Differential contributions of de novo and maintenance DNA methyltransferases to object memory processing in the rat hippocampus and perirhinal cortex - a double dissociation
Epigenetic mechanisms are increasingly acknowledged as major players in memory formation. Specifically, DNA methylation is necessary for the formation of long‐term memory in various brain regions, including the hippocampus (HPC); however, its role in the perirhinal cortex (PRh), a structure critical...
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| Published in | The European journal of neuroscience Vol. 41; no. 6; pp. 773 - 786 |
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| Main Authors | , , , , |
| Format | Journal Article |
| Language | English |
| Published |
France
Blackwell Publishing Ltd
01.03.2015
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| Subjects | |
| Online Access | Get full text |
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| Summary: | Epigenetic mechanisms are increasingly acknowledged as major players in memory formation. Specifically, DNA methylation is necessary for the formation of long‐term memory in various brain regions, including the hippocampus (HPC); however, its role in the perirhinal cortex (PRh), a structure critical for object memory, has not been characterized. Moreover, the mnemonic effects of selective DNA methyltransferase (DNMT) inhibition have not yet been investigated systematically, despite distinct roles for de novo (DNMT3a, 3b) and maintenance (DNMT1) methyltransferases. Consequently, we assessed the effects of various DNMT inhibitors within the HPC and PRh of rats using the object‐in‐place paradigm, which requires both brain regions. The non‐nucleoside DNA methyltransferase inhibitor RG‐108 impaired long‐term object‐in‐place memory in both regions. Furthermore, intracranial administration of Accell short‐interference RNA sequences to inhibit the expression of individual DNMTs implicated DNMT3a and DNMT1 in the HPC and PRh effects, respectively. mRNA expression analyses revealed a complementary pattern of results, as only de novo DNMT3a and DNMT3b mRNA was upregulated in the HPC (dentate gyrus) following object‐in‐place learning, whereas DNMT1 mRNA was selectively upregulated in the PRh. These results reinforce the established functional double dissociation between the HPC and PRh and imply the operation of different epigenetic mechanisms in brain regions dedicated to long‐term memory processing for different types of information.
This study has demonstrated, for the first time, the involvement of DNA methyltransferase enzymes in perirhinal cortex‐mediated memory. Furthermore, we reveal a functional double dissociation between the maintenance and de novo DNA methyltransferase families in perirhinal cortex‐ and hippocampus‐mediated object memory, respectively. We hypothesize that these enzymes differentially regulate memory consolidation in these regions for two distinct forms of memory. |
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| Bibliography: | NSERC grant National Sciences and Engineering Research Council (NSERC) of Canada Discovery Grant Fig. S1. (A) Photomicrograph (5× magnification) illustrating FAM-labeled control siRNA within a section of the dorsal HPC overlaid upon the adjacent Nissl-stained section. (B) Photomicrograph (5× magnification) illustrating FAM-labeled control siRNA within a section of the PRh overlaid upon the adjacent Nissl-stained section. Fig. S2. Intra-HPC RG-108 did not impair short-term OiP memory when a 2-h delay was employed, suggesting that DNMTs are not necessary in the HPC for short-term object memories. Fig. S3. Intra-HPC infusions of the non-selective nucleoside DNMT inhibitor, 5-AZA, had no effect on OiP performance when a short-term memory delay (20 min) was employed, but significantly impaired performance with a 24-h retention delay. Data are mean discrimination ratio ± SEM (*P < 0.05). Fig. S4. Two-day pre-sample DNMT3a, DNMT3b and DNMT1 siRNA administration selectively knocks down only the targeted DNMT in the DG (A), CA regions (B) and PRh (C), in comparison with non-targeting control siRNA administration. These results additionally demonstrate that these siRNAs are active 2 days following learning; therefore, the absence of short-term mnemonic impairments noted in our behavioral experiments demonstrate the lack of DNMT involvement in short-term object memories, rather than ineffectiveness of the DNMT siRNAs. Data are mean relative mRNA expression levels as a percentage of controls ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001). Fig. S5. Immediate post-sample intra-HPC administration of DNMT3a siRNA had no effects on long-term OiP memory; however, when administration occurred 1 day prior to OiP acquisition, significant mnemonic impairments were noted 24 h later. This demonstrates that Accell siRNAs are functionally active, in vitro, both 1 and 2 days (Exps 2 and 5) following administration. Fig. S6. Intra-PRh RG-108 did not impair short-term OiP memory when a 2-h delay was employed, suggesting that DNMTs are not necessary in the PRh for short-term object memories. Fig. S7. Intra-PRh infusions of 5-AZA did not impair OiP performance with a long-term retention delay (24 h). Intra-PRh infusions of lidocaine, however, did disrupt long-term OiP memory, suggesting that the PRh is indeed necessary for OiP memory. Data are mean discrimination ratio ± SEM (*P < 0.05, *** P < 0.001). Fig. S8. Spontaneous object recognition (SOR) task in the Y-apparatus. The high opaque walls discourage the use of spatial cues during both acquisition and retrieval. As such, this is an HPC-independent task but relies heavily on the PRh. Fig. S9. Neither immediate post-sample nor pre-sample intra-PRh administration of 5-AZA impaired long-term SOR performance in a Y-apparatus, a canonical PRh-dependent paradigm. This suggests that the mechanism through which 5-AZA inhibits DNMT function, in contrast to the effects of RG-108, does not affect PRh-dependent long-term OiP memory. Data are mean discrimination ratio ± SEM. Fig. S10. (A) Detailed analysis of the behaviors exhibited in the mRNA expression experiments (Exps 9 and 10) found no differences in locomotion (line crossings) or defecation, (B) the amount of time spent in different areas of the open field, or (C) grooming or exploring the external environment. The control animals did, however, spend more time inactive, as well as exploring the walls/corners and floor of the open field. Data are mean discrimination ratio ± SEM (*P < 0.05, ***P < 0.001). Table S1. Accell siRNA. Table S2. Primer sequences for mRNA analysis. Table S3. Amount of exploration time (s) during each experiment. Values are reported as the mean ± SEM. Table S4. Average discrimination ratio (DR) of the total sample exploration (5 min). Values are reported as the mean ± SEM. Experiment S1. Non-targeting siRNA spread analysis. Experiment S2. RG-108 within the HPC does not impair STM with a 2 h retention delay. Experiment S3. 5-AZA within the HPC impairs long-term OiP memory. Experiment S4. Accell DNMT siRNAs cause a selective knock-down 2 days following intra-cranial administration. Experiment S5. 1d pre-sample administration of DNMT3a siRNA to the HPC caused LTM impairments, but not immediate post-sample administration. Experiment S6. RG-108 within the PRh does not impair OiP STM with a 2 h retention delay. Experiment S7. 5-AZA within the PRh does not impair long-term OiP memory. Experiment S8. 5-AZA within the PRh does not impair long-term HPC-independent SOR memory. Ontario Graduate Scholarship ark:/67375/WNG-CL7792KH-J istex:F46B65DFA953165FB4397BFFA4596E2107E45EF5 ArticleID:EJN12819 ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
| ISSN: | 0953-816X 1460-9568 |
| DOI: | 10.1111/ejn.12819 |