Investigation Of Induced Division In Primary Neurons From Rat Cerebral Cortex

It is generally believed that neurons are end-differentiated cells, which have withdrawn from the cell cycle and are no longer capable of division. However, utilizing the method of immuno-double label, serial microphotography and patch-clamp in our previous work, neurons either differentiated from the neural stem cells or isolated from cerebral cortex in vitro were observed still having the ability to divide under certain conditions. Several proteins and neuro-transmitters specific for differentiated neurons were expressed in such dividing cells. These dividing neurons possessed the typical electro-physiological characteristics of differentiated neurons. The results of previous work primarily demonstrated that the differentiated neurons could divide. The question about neuron-division has been further investigated through modem biological and medical methods in the following experiment.In the first part of our experiment, we initially cultured mixed neurons from rat cerebral cortex in vitro. And the expression of PCNA and nestin in the mix-cultured neurons were tested with the method of immunocytochemistry. The results showed that there were PCNA-ir (immuno-reactivity) cells, but there were no nestin-ir cells. Neural stem cells and progenitors were not present in the mixed culture neurons in addition to other dividing neurons, such as glial cells and fibroblast. The mix cultured neurons could be used for research about neuron-division. In order to investigate the molecular mechanism of neuron division, highly pure neuron must be utilized for experiment. It was very difficult to obtain highly pure neurons. On the basis of methods in literature, we optimized the culture condition and purification methods. The purity of cultured neurons was tested through cell counting and flow cytometry. Before cell counting and flow cytometric analysis, the neurons were stained with MAP2 antibody immunocytochemically. MAP2-ir neurons accounted for more than 99% in all the total cells, which were displayed by nucleus with PI chemical staining. And that the results of flow cytometric analysis showed that MAP2-ir cells accounted for about 99.8% of allthe cells. The above-mentioned results demonstrated either mixed or pure neurons could be used for the research work about neuron-induced division.In the second part of the experiment, utilizing the mixed neurons, we further investigate the phenomenon of neuron-division. Firstly, a lot of neuron-like cells at different mitotic phases were observed under phase-contrast microscope in the induced-division cultures by EGF and bFGF. These dividing cells had the typical morphological characteristics of neurons. Secondly, immune-fluorescence against MAP2/NSE and PI staining were acted on induced-dividing neurons. Under fluorescence microscope and laser confocal microscope, many MAP2/NSE-ir neurons at different mitotic phases were observed and recorded. And these dividing cells also possessed the typical morphological characteristics of differentiated neurons. ShcC is a kind of protein, which is specifically expressed in differentiated and mature neurons, but is not expressed in neural stem/progenitor cells. In order to further demonstrate that the dividing neurons were differentiated neurons, but not neural stem/progenitor cells, ShcC was used again as a specific marker of differentiated neurons. The results showed that ShcC-ir neurons at different mitotic phase were also present in the culture after induced-division. In our electron microscopic investigation of NSE immuno-gold labeling, some neurons at different mitotic phases were also observed under electron microscope. The dividing cells possessed the typical fine structure of neurons and a large number of NSE-colloidal gold particles were dispersed in the cytoplasm. Some typical synapses were observed to be present between the cell body of dividing neurons at telophase and the process emitted from other neurons. The results of immune-fluorescence labelling and immuno-electron microscopy further demonstrated that neuron could divide.