Transcription of genes required for long-term memory not only involves transcription factors, but also enzymatic protein complexes that modify chromatin structure. Chromatin-modifying enzymes, such as the histone acetyltransferase (HAT) CREB (cyclic-AMP response element binding) binding protein (CBP), are pivotal for the transcriptional regulation required for long-term memory. Several studies have shown that CBP and histone acetylation are necessary for hippocampus-dependent long-term memory and hippocampal long-term potentiation (LTP). Importantly, every genetically modified Cbp mutant mouse exhibits long-term memory impairments in object recognition. However, the role of the hippocampus in object recognition is controversial. To better understand how chromatin-modifying enzymes modulate long-term memory for object recognition, we first examined the role of the hippocampus in retrieval of long-term memory for object recognition or object location. Muscimol inactivation of the dorsal hippocampus prior to retrieval had no effect on long-term memory for object recognition, but completely blocked long-term memory for object location. This was consistent with experiments showing that muscimol inactivation of the hippocampus had no effect on long-term memory for the object itself, supporting the idea that the hippocampus encodes spatial information about an object (such as location or context), whereas cortical areas (such as the perirhinal or insular cortex) encode information about the object itself. Using location-dependent object recognition tasks that engage the hippocampus, we demonstrate that CBP is essential for the modulation of long-term memory via HDAC inhibition. Together, these results indicate that HDAC inhibition modulates memory in the hippocampus via CBP and that different brain regions utilize different chromatin-modifying enzymes to regulate learning and memory.

Long-term memory requires the coordinated effort of transcription factors and numerous enzymes and coregulators that modify and remodel chromatin structure (for review, see Barrett and Wood 2008). One mechanism by which chromatin structure can be regulated is via the addition of functional groups to histone proteins, referred to as histone modifications, which serve two main purposes: first to provide recruitment signals for proteins involved in transcriptional activation and silencing (Kouzarides 2007; Taverna et al. 2007) and second to regulate chromatin structure by disrupting contacts between histone tails and genomic DNA, as well as between nucleosomes (Kouzarides 2007). The best-studied histone modification in learning and memory is histone acetylation and the enzymes that are associated with it, histone deacetylases (HDACs) and histone acetyltransferases (HATs).

A well known HAT involved in learning and memory is the CREB (cAMP response element binding protein) binding protein (CBP). Cbp mutant mice exhibit specific forms of impaired long-term potentiation and long-term memory (Bourtchouladze et al. 2003; Alarcon et al. 2004; Korzus et al. 2004; Wood et al. 2005, 2006; Vecsey et al. 2007). Interestingly, all five types of genetically modified Cbp mutant mice exhibit deficits in long-term memory for object recognition (Bourtchouladze et al. 2003; Alarcon et al. 2004; Korzus et al. 2004; Wood et al. 2006; Oliveira et al. 2007; Barrett and Wood 2008; Stefanko et al. 2009). This evidence suggests that the brain regions required for object recognition memory may be particularly sensitive to alterations in histone acetylation and CBP activity. Thus, the object recognition task provides a particularly useful behavioral paradigm for studying the role of histone-modifying enzymes in long-term memory processes.

In contrast to the genetic studies examining the role of CBP in memory, the majority of the studies examining HDACs in memory have been carried out using a pharmacological approach (for review, see Barrett and Wood 2008). HDAC inhibition experiments have shown that HDACs are critical negative regulators of long-term memory formation (Levenson et al. 2004; Vecsey et al. 2007; Stefanko et al. 2009) and a study examining individual HDACs has revealed that HDAC2, but not HDAC1, to be a key HDAC in regulating memory formation (Guan et al. 2009). More recently, a study has shown that HDAC3 is also a critical negative regulator of memory formation (McQuown et al. 2011). However, the underlying mechanism by which HDAC inhibition modulates long-term memory formation remains unclear.

A study by Vecsey et al. (2007) demonstrated that hippocampal long-term potentiation could be significantly enhanced by HDAC inhibition and that this effect was entirely dependent on CBP and its interaction with CREB. However, the same did not appear to be true at the level of behavior when examining long-term memory. Stefanko et al. (2009) found that HDAC inhibition could facilitate long-term memory for object recognition in CBP mutant mice. This suggested that HDAC inhibition could facilitate long-term memory independently of CBP. In the discussion of Stefanko et al. (2009), the investigators suggest that the object recognition task used may not have engaged the hippocampus, which in turn would not engage CBP-dependent mechanisms in the hippocampus. Thus, the prediction is that in an object recognition task that does engage the hippocampus, HDAC inhibition will modulate long-term memory in a CBP-dependent manner.

To test this prediction and to better understand the histone-modifying mechanisms regulating long-term memory formation in the hippocampus, we first examined the role of the hippocampus during retrieval of long-term memory for object recognition and object location by utilizing muscimol as the inhibitor of neuronal activity. We then used similar forms of training paradigms in CBP-mutant mice, as well as HDAC inhibitor treatment, to study the basic underlying mechanisms of HDAC inhibition-induced memory enhancement. The results have important implications for the basic understanding of the role of CBP and histone acetylation in different brain regions for long-term memory formation as well as the role of the hippocampus in the retrieval of long-term memory for object recognition vs. object location.

Results

Muscimol inactivation of the dorsal hippocampus in consolidation and retrieval of long-term memory for object recognition To examine the role of the hippocampus in consolidation of long-term memory for object recognition, we delivered the GABA agonist muscimol directly to the dorsal hippocampus immediately after training. First, we indirectly examined the spread of muscimol using c-fos immunohistochemistry. Mice were cannulated with hippocampal cannulae, handled for 2 min a day for five consecutive days, and then habituated to the chamber for 5 min a day for four consecutive days. Mice then received dorsal hippocampal infusions of muscimol or vehicle 1 h prior to a 10-min training session, and brains were collected 90 min following training. Figure 1 shows that muscimol infusion results in a significant decrease in hippocampal c-fos expression (44 ± 4.0% of vehicle; t-test – t (6) = 8.027; P < 0.001). There was no significant difference in the number of c-fos-positive cells in the cortex surrounding the cannula tract in muscimol-infused mice as compared to vehicle (t-test − t (6) = 0.331; P = 0.752), which demonstrates that this reduction in c-fos immunoreactivity is confined to the hippocampus. View larger version: Download as PowerPoint Slide Figure 1. Intrahippocampal muscimol injection spread indirectly examined by c-fos immunoreactivity. (A) images are 4X magnification on the right and 20X magnification on the left. Histograms depict quantification of cell counts as a percent of vehicle. (A) Representative images showing c-fos immunoreactivity in sections of vehicle (top row) and muscimol-infused mice (bottom row). (B) Quantification shows that c-fos-immunoreactive cells are not changed in the cortex surrounding the cannula, but it is significantly decreased by 56% in the dorsal hippocampus. ***, P < 0.001. Numbers inside bars indicate sample size (n). To examine the effect of muscimol in the dorsal hippocampus on consolidation, mice were cannulated with hippocampal cannulae, handled for 2 min a day for five consecutive days, and then habituated to the chamber for 5 min a day for four consecutive days. Mice received a 10-min training period (Fig. 2A), which we have shown in previous studies results in long-term memory for the familiar object (Stefanko et al. 2009). Neither the total time exploring the objects (one-way ANOVA − F (2,22) = 2.148; P > 0.05), nor the preference between the different objects (discrimination index) during training differs significantly between groups (Kruskal-Wallis one-way ANOVA − H (2) = 1.055; P > 0.05). Exploration times are presented in Supplemental Table S1. Immediately after training, mice received bilateral hippocampal delivery of 0.5 µL of muscimol (1 µg/µL in PBS) or vehicle (PBS). A Kruskal-Wallis one-way ANOVA revealed a significant difference between groups (Fig. 2B) (H (2) = 10.789; P = 0.005). A pairwise multiple comparison revealed animals receiving muscimol (n = 9) exhibited no preference for the novel object as compared to control animals receiving vehicle (n = 8; Q = 3.014, P < 0.05; Dunn's Method) or noncannulated animals (n = 10; Q = 2.632; P < 0.05). Vehicle and noncannulated animals were not significantly different from each other, showing that the surgery had no negative impact on performance. The total time spent exploring the objects during testing did not differ between groups (one-way ANOVA – F (2,24) = 0.094; P > 0.05; see Supplemental Table S1). This experiment demonstrates that the hippocampus is necessary during consolidation to form long-term memory for the familiar object. View larger version: Download as PowerPoint Slide Figure 2. The hippocampus is engaged during object location memory retrieval. (A,C,E) Schematic diagrams for object recognition tasks. Letters (A, B, and C) in the boxes indicate objects. Gray arrow indicates a moved familiar object compared to the training. In each experiment, mice were fitted with bilateral hippocampal cannulae, allowed to recover from surgery, handled, and habituated to the context prior to a 10-min training. Animals received a bilateral injection (0.5 µL at 15 µL/h) of 1 µg/µL muscimol dissolved in PBS or PBS as a control (vehicle). Noncannulated animal did not undergo surgery or injection. (B) During a 24-h retention test, mice that received muscimol immediately after the training displayed no preference for the novel object in contrast to vehicle or noncannulated mice. (D) During a 24-h retention test, mice that received muscimol 1 h prior to the retention test displayed similar preference for the novel object compared with vehicle-treated mice. (F) During a 24-h retention test, mice that received muscimol 1 h prior to the retention test in the OLM task (moved familiar object—gray arrow) displayed a significant preference for the novel object compared to vehicle-treated mice. *, P < 0.05. Numbers inside bars indicate sample size (n). To examine the role of the hippocampus in retrieval of long-term memory for object recognition, we delivered the GABA agonist muscimol directly to the dorsal hippocampus 1 h before the retention test (Fig. 2C,D). Neither the total time exploring the objects (t-test − t (17) = 0.148; P > 0.05), nor the preference between the different objects (discrimination index) during training differs significantly between groups (t-test − t (17) = 0.520; P > 0.05; for times see Supplemental Table S1). As shown in Figure 2D, animals receiving muscimol (n = 10) exhibited similar long-term memory for the familiar object as compared to control animals receiving vehicle (n = 9; t-test − t (17) = 1.352; P = 0.194). The total time spent exploring the objects during testing did not differ between groups (t-test – t (17) = −1.851; P > 0.05; see Supplemental Table S1). This experiment demonstrates that the hippocampus is not necessary for the retrieval of long-term memory for the familiar object.

Inactivation of dorsal hippocampus reveals intact long-term memory for object recognition when familiar object has been moved to a novel location We hypothesized that if a familiar object changes location between training and testing it would impart novelty to the familiar object during the retention test. Thus, in a retention test in which a familiar object (see Fig. 2E, object B) has been moved to a novel location and a novel object (see Fig. 2E, object C) is introduced, a mouse is predicted to spend equal time exploring both objects, resulting in a discrimination index of zero. Because object location is thought to be encoded by the hippocampus, we predicted that muscimol inactivation of the hippocampus during the retention test would prevent processing of object location information, resulting in a mouse spending more time exploring the novel object (object C) as compared to the familiar object (object B). As shown in the previous experiment (Fig. 2D) a mouse can distinguish between a novel and familiar object without an active hippocampus. To test this prediction, mice were treated and handled as in the previous experiment except that during the test the familiar object (object B) was moved to a novel location (as indicated by the gray arrow in Fig. 2E) and the novel object (object C) was introduced in the former spot of the familiar object. Neither the total time exploring the objects (Mann–Whitney U = 16.000; n (muscimol) = 6; n (vehicle) = 8; P > 0.05), nor the preference between the different objects (discrimination index) during training differs significantly between groups (t-test – t (12) = 0.029; P > 0.05; for times see Supplemental Table S1). As shown in Figure 2F, animals receiving muscimol (n = 6) 1 h prior to the retention test exhibited significant preference for the novel object (object C) as compared to control animals receiving vehicle (n = 8; t-test – t (12) = 4869; P < 0.001). The total time spent exploring the objects during testing did not differ between groups (t-test – t (12) = 0.212; P > 0.05) (see Supplemental Table S1). Thus, vehicle-treated mice explored both objects similarly, resulting in a near zero discrimination index. However, muscimol-treated mice preferentially explored the novel object (object C), resulting in a high discrimination index, which demonstrates a significant long-term memory for the familiar object but no memory of object location. These results suggest that the hippocampus becomes engaged during retrieval of long-term memory for a familiar object when the location of that object is different between training and testing. These data also suggest that if the hippocampus is engaged during retrieval, then behavior based on the existing long-term memory for the familiar object is masked by the competing memory for object location.

Inactivation of dorsal hippocampus reveals intact long-term memory for object recognition when familiar object is placed in a novel context To demonstrate that the effects of muscimol on the hippocampus during memory retrieval are not specific to object location, we also examined the effect of changing the context between training and testing. Similar to the results shown in Figure 2D, if the context is not changed between training and testing (see schematic in Supplemental Fig. S1A), delivery of muscimol to the dorsal hippocampus has no effect on long-term memory for the familiar object (Supplemental Fig. S1B). As shown in Supplemental Figure S1B, vehicle-treated (n = 10) as well as muscimol-treated animals (n = 10) both spent more time exploring the novel object (no statistical significant differences between groups; t-test – t (18) = 1.533; P > 0.05). In contrast, if the context is switched between training and testing (see schematic in Supplemental Fig. S1C), vehicle-treated animals (n = 7) showed no preference for the novel object, whereas muscimol-treated animals (n = 7) exhibited a strong preference for the novel object (t-test – t (12) = 6.432; P < 0.001) (Supplemental Fig. S1D). There was no statistical significant difference in exploratory behavior neither during training (Supplemental Fig. S1B: t-test – t (18) = 0.475; P > 0.05; Supplemental Fig. S1D: t-test – t (12) = 1.863; P > 0.05), nor during testing (Supplemental Fig. S1B: t-test – t (18) = 0.716; P > 0.05; Supplemental Fig. S1D: t-test – t (12) = 0.414; P > 0.05), as well as no preference for either object (discrimination index) during the training (Supplemental Fig. S1B: t-test – t (18) = 0.455; P > 0.05; Supplemental Fig. S1D: t-test – t (12) = 0.189; P > 0.05). These results are very similar to those in Figure 2, suggesting that if either object location or contextual information changes between training and testing, then the hippocampus becomes engaged, resulting in masking of long-term memory for the object itself.

Enhanced long-term memory for object recognition via HDAC inhibition is masked by engaging the hippocampus during retrieval In the next experiment, we examined whether histone deacetylase (HDAC) inhibition can enhance long-term memory for object recognition, even when a familiar object has been moved to a novel location. One group of mice were given a 3-min training period (see schematic in Fig. 3A), which we have previously shown is not sufficient for a mouse to form an observable long-term memory for the familiar object 24 h after training (Stefanko et al. 2009). Immediately after training, one-half of the mice received a systemic i.p. injection of NaBut (1.2 g/kg) and the other half vehicle. These two treatment groups then were split in half to test them in different paradigms. The first set of mice (vehicle and NaBut treated) was given a retention test in which the familiar object (object B) remained in the familiar location (object recognition memory [ORM]; see left-hand side of schematic in Fig. 3A). The other set was given a retention test in which the familiar object (object B) was moved to a novel location (object location memory [OLM]; see right-hand side of schematic in Figure 3A [gray arrow indicates a moved object B]). Neither the total time exploring the objects (two-way ANOVA – experiment type [ORM, OLM]: F (1,31) = 1.511; P > 0.05; Treatment [Vehicle, NaBut]: F (1,31) = 0.132; P > 0.05; experiment type × treatment: F (1,31) = 0.094; P > 0.05), nor the preference between the different objects (discrimination index) during training differs significantly between groups (two-way ANOVA – experiment type [ORM, OLM]: F (1,31) = 0.800; P > 0.05; Treatment [Vehicle, NaBut]: F (1,31) = 3.007; P > 0.05; experiment type × treatment: F (1,31) = 0.0175; P > 0.05; see Supplemental Table S1). Immediately after training, mice received a systemic i.p. injection of NaBut (1.2 g/kg) (see Stefanko et al. 2009) or vehicle. As shown in Figure 3B, mice receiving NaBut (n = 9) exhibited significantly greater preference for the novel object than vehicle controls (n = 9; t-test – t (16) = 2.866; P = 0.011). The total time spent exploring the objects during testing did not differ between groups (t-test – t (16) = 0.760; P > 0.05; for times see Supplemental Table S1). These results are similar to our previous findings and support the conclusion that HDAC inhibition can transform a learning event that would not normally result in long-term memory (3-min training period) into an event that does result in significant long-term memory (Stefanko et al. 2009). View larger version: Download as PowerPoint Slide Figure 3. HDAC inhibition enhances preference for the novel object in the ORM task, but does not affect performance in the object location-dependent OLM task. (A) Schematic for ORM task and OLM task. Letters (A, B, C) in the boxes indicate objects. Gray arrow indicates a moved familiar object compared to the training. (B) Mice administered NaBut immediately after training exhibit significant long-term memory for the familiar object in its familiar location. (C) In contrast, in the OLM task where the familiar object is placed in a different location, both vehicle- and NaBut-treated mice exhibit similar preference for both objects during the retention test, resulting in negligible discrimination. *, P < 0.05. Numbers inside bars indicate sample size (n). In contrast, when a familiar object is moved to a novel location (object B) and a novel object is introduced (object C), NaBut-treated animals (n = 9) performed similarly to vehicle-treated animals (n = 8; t-test – t (15) = 1.603; P = 0.130; Fig. 3C). Both NaBut and vehicle-treated groups explored the familiar object in a novel location (object B; see right-hand side of schematic in Fig. 3A) and the novel object (object C) to a similar extent. The total time spent exploring the objects during testing did not differ between groups (Mann-Whitney U = 35.500; P > 0.05; for times see Supplemental Table S1). NaBut-treated animals did not explore the novel object more than the familiar object even though animals in the same experiment receiving the same handling, treatment and training spend more time with the novel object in the ORM task (Fig. 3B). Together, the results from Figure 3B,C suggest that even though HDAC inhibition can enhance long-term memory for the object itself, it is not evident when the hippocampus becomes engaged during retrieval and drives behavior. Similar to Figure 2C, NaBut-treated animals have a memory for both the object location and the object itself, which results in a masking of the long-term memory for the object itself during retrieval as determined by the discrimination index. An alternative explanation is that NaBut simply failed to affect long-term memory for object location, but this is unlikely as we have shown NaBut can enhance long-term memory for object location in a previous study (Roozendaal et al. 2010), and the data shown in the next two figures.

CBPKIX/KIX homozygous knock-in mice exhibit HDAC inhibition-induced long-term memory enhancement for object recognition memory in an OLM task CBPKIX/KIX homozygous knock-in mice express mutant CBP protein carrying a triple point mutation in the CREB-binding (KIX) domain of CBP (Kasper et al. 2002). In a previous study we demonstrated that these mice have impaired hippocampus-dependent long-term memory for contextual fear (Wood et al. 2006). We have also shown that CBPKIX/KIX mice exhibit significantly impaired long-term memory for object recognition using a task similar to the schematic shown in Figure 2A, in which object location is not changed (Stefanko et al. 2009). Further, impaired long-term memory for object recognition in CBPKIX/KIX mice could be rescued by HDAC inhibition (Stefanko et al. 2009), suggesting that HDAC inhibition modulates long-term memory for the object itself independently of CBP. Results from Figures 1 and 2 suggest that the hippocampal dysfunction of CBPKIX/KIX mice should allow one to observe HDAC inhibition-dependent enhancement of long-term memory for object recognition in CBPKIX/KIX mice, but not wild-type littermates. To test this, CBPKIX/KIX mice and CBP+/+ wild-type littermates were handled and trained according to the schematic in Figure 4A using a 10-min training period. Neither the total time exploring the objects (two-way ANOVA genotype: F (1,39) = 0.220; P > 0.05; treatment: F (1,39) = 0.037; P > 0.05; genotype × treatment: F (1,39) = 0.147; P > 0.05), nor the preference between the different objects (discrimination index) during training differs significantly between groups (two-way ANOVA genotype: F (1,39) = 0.632; P > 0.05; treatment: F (1,39) = 1.159; P > 0.05; genotype × treatment: F (1,39) = 2.147; P > 0.05; for times see Supplemental Table S1). Immediately after training, mice received a systemic i.p. injection of NaBut (1.2 g/kg) or vehicle. A two-way ANOVA revealed an effect of treatment (F (1,39) = 8.280; P = 0.007) and genotype (F (1,39) = 57.643; P < 0.001) with a strong trend toward an interaction (F (1,39) = 3.911; P = 0.055). Bonferroni post-hoc comparisons showed that CBPKIX/KIX mice treated with NaBut (n = 11) have significantly enhanced object memory compared to vehicle-treated CBPKIX/KIX mice (n = 11; t (20) = 3.203; P = 0.004; Fig. 3B). In contrast, wild-type mice treated with either NaBut or vehicle failed to exhibit long-term memory for the familiar object. CBPKIX/KIX mice treated with NaBut showed a significant increase in the preference for the novel object as compared to wild-type animals treated with NaBut (KIX, n = 11; WT, n = 10; t (19) = 9.236; P < 0.001). The total time spent exploring the objects during testing did not differ between groups (two-way ANOVA genotype: F (1,39) = 0.000; P > 0.05; treatment: F (1,39) = 0.857; P > 0.05; genotype × treatment: F (1,39) = 0.504; P > 0.05; for times see Table S1). The data from CBP+/+ wild-type littermate mice, showing that both vehicle- and NaBut-treated groups failed to exhibit long-term memory for the familiar object, replicate the results shown in Figure 3C. In contrast, CBPKIX/KIX mice treated with NaBut exhibited significant long-term memory for the familiar object, presumably due to impaired hippocampus-dependent memory formation in these mice. This finding will be carefully addressed in the Discussion. View larger version: Download as PowerPoint Slide Figure 4. HDAC inhibition enhances memory for the object itself in CBPKIX/KIX homozygous knock-in mice. (A) Schematic of OLM task. Letters (A, B, C) in the boxes indicate objects. Gray arrow indicates a moved object compared to the training. (B) CBPKIX/KIX mice and wild-type littermates received 10-min training period followed immediately by i.p. injection of either NaBut (1.2 g/kg in water) or vehicle (water). During the retention test, in which the familiar object is in a different location (gray arrow), wild-type mice exhibited no preference for the novel object regardless of treatment. In contrast, CBPKIX/KIX mice displayed a poor preference for the novel object, which was significantly enhanced by NaBut treatment. **, P < 0.01; ***, P < 0.001. Numbers inside bars indicate sample size (n).