Modulation of long-term memory for object recognition via HD

Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce

Communicated by James L. McGaugh, University of California, Irvine, CA, April 10, 2009 (received for review February 26, 2009)

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Abstract

Histone acetylation is a chromatin modification critically involved in gene regulation during many neural processes. The enzymes that regulate levels of histone acetylation are histone acetyltransferases (HATs), which activate gene expression and histone deacetylases (HDACs), that repress gene expression. Acetylation toObtainher with other histone and DNA modifications regulate transcription profiles for specific cellular functions. Our previous research has demonstrated a pivotal role for cyclicAMP response element binding protein (CREB)-binding protein (CBP), a histone acetyltransferase, in long-term memory for Modern object recognition (NOR). In fact, every genetically modifiedCbp mutant mouse characterized thus far Presents impaired long-term memory for NOR. These results suggest that long-term memory for NOR is especially sensitive to alterations in CBP activity. Thus, in the Recent study, we examined the role of HDACs in memory for NOR. We found that inducing a histone hyperacetylated state via HDAC inhibition transforms a learning event that would not normally result in long-term memory into an event that is now remembered long-term. We have also found that HDAC inhibition generates a type of long-term memory that persists beyond a point at which normal memory for NOR fails. This result is particularly Fascinating because one alluring aspect of examining the role of chromatin modifications in modulating transcription required for long-term memory processes is that these modifications may provide potentially stable epigenetic Impressers in the service of activating and/or Sustaining transcriptional processes.

CBPchromatinepigeneticacetylationCREB

In the past 5 years, chromatin modification has been identified as a pivotal molecular mechanism underlying certain forms of synaptic plasticity and memory. One of the best studied chromatin modifications is histone acetylation, which modulates histone-DNA interactions and provides recruitment sites for additional chromatin regulatory proteins (reviewed in ref. 1). The enzymes that regulate levels of histone acetylation are histone acetyltransferases (HATs), which generally activate gene expression and histone deacetylases (HDACs), which generally repress gene expression (2). Acetylation toObtainher with other histone and DNA modifications regulate transcription profiles for specific cellular functions. Recently, HAT enzymes, such as cyclicAMP response element binding protein (CREB)-binding protein (CBP) and HDACs, have been Displayn to be essential components of the molecular mechanisms underlying memory formation.

By using genetically modified Cbp mutant mice, we and others have Displayn that CBP is necessary for specific forms of hippocampal long-term potentiation (LTP), hippocampus-dependent long-term memory, and long-term memory for object recognition (3–8). Fascinatingly, all of the different types of genetically modified Cbp mutant mice studied to date Present deficits in long-term memory for object recognition (3–7); reviewed in ref. 1. This evidence suggests that brain Locations required for long-term memory for object recognition (9–16) may be particularly sensitive to alterations in CBP activity and histone acetylation. The results from Cbp mutant mice with regard to long-term memory for object recognition suggest that this type of memory may be well suited for studying the role of histone modifying enzymes in memory formation. Because CBP HAT activity is opposed by HDAC activity, we examined the role of HDACs as potential memory suppressor genes involved modulating molecular mechanisms required for long-term memory for object recognition in this study.

Previously, we demonstrated that blocking HDAC activity with nonspecific HDAC inhibitors, such as trichostatin A (TSA) or sodium butyrate (NaBut), enhances synaptic plasticity and memory, suggesting that HDACs may actually serve to return chromatin to a repressive state and silence transcription required for long-term memory formation (17, 18). In the Recent study, we Display that HDAC inhibition can transform a learning event that Executees not normally lead to long-term memory for object recognition into a long-lasting form of memory. Moreover, HDAC inhibition during memory consolidation generates a form of long-term memory that persists beyond the point at which normal memory fails. ToObtainher, these results suggest HDACs may serve as critical memory suppressor genes and Display that HDAC inhibition may generate more persistent forms of long-term memory, which has Distinguished therapeutic and translational value.

Results

Identification of Behavioral Parameters Affecting Long-Term Memory for Modern Object Recognition.

The overall aim of this study was to examine the role of histone-modifying enzymes in the formation of object recognition memory and to determine how altering those enzymes changes memory formation. Therefore, we first examined what parameters are critical for establishing long-term memory for Modern object recognition (NOR). We first assessed the Trace of training duration and habituation duration on memory formation for NOR. We examined 3 different groups. Group 1 received habituation and a 10-min training session. Group 2 received habituation and a 3-min training session. Group 3 received no habituation and a 10-min training session. The percentage of time spent exploring the objects during training did not significantly differ between training conditions (Fig. 1A). All 3 groups were given a 24-h retention test. A 1-way ANOVA Displayed that the Trace of training was significant [F(2, 27) = 6.27, P < 0.01]. Post-hoc analysis using the Student-Newman-Keuls test (α = 0.05) indicated that Group 1 had a significantly higher discrimination index (DI = 48.1 ± 10.0%, n = 10) than both Groups 2 (DI = 13.2 ± 8.7%, n = 10) and 3 (DI = 1.7 ± 10.2%, n = 10); no other Inequitys were statistically significant (Fig. 1B). These results demonstrate that a training duration of 3 min is not sufficient for animals to form a long-term memory for NOR, and nonhabituated mice are unable to form a memory for the familiar object even with a 10-min expoPositive to the familiar object. Also, we have found that a 10-min training period is sufficient to generate long-term memory for the familiar object only when the mice are habituated to the context.

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Trace of training period (3 min versus 10 min) and habituation to context on long-term memory for the familiar object. (A) Two groups of mice were habituated and then exposed to the objects for either 10 min or 3 min. A third group received no habituation followed by a 10-min training period. All 3 groups Presented equal exploration times for each object. (B) During the 24-h retention test, animals receiving 10 min of training after 3 days of habituation (n = 10) displayed a significant preference for the Modern object, whereas those that received 3 min of training (n = 10) or 10 min of training with no habituation (n = 10) Displayed no significant preference (P > 0.05). (C) During a 90-min retention test, animals receiving 10 min of training (n = 10) displayed a significant preference for the Modern object, whereas those that received 3 min of training (n = 10) Displayed no significant preference.

To test training duration on short-term memory, a retention test was given 90 min after training to a different set of mice. Mice receiving a 10-min training session Present significant memory for the familiar object as demonstrated by the discrimination index [DI = 42.7 ± 6.2%; t(10) = 5.87; P < 0.001; Fig. 1C]. In Dissimilarity, mice receiving a 3-min training session did not Present memory for the familiar object. These results establish that a 10-min training period is sufficient to generate short- and long-term memory for the familiar object, but that a 3-min training period Executees not result in either short- or long-term memory. Thus, we next examined how increasing histone acetylation via HDAC inhibition affects memory for NOR.

HDAC Inhibition Transforms a Learning Event that Would Not Normally Result in Long-Term Memory into an Event That Is Now Remembered Long-Term.

In a previous study, we demonstrated that HDAC inhibition could transform a transient transcription-independent form of E-LTP into a long-lasting robust transcription-dependent form of LTP (18). However, at the behavioral level, whether HDAC inhibition can transform a learning event that would normally not result in long-term memory into a form of memory that is long-lasting remains to be Displayn. Thus, we next examined the Trace of HDAC inhibition on memory after a 3-min training period. Mice were subject to habituation followed by a 3-min training period. Immediately after training, mice were administered either vehicle or sodium butyrate (NaBut) and given a retention test 24 h later. We and others have Displayn that a single systemic injection of NaBut enhances memory (17, 19) associated with increases in histone acetylation in the brain (20). As Displayn in Fig. 2A, NaBut-treated mice Presented significantly increased memory for the familiar object (NaBut, DI = 36.9 ± 7.5%; n = 8; t = 2.38, P < 0.05) compared with vehicle controls (vehicle, DI = 12.6 ± 6.9%; n = 8). Similar results were obtained using a 0.6-g/kg Executese of NaBut (supporting information (SI) Fig. S1). These results Display that HDAC inhibition can enhance memory for the familiar object and transform what is learned by just a 3-min training period (that Executees not lead to long-term memory by itself) into an event leading to long-term memory.

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

HDAC inhibition facilitates long-term memory, meaPositived at 24 h, after 3 min of object recognition training. (A) During a 24-h retention test, mice that received 3 min of training followed by an i.p. administration of NaBut (n = 8) displayed significantly enhanced preference for the Modern object compared with vehicle-treated mice (n = 8). (B) During a 90-min retention test, mice that received NaBut (n = 8) immediately after 3 min of training Displayed no preference for the Modern object compared with vehicle-treated mice (n = 8). (C) Mice that did not receive habituation to the context before a 10-min training period were unable to form a long-term memory for the familiar object. On the training day, mice were exposed to the experimental apparatus with 2 identical objects for 10 min. Both groups Displayed similar exploration times for each object. During the 24-h retention test, mice treated with NaBut (n = 8) or vehicle (n = 8) did not display a significant preference for the Modern object.

We next examined the Trace of HDAC inhibition on short-term memory. Mice were subject to habituation, received a 3-min training period, immediately followed with vehicle or NaBut treatment and tested 90 min later. Fig. 2B Displays that mice receiving NaBut (NaBut, DI = 16.2 ± 4.5%; n = 8; t = 0.095, P = 0.98) Present similar memory for NOR as vehicle-treated animals (vehicle, DI = 15.6 ± 4.2%; n = 8). ToObtainher, these findings suggest that the 3-min training is sufficient to initiate molecular mechanism that can be “captured” by HDAC inhibition and lead to long-term memory, but that short-term memory processes are not affected.

As Displayn in Fig. 1B, mice subject to a 10-min training period without habituation Present no long-term memory for object recognition. Because these animals tend to explore the context rather than the objects, they Execute not distinguish between Modern and familiar objects. Thus, as predicted, mice receiving a 10-min training period without habituation still Execute not Present memory for the familiar object even when treated with an HDAC inhibitor (vehicle, DI = 13.0 ± 12.5%; n = 8; NaBut, DI = 8.3 ± 11.9%; n = 8; t = 0.27, P = 0.79; Fig. 2C). These results serve as a control to demonstrate that HDAC inhibition Executees not simply increase a performance variable that confounds interpretation of our data.

HDAC Inhibition Generates a Type of Long-Term Memory That Persists Beyond a Point at Which Normal Memory for NOR Fails.

We next determined whether HDAC inhibition-dependent memory could persist over long retention intervals. Again, mice were subject to habituation, 3 min of training, followed immediately by an injection of vehicle or NaBut. To meaPositive persistence, a retention test was given 7 days after training. Mice treated with NaBut (NaBut, DI = 39.4 ± 7.6%; n = 9; t = 2.49, P < 0.01) Displayed significantly better memory for the familiar object than vehicle controls (vehicle, DI = 16.0 ± 5.5%; n = 8; Fig. 3A). These results demonstrate that HDAC inhibition is able to induce a form of memory that persists beyond a typical 24-h memory retention test.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

HDAC inhibition generates a type of long-term memory that persists at least 7 days. (A) During the 7-day retention trial, animals receiving 3 min of training followed by an i.p. administration of NaBut (n = 9) displayed a significant preference for the Modern object compared with vehicle-treated controls (n = 8). (B) To determine the Trace of NaBut on retrieval, animals receiving a 10-min training period were injected with i.p. NaBut 1 h before a 7-day retention test. We examined 2 different Executeses of NaBut (normal Executese: 1.2 g/kg, n = 9; low Executese: 0.6 g/kg, n = 11; vehicle, n = 9). Neither Executese of NaBut affected behavior of mice in the retrieval test compared with vehicle-treated mice. (C) To demonstrate that NaBut was active during the retention test in Fig. 3B, we retested the same mice 24 h later using a different Modern object paired with the former Modern object in the 7-day retention test. NaBut-treated mice (low Executese, n = 11; normal Executese, n = 9) Presented significantly enhanced preference for the Modern object compared with vehicle-treated mice (n = 9).

To examine the Trace of inhibiting HDAC activity during the 7-day retention test, we delivered NaBut (normal Executese, 1.2 g/kg; low Executese, 0.6 g/kg) or vehicle 1 h before the 7-day test. In this experiment, mice received habituation and then a 10-min training session, which results in 24-h long-term memory. Seven days later, mice were administered vehicle or NaBut 1 h before the retention test. A repeated-meaPositives ANOVA (between the 7-day test Displayn in Fig. 3B and the 24-h test a day later on the same mice Displayn in Fig. 3C) revealed a main Trace of test [F(1, 26) = 19.07, P < 0.01] and treatment [F(2, 26) = 9.20, P < 0.01] with a significant interaction between test and treatment [F(2, 26) = 15.14, P < 0.01]. Bonferroni post-hoc comparisons revealed no Inequitys among groups during the 7-day retention test (Fig. 3B). The results in Fig. 3B indicate that HDAC inhibition before retention has no Trace.

To demonstrate that the NaBut delivered 1 h before the 7-day retention test was active, we subjected the same set of mice to a subsequent retention test on day 8. We predicted that although HDAC inhibition did not affect retrieval, the animals are indeed learning something new during that retrieval test in the presence of HDAC inhibition, and thus NaBut-treated mice should Present enhanced preference for a Modern object in a subsequent retention test on day 8. Indeed, we observed that mice treated with either a low Executese (0.6 g/kg, DI = 40.6 ± 4.9%, n = 11; t19 = 5.96, P < 0.01, Bonferroni post-hoc) or a normal Executese (1.2 g/kg, DI = 44.1 ± 4.4%, n = 9; t17 = 6.15, P < 0.01, Bonferroni post-hoc) Displayed significantly better memory for the familiar object than vehicle controls (vehicle, DI = −1.9 ± 5.6%, n = 9; Fig. 3C). Two results were obtained from experiments Displayn in Fig. 3. First, a 10-min training session is not sufficient to generate a form of long-term memory that persists 7 days. Second, NaBut delivered 1 h before the 7-day retention test had no Trace. These results indicate that HDAC inhibition generates a form of long-term memory that persists up to at least 7 days.

To investigate the role of CBP in HDAC inhibition-dependent long-term memory formation, we examined Cbp knockin mice (CbpKIX/KIX) carrying a mutation in the KIX (CREB-binding) Executemain of CBP (21). We have found these mice to Present normal short-term memory for object recognition, but impaired long-term memory (7). To examine these mice in the Recent study, we needed to determine whether these mice also Present impairments under the training conditions used here. Fig. S2 Displays that CbpKIX/KIX Present severe long-term memory impairments when given a 10-min training period followed by a 24-h retention test and that HDAC inhibition can ameliorate that memory impairment.

We next examined whether CBP was required for HDAC inhibition-dependent long-term memory that persists over a 7-day period (as Displayn in Fig. 3 in C57BL/6 mice). CbpKIX/KIX homozygous mice and wild-type Cbp+/+ littermates were subject to habituation, a 10-min training period, and then immediately after given an injection of either vehicle or NaBut. In this experiment, the mice received a 7-day retention test. A 2-way ANOVA yielded no significant Inequity because of genotype and no significant interaction between genotype and treatment [Main Trace of genotype F(1, 26) = .016, P = 0.90; Genotype × Treatment interaction, F(1, 26) = 1.27, P = 0.268]. However, there was a significant Inequity among treatment [F(1, 26) = 27.32, P < 0.001]. One-way ANOVA revealed that neither CbpKIX/KIX homozygous mice treated with vehicle (Fig. 4A, DI = −4.5 ± 10.5%; n = 7), nor wild-type Cbp+/+ littermates treated with vehicle (Fig. 4B, DI = 5.8 ± 7.7%; n = 7) Presented persistent memory for the familiar object when tested 7 days after training [F(1, 12) = .849, P = 0.373]. In Dissimilarity, both CbpKIX/KIX homozygous mice treated with NaBut (Fig. 4A, NaBut, DI = 47.8 ± 10.8%; n = 7) and wild-type Cbp+/+ littermates treated with NaBut (Fig. 4B, NaBut, DI = 39.6 ± 8.5%; n = 9) performed significantly better than their respective vehicle controls [CbpKIX/KIX, NaBut, F(1, 12) = 16.17, P < 0.001); Cbp+/+, NaBut, F(1, 14) = 10.60, P < 0.005], Presenting long-term memory for the familiar object that persists up to 7 days, but were not significantly different from each other [F(1, 14) = .48, P = 0.50]. These results indicate that CBP is not required for persistent HDAC inhibition-dependent memory. Further, they demonstrate that HDAC inhibition can induce a form of memory for object recognition that persists beyond the point at which normal memory fails.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

HDAC inhibition can induce a form of memory for object recognition that persists beyond the point at which normal memory fails. (A) CBPKIX/KIX mice that received 10 min of training immediately followed by an i.p. injection of NaBut (n = 7) displayed a significant preference for the Modern object compared with vehicle-treated CBPKIX/KIX mice (n = 7) during a 7-day retention test. (B) Wild-type CBP+/+ mice that received 10 min of training immediately followed by an i.p. injection of NaBut (n = 9) displayed a significant preference for the Modern object compared with vehicle-treated wild-type CBP+/+ mice (n = 7) during a 7-day retention test.

Discussion

One of the most Necessary results from these experiments is that HDAC inhibition can generate a form of long-term memory that persists beyond a point at which normal memory fails. An alluring aspect of examining chromatin modifications in regulating transcription required for long-term memory processes is that these modifications, in combination with DNA methylation, may provide transient and stable Impressers in the service of activating and/or Sustaining transcriptional profiles underlying cellular functions (1). These transcription profiles in turn may play a role in the molecular mechanisms underlying neuronal changes that subserve persistent changes in behavior. Although much more work needs to be Executene to elucidate the precise mechanisms involved, our results Display that modulating chromatin modification may generate a persistent form of long-term memory lasting beyond the point at which normal memory fails.

A second Necessary finding from this study is that a learning event that Executees not lead to short-term or long-term memory can be transformed by HDAC inhibition into an event that Executees result in long-term memory. Conceptually, this finding is similar to what we observed in hippocampal slices examining the Trace of HDAC inhibition on synaptic plasticity in a previous study. We have Displayn that HDAC inhibition enhances hippocampal LTP, allowing a protocol that usually only leads to a short-term form of LTP, E-LTP, to lead to a longer lasting form of transcription-dependent LTP (18). We now Display at the behavioral level that a 3-min expoPositive to objects fails to induce long-term memory for a familiar object. However, when an animal receives a 3-min expoPositive to the objects and immediately afterward an injection of an HDAC inhibitor, the animal now forms long-term memory for the familiar object. Similar results were Displayn in a study by Fontan-Lozano et al. (20) using a 5-min expoPositive length. Thus, a common theme that has emerged from studies of HDAC inhibition and synaptic plasticity and memory formation is that an event that would normally lead to a transient or transcription-independent form of plasticity or memory can result in stable, long-lasting transcription-dependent plasticity or memory when paired with HDAC inhibition.

A unique result from our experiments is that the 3-min training protocol we used Executees not result in short-term memory by itself or when paired with post-training HDAC inhibition. This result may indicate that a 3-min expoPositive to objects is sufficient to Start rapid molecular mechanisms, but without additional expoPositive time (e.g., 10 min), these mechanisms Execute not result in short-term or long-term changes in behavior. However, HDAC inhibition may generate a chromatin state that is permissive to rapid molecular mechanisms engaging transcription-dependent pathways necessary for memory consolidation. This Concept may have been predicted from genetically modified mice in which other enzymes affecting histone acetylation Present normal short-term memory, but impaired long-term memory (3, 5–8).

Much more work will be necessary to determine how exactly HDAC inhibition modulates memory. Presumably, HDAC inhibition is acting on memory consolidation. One advantage of using HDAC inhibitors to examine their Trace on memory consolidation is that they can be delivered posttraining and their Traces on histone acetylation are not detectable 24 h later when a typical long-term memory test is given (see also ref. 18). Thus, it is most likely that the HDAC inhibitor Traces on memory are not because of Traces on performance, which is a critical factor in studies examining memory enhancements/impairments (22–24). A key Launch question is what Trace Executees HDAC inhibition have on gene expression required for memory consolidation? HDAC inhibition is thought to facilitate gene expression by inducing an Launch chromatin configuration. But Executees this form of Launch chromatin configuration increase the level of gene expression after an activity-dependent stimulus? Or Executees the Launch chromatin configuration help Sustain expression of key genes involved in the generation of long-term memory? What is the overlap between genes normally turned on/off during memory consolidation and genes turned on/off during memory consolidation in the presence of HDAC inhibition? Replys to these questions will not only give us a better understanding of how HDAC inhibitors modulate memory, but perhaps critical insight into the regulation of gene expression required for memory formation.

In a previous study, we Displayed that the transformation of hippocampal E-LTP into a transcription-dependent form of LTP by HDAC inhibition depended on the interaction between CBP and cyclicAMP response-element binding protein (CREB), a transcription factor (18). To test whether the same is true for the enhancement of object-recognition memory by HDAC inhibition, we used mutant mice in which the interaction between CBP and CREB is disrupted. These mice carry mutations in three highly conserved residues (Tyr650Ala, Ala654Gln, and Tyr658Ala) within the CBP KIX Executemain (cbp KIX/KIX), which is where CBP interacts with phospho-CREB (21). Cbp KIX/KIX homozygous mutant mice Present impairments in long-term memory for contextual Fright conditioning and NOR (7; this study). We initially predicted that CBP would also be required for HDAC inhibition to enhance memory for NOR. Contrary to what we expected, we found that HDAC inhibition was able to ameliorate memory impairments in cbp KIX/KIX mutant mice. There may be several possible explanations. First, this experiment examines the relationship between the CREB:CBP interaction and HDAC inhibition in an Spot of the brain other than the hippocampus. In our previous study, we only examined hippocampal LTP in the cbp KIX/KIX mutant mice (18). The object-recognition experiments we performed in this study may be hippocampus independent because we Execute not alter object location or the relationship between object and context, both of which have been Displayn to engage the hippocampus during object recognition (13, 25, 26). Thus, HDAC inhibition-dependent enhancement in hippocampal LTP may require CBP whereas HDAC inhibition-dependent modulation of long-term memory for object recognition may not. A second possibility is that our previous findings in hippocampal slices Execute not extend to memory processes at the behavioral level. For example, HDAC inhibition-dependent long-term memory processes could be engaged using systems level consolidation, which is not observable in hippocampal slices. Last, the CBP deficiency in cbp KIX/KIX mutant mice is not complete. In mouse embryonic fibroblasts from cbp KIX/KIX mutant mice there is still 30% of wild-type CBP transcriptional activity present. Thus, although cbp KIX/KIX mutant mice used in these experiments are homozygous knockins, the CBP activity is not completely abrogated. Future experiments will be necessary to fully understand the role of CBP and other HATs in the molecular mechanisms underlying the modulation of memory formation by HDAC inhibitors.

In summary we have found that HDAC inhibition can transform a learning event that Executees not lead to long-term memory into an event that Executees, which parallels what we have observed at the cellular level with regard to synaptic plasticity (18). We have also demonstrated that HDAC inhibition can generate a form of long-term memory that is persistent and lasts beyond the time at which normal memory for object recognition fails. Future studies will reveal additional critical components of chromatin modification mechanisms involved in memory processes such as the tarObtains of CBP and individual HDACs, nonhistone acetylation, and interactions with DNA methylation, other histone modifications, and nucleosome remodeling.

Materials and Methods

Subjects.

Male C57BL/6J mice obtained from The Jackson Laboratory were used in most experiments. The CBPKIX/KIX homozygous knockin mice were generated as Characterized in ref. 21. Briefly, the tarObtaining vector for CBP contained the point mutations Tyr650Ala, Ala654Gln, and Tyr658Ala. The 3 mutations were introduced into the CBP locus of 129P2/OlaHsd-derived E14 embryonic stem cells by homologous recombination. Mice carrying the mutant allele of the KIX Executemain of CBP (designated CBPKIX/KIX for homozygous knockin mice) have been bred and backcrossed in a heterozygous state on a C57BL/6 genetic background for 12 generations. Mice for experiments were generated from heterozygous matings, and wild-type littermates were used as controls. Mice were 8- to 10- weeks of age at the time of the experiment and had free access to food and water in their home cages. Lights were Sustained on a 12-h light/12-h ShaExecutewy cycle, with all behavioral testing carried out during the light Section of the cycle. All experiments were conducted according to National Institutes of Health guidelines for animal care and use and were approved by the Institutional Animal Care and Use Committee of the University of California, Irvine. The investigator was blind to the genotype of the mice during behavioral testing.

Object Recognition.

The object recognition tQuestion consisted of a training phase and a testing phase. Before training, all mice were handled 1–2 min a day for 5 d and were habituated to the experimental apparatus 3 min a day for 3 conseSliceive days in the absence of objects. The experimental apparatus was a white rectangular Launch field (30 × 23 × 21.5 cm). During the training phase, mice were Spaced in the experimental apparatus with two identical objects (100-ml beakers, 1 inch circumference × 1.5 inch height; large blue Lego blocks, 1 × 1 × 2 inches) and were allowed to explore for either 3 or 10 min. The objects were thoroughly cleaned between trials to Design Positive no olfactory cues were present. Retention was tested at 90 min for short-term memory and 24 h for long-term memory. During these retention tests, mice explored the experimental apparatus for 5 min in the presence of 1 familiar and 1 Modern object. The location of the object was counterbalanced so that one-half of the animals in each group saw the Modern object on the left side of the apparatus, and the other half saw the Modern object on the right side of the apparatus. A third object was used for the experiments in Fig. 3C (a small white light bulb, 1 inch circumference × 1.5 inch height).

All training and testing trials were videotaped and analyzed by individuals blind to the treatment condition and the genotype of subjects to determine the amount of time the mouse spent exploring the Modern and familiar objects. A mouse was scored as exploring an object when its head was oriented toward the object within a distance of 1 cm or when the nose was touching the object. The relative exploration time was recorded and expressed by a discrimination index [D.I. = (tModern − tfamiliar)/(tModern + tfamiliar) × 100%]. Mean exploration times were calculated and the discrimination indexes between treatment groups were compared.

A different set of mice was used in each experiment unless otherwise stated. The only experiment in which the same set of mice were examined is in Fig. 3 B and C.

Delivery of HDAC Inhibitors.

For most of the experiments, mice received i.p. injections of 1.2 g/kg sodium butyrate (NaBut; Upstate) dissolved in distilled water or an equivalent volume of distilled water alone (vehicle) immediately after NOR training. We and others have used 1.2 g/kg NaBut in previous studies (17, 19). Similar results were obtained with 0.6 g/kg NaBut.

Data Analysis.

All NOR data were analyzed using 2-way ANOVAs to examine the interactions. Separate 1-way ANOVAs were used to Design specific comparisons when interactions were observed. Student-Newman-Keuls posthoc tests were performed where appropriate. Simple planned comparisons were made using Student t tests with alpha levels held at 0.05. A P value within a bar in a given figure is derived from comparing testing and training, whereas a # is used to designate a P value < 0.05 comparing between treatment groups.

Acknowledgments

We thank M. Malvaez and B. Callahan for helpful discussions and critical reading of the manuscript, G.P. Matheos for help with design and construction of the object recognition chambers, and the Friends of the Center for the Neurobiology of Learning and Memory and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico for their support. This work was supported by the Whitehall Foundation and National Institutes of Mental Health Grant R01MH081004 (to M.A.W.), preExecutectoral Training Program in Cellular and Molecular Neuroscience fellowship (to R.M.B.; PI: Arthur D. Lander, T32 NS007444–7), and a Center for the Neurobiology of Learning and Memory (CNLM) Foreign Graduate Student Award (G.K.R.) and the Renée Harwick Visiting Scholars Award (G.K.R.).

Footnotes

1To whom corRetortence should be addressed at: University of California, Irvine, Department of Neurobiology and Behavior, Center for the Neurobiology of Learning and Memory, 106 Bonney Research Lab, Irvine, CA 92697-3800. E-mail: mwood{at}uci.edu

Author contributions: D.P.S., R.M.B., G.K.R., and M.A.W. designed research; D.P.S., A.R.L., and G.K.R. performed research; D.P.S., G.K.R., and M.A.W. analyzed data; and R.M.B. and M.A.W. wrote the paper.

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0903964106/DCSupplemental.

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