Developmental Regulation Of The Neurite Of Cerebral Cortical Neurons Of Mouse By BRD2

Cortical neuron is the essential unit in the functional regulation of the cerebral cortex, which participate in maintaining various physiological functions in the central nervous system in mammalian. The dendrites and axons of the cortical neurons form local circuits by synaptic connections, in charge of the reception of peripheral afference. While the changes of dendrites and axons, such as morphological characteristics, arborization numbers, and extension directions, can affect the development of the cerebral cortex, as well as influence the physiological functions. A great of molecular signals can affect the characteristics of dendrites and axons, which include Septin 7. Bromodomain containing protein 2 (BRD2), a kind of conservative BRD, plays an important role in the cell cycle, the deficiency of BRD2 can largely increase the proliferation of precursor in the nervous system. Functional studies showed that BRD2 plays an important role in embryonic development, yet until recently there is no report about the role of BRD2 in the development of cerebral cortex.Gene transfer is an essential way in the study of cortical neurons, while the traditional calcium phosphate precipitation and liposome infection protocol only show the lower transfection efficiency, which can be used to morphological observations, but not for the molecular biological study. However, the transfer rate of electroporation transfection is higher, and can be applied in the study of molecular biology.This study began with the separation of cortical neurons, and then optimized the electroporation parameters. Based on this, we further studied the regulation mechanisms of BRD2 in the cultured cortical neurons. This study contains the following two experiments:Part 1. The isolation and optimization of electroporation parameters for cerebral cortical neurons of mouseThe newborn C57 mice within 24 hours were used in this part of experiments. We collected the bilateral cerebral cortex under sterile conditions, then isolated the cerebral cortical neurons. Green fluorescent protein (GFP) expression plasmids shNC were transferred into these cortical neurons with different electroporation voltages and impulse times by using ECM830 electroporation system. Expression of GFP was observed after 24 hours of transfection and the electroporation efficiencies were analyzed to determine the optical electrictransfection conditions by using immunofluorescence double staining method. Results:①Under the condition of isopulse 15 ms pulse duration, the electroporation efficiencies gradually increased as well as the voltages, and reached to the peak value (25.28±2.39%) under the condition of voltage of 250 v, yet the efficiencies decreased as the voltages continued increase.②Under the condition of constant voltage of 200 v, the electroporation efficiencies also showed the increase-then-decrease tendency according to the pulse durations, and the summit was 27.10±2.05% under the conditon of 25 ms pulse duration.③By using two-ways regulations, the highest electroporation efficiencies for the mouse cortical neurons were achieved, and the values were 31.15±1.89% under the condition of 200 v/25 ms and 29.10±1.93% under the condition of 250 v/15 ms, respectively. Conclusion: Electroporation is a highly efficient gene transfection method in the study of mouse cortical neurons. And by optimizing electroporation parameters (appropriate voltage and pulse duration) , we can reach to the maximum transfection efficiency, and provide technical support for the further molecular biological study.Part 2. Regulation of neurite outgrowth in the cerebral cortical neurons by BRD2.The newborn C57 mice within 24 hours were also used in this part of experiments. We isolated and cultured the cerebral cortical neurons under sterile conditions. Then transferred the GFP expression BRD2 RNAi plasmid and shNC plasmid into these cortical neurons by using electroporation or liposome infection protocol, respectively. The expression of BRD2 and Septin 7 in the cortical neurons were analyzed by using the Real-Time PCR method, while the growth of neurite of these neurons were observed by using immunofluorescence double staining method. Results:①After transfection of the BRD2 RNAi plasmid, the expression of BRD2 in the cortical neurons was significantly reduced compared with the shNC plasmid group (P<0.05).②Morphological study showed that after transfection of the BRD2 RNAi plasmid, both the branches (P<0.05) and total lengthes (P<0.01) of axon and dendrite were increased compared with the shNC plasmid group.③After transfection of the BRD2 RNAi plasmid, the expression of Septin7 in the cortical neurons was significantly increased compared with the shNC plasmid group (P<0.05). Conclusion: The BRD2 RNAi plasmid can successfully reduce the expression of BRD2 in the cortical neurons, BRD2 affected the neurite growth of cortical neurons and Septin7 might participant in these regulations as the downstream target of BRD2.