Development and Differentiation of Neural Stem Cells co-cultured with Epileptic Neurons in vitro in Rats

BACKGROUND: Can neural stem cells (NSCs) differentiate into ”epileptic neurons” in epileptic microenvironment in vitro? Epileptic microenvironment includes magnesium-free media and co-culture with ”epileptic neuron”, the former is stronger than the latter to induce epilepsy. OBJECTIVE: To model the microenvironment in vivo, and to co-culture the NSCs with normal hippocampal neuron and ”epileptic neuron” for the observation of NSCs development. DESIGN: Repeated measurement and observation. SETTING: The First Affiliated Hospital of Harbin Medical University (Harbin, Heilongjiang Province, China). MATERIALS: Experiment was done in Department of Microorganism and Department of Pharmacology in Harbin Medical University from August 2005 to April 2007. A total of 150 neonatal Wistar rats, irrespective of genders, were applied by the Experimental Animal Center of the Second Affiliated Hospital of Harbin Medical University. All the procedures were in line with ethical standards of animal. Rabbit anti-rat synaptophysin antibody was purchased from Lab Vision Company (USA). Adeno-associated virus containing enhanced green fluorescent protein gone was applied by Beijing Vector Geue Technology Company., Ltd (China). Axopatch 200B magnification was the product of Axon (USA). 5111A oscillograph was the product of Tektronix (USA). METHODS: Rat hippocampal neurons were isolated and cultured, magnesium-free media treatment was applied to establish the model of ”epileptic neuron”. NSCs were cultured according to regular me, thods. After labeled by green fluorescence protein, NSCs were co-cultured with normal hippocampal neuron or ”epileptic neuron” for 14 days, respectively. Patch clamp was used to record the electrophysiology of NSCs in co-culture; immunocytochemistry was used to demonswate the expression of synaptophysin antibody of NSCs; patch clamp was also used to record the postsynaptic potential of the neurons differentiated from NSCs in magnesium-free medium. MAIN OUTCOME MEASURES: Postsynaptic potential and the expression of synaptophysin antibody of NSCs in co-culture with ”epileptic neuron” and normal neuron for 14 days. The postsynaptic potential and epileptic discharge of the neurons differentiated from NSCs in magnesium-free medium. RESULTS: After NSCs was co-cultured with normal hippocampal neuron, 14 beats/5 minutes excitatory postsynaptic potential was recorded in 60%NSCs (6/10) by patch clamp; After co-culture with ”epileptic neuron”, 12 beats/5 minutes excitatory postsynaptic potential of NSCs was recorded. Immunocytochemistry revealed that 80% NSCs (12/15) was observed to express the synaptophysin in co-culture with normal neuron or ”epileptic neuron”. In magnesium-flee medium, 14 beats/5 minutes excitatory postsynaptic potential with a duration of about 10 seconds was found in 60%neurons differentiated from NSCs (9/15), and no epileptic discharge was recorded. CONCLUSION: Rat hippocampal NSCs can form functional synapse in the co-culture with ”epileptic neuron” in vitro. The possibility that NSCs develop into ”epileptic neuron” is minimal.