A Study Of Synaptic Morphology And Function Of Differentiated Neurons From Neural Progenitor Cells Of From Embryonic Rats Hippocampus In Vitro

PARTⅠOBSERVATION OF THE DIFFERENTIATION AND ULTRASTRUCTURE OF NEURAL PROGENITOR CELLS FROM EMBRYONIC RATS HIPPOCAMPUS IN VITROObjective: To explore the methods of isolation and culture of neural progenitor/stem cells (NPCs) from hippocampus of embryonic rats, and observe the characteristics of induced differentiation and ultrastructure of NPCs.Methods: To isolate the primary NPCs from Wistar rats (E15~16 days) hippocampus tissues, mechanically dissociated into single cell suspension, the cells are suspended and cultured in serum free medium (containing bFGF and EGF) for long-term survival in vitro. and induced to differentiate in medium containing fetal calf serum; Immunocytochemical staining was applied to identify the NPCs and their potential of differentiation, ultrastructure of the differentiated cells was observed by electron micro- scopes.Results: The cells isolated from hippocampus of embryonic rats could be continuously passage cultured in vitro and form neurosphere and were nestin immunostaining positive which is the marker of NPCs. The cells after differentiation could express the specific antigens-βⅢtubulin (tuj-1) and glial fibrillary acidic protein (GFAP) in neuron and astrocyte, respectively. Nervous processes formed network and various kinds of develoed organelles structure were observed in differentiated cells under electron microscopes; At the same time, structure of junction-like between the cells and intact synapse in some mature differentiated cells cultured were observed.Conclusion: These results indicated that the NPCs isolated and cultured by using the present methods had the ability of proliferation and multi-potential differentiation,The differentiated cells from NPCs can form synapse and junctions.PARTⅡEFFECT OF BDNF ON THE SYNAPTIC MORPHOLOGY AND FUNCTION IN DIFFERENTIATED NEURONS FROM NEURAL PROGENITOR CELLS OF EMBRYONIC RATS HIPPOCAMPUS IN VITRO Objective: To explore the effect of brain-derived neurotrophic factor (BDNF) on the synaptic morphology and function in differentiated neurons of neural progenitor cells (NPCs) from embryonic rats hippocampus.Methods: NPCs were isolated from Wistar rats (E15~16 days) and cultured in DMEM/F12 medium without serum, neurosphere were harvested and fixed, then immunostained with nestin which is the marker of NPCs; NPCs were induced and differentiated in culture medium containing 40 ng/mL BDNF (experimental group) and 1% fetal calf serum (control group) for 14 days, respectively; the morphological changes of differentiated cells were observed daily under the phase-contrast microscope. Expression of synaptophysin (SYN) was observed using Western blot analysis, and the distribution of SYN was analyzed using immunocytochemical staining technique; at the same time, real-time imaging technology was applied to monitor dynamicly the fluorescence intensity change of cells pre and after high K+ stimulation in the laser scanning confocal microscope.Results:The expression level of SYN from differentiated neurons of NPCs in the BDNF-treatment group was higher than that in the serum control group by using Immunocytochemical staining and Western blot; Further, real-time imaging technology also showed that higher intensity were found as compared to the control group.Conclusions: These results indicated that BDNF could promote the expressinon of synaptic morphology and function in differentiated neurons of neural progenitor cells from embryonic rats hippocampus in vitro.PARTⅢTHE ELECTROPHYSIOLOGICAL FEATURES OF DIFFERENTIATED NEURONS FROM NEURONSAL PROGENITOR CELLS CO-CULTURED WITH ASTROCYTESObjective: To study the effect of astrocytes (AST) on the fuctional maturation of differentiated neurons from neural progenitor cells (NPCs).Methods: NPCs were isolated from the hippocampus of Wistar rats (E15~16 days) and cultured in DMEM/F12 medium without serum, AST were isolated from the hippocampus of neonatal Wistar rats and cultured in DMEM/F12 medium with serum, purified by a chemical and standard shaking method and differential adhesion. Immunocytochemical method was used to detect the expressions of glial fibrillary acidic protein (GFAP) of the differentiated cells. AST and NPCs were co-cultured of without contact. AST of co-culture group was digested into monoplast suspension with trypsin, and then mixed with complete culture medium. In order to support the coverslip, four drops of paraffin about 0.1 mm high were made by soldering ion in the bottom of dish. When AST covered 70%～80% of the whole bottle bottom. NPCs'culture medium was used for 24 hours. Cover slip with polylysine was put on 6-well plate, then the dissociated NPCs were co-cultured on the coverslip. Simultaneously, NPCs'culture medium was utilized. Four hours later, the coverslip was inverted and transferred to the dish containing AST. The half of medium was exchanged every 4 days to 5 days, NPCs were inoculated on 6-well plate where cover slip was with polylysine in the control group which were cultured respectively in DMEM/F12 medium with 40 ng/mL BDNF and 1% fetal calf serum. The ion channels, action potential (AP) and post synapse current (PSC) were recorded by using whole-cell patch-clamp technique.Results: The purified AST were GFAP positive. Co-culturing the neurospheres with AST promoted more rapidly differentiation as compared with the control group which were cultured respectively in DMEM/F12 medium with 40 ng/mL BDNF and 1% fetal calf serum, and have ion channels, AP and PSC. ion channel was only recorded in the control group. Conclusions: Under the conditions of co-culture, AST can induce the differentiation of NPCs a certain electrophysiological characteristics of neurons.