Study On Rara Subcellular Expression Pattern In The Hippocampus Related With Adult Rat’ Spatial Learning And Memory Function

Part1 Effects of morris water maze training and neurons’induce mature on the endogenous RARa subcellular localizationObjective To investigate the relevance of endogenous retinoic acid receptors a (RARa) subcellular localization and its biological following the morris water maze training or induced PC 12 cells differentiation by nerve growth factor (NGF).Methods Male SD (Spraque-Dawley) rats (about 7 weeks old,200g weight) were used with Morris water maze training for 6 days. The levels of RARa mRNA expression in the hippocampus were tested by real-time PCR, and the changes of RARa subcellular localization in the hippocampus were detected by western blot. After the PC12 cells were induced into neural-like differentiation by 50ng/ml NGF for 7 days, the changes of RARa subcellular localization in the induced PC 12 cells were detected by immunofluorescence and western blot. Whole-cell patch clamp technique was used to test the levels of resting membrane potential in the induced PC 12 cells. Moreover, the changes of intracellular calcium concentration were observed via calcium imaging detection system.Results After Morris water maze training 6-day, the levels of RARa expression in the rat hippocampus were significantly increased compared with that of the rats without training. Further analysis found that the expression level of RARa protein in the cytoplasm was statistically increased in the training group, while it was markedly decreased in the nucleus. The PC 12 cells induced by NGF displayed many synaptic, which were cross-linked with each other to form a network. The value of resting membrane potential (BMP) in the NGF-induced PC 12 cells were more similar to the neuron’s BMP threshold. And the PC 12 cells were more sensitive to the calcium ion agonist following NGF treatment. Moreover, the level of RARa protein expression was significantly higher in the NGF-treated group PC 12 cells than that of the untreated groupcells. The expression level of RARa protein was statistically increased in the cytoplasm and synapses of the induced PC 12 cells, whereas it was markedly decreased in the nucleus of the PC 12 cells treated by NGF.Conclution The translocation of RARa expression from the nucleus to the cytoplasm is closely related with the ability of spatial learning and memory function and the differentiation of neural-like cells mature.Part2 Effects of RARa expression in the rat hippocampus on its spatial learning and memory functionObjective To investigate the level of RARa expression in the rat hippocampus to directly impact on the spatial learning and memory ability.Methods The hippocampus of male SD (Spraque-Dawley) rats of 7 weeks old were injected with AAV-RARa shRNAs, and injected by AAV-control shRNA or PBS as its control, respectively. After 14 days, the AAV infection ratio in the brain slices of hippocampus were examined under a fluorescent microscope, and rats’ learning-memory function were detected by Morris water maze. The levels of RARa mRNA expression in the hippocampus were tested by real-time PCR, and the changes of RARa protein expression in the hippocampus were detected in both cytoplasm and nucleus by western blot.Results There were no statistical differences of in the levels of RARa mRNA level expression and rats’ learning-memory ability in between the PBS and AAV-control shRNA groups, suggesting that AAV injection don’t affect learning and memory function of hippocampus. However, the levels of RARa mRNA and protein expression were significantly decreased in the AAV-RARa shRNAs rats hippocampus compared with than that of the AAV-control shRNA group. Moreover, the decrease of RARa expression was more obvious in the cytoplasm, and no significant difference in the nucleus. Meanwhile, we found that the escape latencies in the AAV-RARa shRNAs rats were longer than that of AAV-control shRNA rats, and the ability of learning memory was decreased in the AAV-RARa shRNAs rats compared with that of the AAV-control shRNA rats.Conclution The level of RARa expression in the hippocampus can directly regulate spatial learning and memory function in adult rats.