 2 days and 14 days after learning. To examine this issue, we inactivated the dorsal hippocampus with the AMPAR antagonist CNQX prior to testing. Memory was assessed by measuring the freezing response, a species-specific defensive behavior observed in rodents []. TetTag littermates lacking H2B-GFP were trained in context A and tested 2 days or 14 days later in the same environment. Infusion of CNQX into the dorsal hippocampus prior to testing impaired memory retrieval at 2 days (main effect of group F (1, 24) = 10.23, p < 0.05) but had no effect 14 days after training (no effect of group F (1, 22) < 1) ( Figure 3 A). These results suggest that the systems mediating context fear are reorganized within 2 weeks of learning.

(D) The percentage of H2B-GFP-positive neurons that were reactivated (i.e., colabeled with c-fos) is shown for mice tested 2 days (recent, n = 5) or 14 days (remote, n = 4) after training. The percentage of reactivated neurons decreased over time in the BLA and DG, increased in CA3, and remained unchanged in CA1. Error bars represent ± SEM. ∗ p < 0.05.

(C) A separate group of TetTag mice were trained in context A and tested in the same environment 14 days later (n = 4). Significant reactivation was observed in CA3 and CA1 but not in DG or BLA.

(B) TetTag mice were trained off DOX in context A and tested in the same environment 2 days later (n = 5). The percentage of double-labeled neurons is shown relative to chance (percent H2B-GFP × percent c-fos). Significant reactivation was observed in DG, CA1, and BLA but not CA3.

(A) Control mice were trained in context A and tested 2 days (recent, saline n = 13, CNQX n = 13) or 14 days (remote, saline n = 12, CNQX n = 12) later in the same environment. Infusion of CNQX into the dorsal hippocampus prior to testing impaired memory retrieval at 2 days but not 14 days after training.

We next examined reactivation of H2B-GFP neurons during memory retrieval in several brain regions ( Figure 2 A) 2 days and 14 days after learning ( Figure 2 B). Regions were analyzed in coronal sections that ranged from −2.05 mm to −2.25 mm posterior to bregma. Neurons activated during learning were labeled with H2B-GFP by removing DOX prior to training. The animals were put back on DOX immediately afterward to prevent further H2B-GFP expression. Two days or 14 days later, the mice were tested in the same context and c-fos expression was used to index cellular activity. H2B-GFP and c-fos expression were quantified in DG, CA3, CA1, and the basolateral nucleus of the amygdala (BLA). The BLA receives direct projections from the hippocampus and is essential for context fear learning and expression []. Within the same AP coordinates, we also identified regions of interest (ROIs) in the lateral entorhinal cortex (ENTl), retrosplenial cortex (RSPv), and posterior parietal cortex (PTLp). These regions were selected based on their contributions to spatial learning and their anatomical connections with the hippocampus []. We also quantified reactivation in a control region (supplementary motor cortex [MOs]) that is not involved in context fear learning.

(D) c-fos expression was the same in most brain regions at 2 days and 14 days. It differed in CA1 and RSPv in which expression was increased 14 days after training. Error bars represent ± SEM. ∗ p < 0.05.

(B) Behavioral procedures for the reactivation experiments. Mice underwent fear conditioning in context A off DOX to tag activated neurons with H2B-GFP. After training, animals were put back on DOX and tested 2 days (n = 5) or 14 days (n = 4) later. c-fos expression during testing was used to identify activated neurons. Neurons double labeled with GFP and c-fos were activated during training and testing.

(A) A coronal section (−2.05 mm posterior to bregma) illustrating the brain regions analyzed for reactivation: dorsal hippocampus (DG, CA3, CA1), lateral entorhinal cortex (ENTI), basolateral amygdala (BLA), posterior parietal association area (PTLp), and retrosplenial cortex (RSPv). Image is adapted from the Allen Reference Atlas.

In the next experiment, we verified that the H2B-GFP signal is stable over time. TetTag mice were trained off DOX to label activated neurons with GFP. After learning, the animals were put back on DOX for 2 days or 14 days prior to brain extraction and quantification. We found equivalent H2B-GFP expression 2 days and 14 days after learning (no effect of day F (1, 7) < 1) ( Figure 1 D). These data indicate that the activity of tagged neurons can be followed for several weeks after context fear conditioning.

We first determined whether H2B-GFP TetTag mice could be used to selectively label neurons in the hippocampus that were active during context fear conditioning. Mice fear conditioned off DOX (OFF) showed greater H2B-GFP expression than homecage (HC) control animals (main effect of group F (1, 7) = 25.28, p < 0.05). Post hoc tests (Fisher’s PLSD) revealed that H2B-GFP expression was significantly elevated in CA1 and CA3 (p values < 0.05) ( Figure 1 C). The percentage of labeled neurons was similar to that observed in previous studies, indicating that H2B-GFP is a reliable indicator of cellular activity []. These results are consistent with a recent paper that showed that activity-dependent labeling in fos-tTA transgenic mice recapitulates endogenous c-fos expression []. We also observed a 57% increase in the percentage of H2B-GFP-positive neurons in the DG, although this change was not statistically significant (p = 0.1). The lack of an effect was probably due to the small number of neurons that are typically activated in this region during learning (1%–5%) []. No H2B-GFP-positive neurons were observed in DG, CA3, or CA1 in mice that were fear conditioned on DOX (ON).

Sparse, environmentally selective expression of Arc RNA in the upper blade of the rodent fascia dentata by brief spatial experience.

To label cells during learning, we used transgenic mice that express a long-lasting, activity-dependent form of GFP. In these mice, the tetracycline-transactivator (tTA) system for transgene regulationn was combined with the c-fos promoter to tag active neurons ( Figure 1 A). In the absence of doxycycline (DOX), activation of the c-fos promoter leads to expression of an H2B-GFP fusion protein, which is stable for several weeks after induction []. In the presence of DOX, H2B-GFP expression is prevented. Figure 1 B shows widespread H2B-GFP expression after context fear conditioning (top) that was confined to excitatory neurons expressing αCamKII (bottom).

(D) Two groups of mice were fear conditioned in the absence of DOX. Afterward, the animals were put back on DOX for 2 days (n = 5) or 14 days (n = 4) before being sacrificed for immunohistochemistry. We observed robust expression of H2B-GFP in DG, CA3, and CA1 that did not change over time. Error bars represent ± SEM. ∗ p < 0.05.

(C) Mice fear conditioned off DOX (OFF) (n = 4) showed greater H2B-GFP expression than homecage control (HC) animals (n = 5). H2B-GFP expression was significantly elevated in CA1 and CA3. There was a numerical increase in the percentage of H2B-GFP-positive neurons in the DG, but this change did not reach statistical significance. No H2B-GFP expression was observed in mice fear conditioned on DOX (ON) (n = 4).

(B) Robust H2B-GFP expression was observed throughout the brain in mice that underwent context fear conditioning off DOX (top). Expression of H2B-GFP was limited to excitatory neurons labeled with αCAMKII (red) in the hippocampus (bottom). The yellow outline indicates overlap between H2B-GFP-positive nuclei and cytosolic αCAMKII staining.

(A) The tetracycline-transactivator (tTA) system for transgene regulation was combined with the c-fos promoter to tag activated neurons. In the absence of doxycycline (DOX), activation of the c-fos promoter leads to expression of an H2B-GFP fusion protein, which is stable for weeks after induction. In the presence of DOX, H2B-GFP expression is prevented.

Discussion

8 Guzowski J.F.

McNaughton B.L.

Barnes C.A.

Worley P.F. Environment-specific expression of the immediate-early gene Arc in hippocampal neuronal ensembles. 9 Kubik S.

Miyashita T.

Kubik-Zahorodna A.

Guzowski J.F. Loss of activity-dependent Arc gene expression in the retrosplenial cortex after hippocampal inactivation: interaction in a higher-order memory circuit. Figure 6 Exploration Differences during Training and Testing Show full caption (A) Average motion scores for mice trained and tested in context A. Mice were significantly more active during the training session compared to the testing session 2 days later. Activity only decreased during training after shock was presented (bins 10–12). (B) Freezing data for the same training and testing sessions. There was significantly more freezing during the testing session compared to the training session. Error bars represent ± SEM. The current data demonstrate that memory retrieval involves widespread reactivation of neural ensembles that were engaged during learning. Using H2B-GFP TetTag mice, we found significant reactivation in several regions of the hippocampus, amygdala, and cortex during the retrieval of a recently formed context fear memory. Reactivation was not observed when mice were trained and tested in different environments. Similar results have been obtained in FISH studies in which rats explored the same spatial environment twice within a 30 min period []. Our experiments extend these findings to context fear conditioning using memory tests that were conducted days and weeks after learning. Our results are also unique in that reactivation was observed even though behavioral responses were distinct during training and testing (exploration versus freezing). This implies that neural ensembles activated during exploration can be reactivated during subsequent memory retrieval when animals are immobile. To illustrate this point, Figure 6 A shows the amount of activity observed during training and testing in context A (group AA, Recent). Mice were significantly more active during the training session compared to the testing session (main effect of session (1, 4) = 72.43, p < 0.05). The only reduction in activity that was observed during training occurred late in the session after footshocks were presented (bins 10–12). Figure 6 B shows the amount of freezing observed during the same sessions. As expected, mice froze substantially more during the testing session compared to the training session (main effect of session (1, 4) = 8.29, p < 0.05).

38 Kentros C.

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Muller R.V. Abolition of long-term stability of new hippocampal place cell maps by NMDA receptor blockade. 39 Barry J.M.

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Muller R.U. Inhibition of protein kinase mζ disrupts the stable spatial discharge of hippocampal place cells in a familiar environment. It is possible that memory retrieval is not required for reactivation of H2B-GFP-positive neurons (e.g., exposure to the same sensory cues on two different occasions may simply activate a similar population of neurons). However, place cell work has found that repeated exploration of a spatial environment does not reactivate the same neurons unless plasticity mechanisms are engaged during learning. For example, if NMDARs are blocked during exploration of a novel context, place fields form but they are not stable []. When the animal is subsequently returned to the same context, new place fields are observed as if the rat is in a different environment. Therefore, exploring the same physical environment is not sufficient to reactivate the same group of neurons. Instead, learning needs to take place during initial exploration so that the same spatial representation can be reactivated when the animals are returned to the environment. A recent paper found that blockade of PKMζ, which impairs memory retrieval, also results in place cell remapping in a familiar environment []. These results suggest that reactivation of neurons in the hippocampus is contingent on memory formation and retrieval.

19 Liu X.

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40 Kitamura T.

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Roullet P. Pharmacological intervention of hippocampal CA3 NMDA receptors impairs acquisition and long-term memory retrieval of spatial pattern completion task. Within a few weeks of learning, we observed that the degree of reactivation changed in several brain regions. In the hippocampus, the percentage of reactivated neurons decreased in the DG, increased in CA3, and remained stable in CA1. The continuous generation of new neurons in the DG may contribute to the loss of reactivation in this region. Recent data indicate that neurogenesis in the DG plays an essential role in the clearance of previously formed context fear memories []. Based on these data, one would predict a gradual decline in the reactivation of DG neurons after learning. As the percentage of reactivated neurons decreased in the DG, we observed a corresponding increase in CA3. This finding may be related to the role that CA3 plays in pattern completion. Several studies have shown that context memories lose details and become less precise with the passage of time []. This implies that remote memory retrieval requires reactivation of partially degraded information, a process that is known to depend on CA3 []. Therefore, as reactivation of DG neurons decreases over time, memory retrieval may be supported by reactivation of CA3 and CA1 networks.

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Deisseroth K. Dynamics of retrieval strategies for remote memories. Neurons in the CA1 region of the hippocampus were reactivated during the retrieval of recent and remote context fear memories. This result was unexpected given that the hippocampus was not required for memory retrieval 2 weeks after learning ( Figure 3 A). If memory can be retrieved without the hippocampus at this test interval, then why is CA1 reactivated? One possibility is that remote memory retrieval normally involves the hippocampus but can be mediated by other structures if this region is compromised. Two recent studies support this idea. The first showed that, under some conditions, inactivation of the hippocampus does not impair context fear, although it significantly alters the quality of memory that can be retrieved []. This suggests that alternative brain regions can retrieve information that supports freezing if the hippocampus is compromised. The second study found that prolonged inhibition of the dorsal hippocampus produces compensatory changes in the anterior cingulate cortex (ACC) that were sufficient to support memory retrieval []. Together, these data indicate that the hippocampus (1) is required for recent memory retrieval and (2) contributes to, but is not essential for, remote memory retrieval.

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Squire L.R. Memory consolidation and the medial temporal lobe: a simple network model. We also observed robust reactivation of cortical regions involved in spatial and contextual learning (ENTl, RSPv, and PTLp) []. Reactivation of H2B-GFP-positive neurons in these regions was similar during the retrieval of recent and remote context fear memories ( Figure 4 C). These results are consistent with models of memory consolidation that predict stable reactivation of cortical neurons over time. According to these models, consolidation involves a gradual strengthening of intracortical connections between neurons that were coactive during learning []. Shortly after learning, reactivation of these networks is assumed to require input from the hippocampus. However, once intracortical connections have been strengthened, cortical networks are thought to reactivate without the hippocampus. Consequently, cortical neurons should be reactivated during the retrieval of new and old memories as was observed in the current experiments.