Role Of Histone Acetylation In NMDA Receptor Dependent Contextual Fear Memory

The NMDA receptor plays a key role in the course of learning and memory. Recent studies have shown that histone acetylation is crucial to the formation and consolidation of long term memory, and closely related with NMDA receptors. However, it is still not clear whether overexpressing the NR2B subunit of NMDA receptor is correlated with histone acetylation functioning in memory formation. Therefore, in this thesis the following studies are carried out with transgenic mice overexpressing NR2B in the forebrain:In this thesis, using NR2B transgenic mice (NR2B-Tg mice) and the control mice (wt, NR2B negative mice of the same litter) before and after fear conditioning(FC), we detect histone acetylation and ERK phosphorylation variation in the cortex by Western blotting, evaluate the activity histone deacetylase (HDAC) and histone acetyltransferase in the cortex and hippocampus through HDAC and HAT assays and detect the expression of several genes in the same tissues via the real-time PCR technology. NR2B transgenic mice (NR2B-Tg mice) and the control mice (wt, NR2B negative mice of the same litter) are trained by the fear conditioning paradigm, and tissues are collected from the cortex and hippocampus 1 hour (1h),24 hour(24h) and 7 days(7d) after the training, from which nuclear proteins and total RNA are extracted. Nuclear proteins and RNA are also obtained from NR2B-Tg and wt mice without training.Western blotting results show that before training the cortex of NR2B-Tg mice has significant higher levels of H3 acetylation and ERK phosphorylation compared to wt mice. And there is no significant changes in H3 acetyaltion after FC, while ERK phosphorylation drops after FC.HDAC and HAT results show that there is no difference with statistical significance between the two genotypes of mice at any time points in either tissue. While there is no significant change in HDAC activity in the cortex, there is an increase in HDAC activity 1h after FC and then a drastic decrease of that activity 7d after FC. And there is no detectable difference in HAT activity between the two genotypes of mice. HAT activity is reduced 24h after FC in the cortex of wt mice, and so is the HAT activity in the cortex of NR2B-Tg mice 7d after FC; In the hippocampus, there is an increase of HAT activity in the wt mice 7d after FC with no such phenomenon for the NR2B-Tg mice.The Real-time PCR results show that the initial level of BDNF is higher in the cortex of wt mice than NR2B-Tg mice, yet surpased by the latter in both tissues 7d after FC. CREB-1 expression is higher in the cortex of wt than NR2B-Tg mice before FC, with no such difference in the hippocampus. And at 7d after FC CREB-1 expression is increased in both tissues of NR2B-Tg mice, with only the increase in the cortex of wt mice. There is no difference of NeuroDl expression in both tissues at any time point between the two genotypes of mice. And at 7d after FC, there is a significant increase in NeuroDl expression in both tissues for both genotypes. In the cortex SYP expression is initially increased after FC for wt and NR2B-Tg mice with it higher in NR2B than wt at 24h after FC, then dropped 24h after FC; in the hippocampus, SYP expression is significantly reduced at 7d after FC in both genotypes, with it higher in the wt than NR2B-Tg at 1h and 24h after FC. The results suggest that the influence of NR2B overexpression on gene expression is existent, yet the patterns in different tissues are very complex and require further studies.To confirm the role of histone acetylation in NMD A receptor dependent memory formation, we administrated HDAC inhibitor sodium butyrate to NR2B and wild type mice and test its effect on mice performance in novel object recognition and fear conditioning. Results show that administration of HDAC inhibitor one day before has no effect on mice performance in novel object recognition test, while HDAC inhibitor given 7 days in advance can enhance the contextual fear memory of mice, yet the difference has no statistical significance. And there is no effect on cued fear memory.