Neurogenesis Of Immature Brain After Status Convulsion And The Effect Of Brain-Derived Neurotrophic Factor On Electrophysiological Properties Of Voltage-Gated Ion Channels During Neuronal Stem Cell Development

PART I PROLONGED SEIZURES INCREASE PROLIFERATING NEUROBLASTS IN THE IMATURE RAT HIPPOCAMPUS, SUBCALLOSAL ZONE, CORTEX AND THE TRENDS OF MIGRATIONObjective: To explore the dynamic changes of neurogenesis after status convulsion in the immature rat brain.Method: Rats were induced by chemoconvulsants lithium- pilocarpine and were killed 4h, 2 ,7,13,20,27,40d afer. All rats received four injections of bromodeoxyuridine (BrdU) with 2-h intervals 1 day before killed. Animals were perfused and brains were processed for immunocytochemistry antibodies against BrdU. Comparisons of the number of nuclei positive for BrdU which is the marker of proliferating cells at the 7 time points of hippocampus, subcallosal zone and cortex were performed to explore the proliferation and the trends of migration.Results: Our experiments with the lithium-pilocarpine models suggested that acute seizures considerably increase neurogenesis of subgranular zone (SGZ), subcallosal zone (SCZ) and cortex immediately when compared with the age-matched-control groups. Only a few of cells labeled by BrdU were found in SGZ, SCZ and cortex in the age-matched-control groups. SC induced a transitory proliferative surge in the SGZ,CA1,CA3 and cortex with the number of new neurons increasing several folds 2 days after SC which had the trend to migrate to the molecular layer and hilar of the dentate gyrus (DG), but 7 days after in the SCZ with the tendency of migrating toward CA1,CA3 and cortex.Conclusion: Prolonged seizure activity markedly increases neurogenesis of immature brain. Seizure recruits new born cells into abnormal locations and injured hippocampus.PART II THE EXPRESSION CHARACTERISTICS OF VOLTAGE-GATED SODIUM AND POTASSIUM CHANNELS VIA CULTURED NEURAL STEM CELLS DIFFERENTIATION.Objective: To exam the expression of voltage-gated sodium and potassium channels of neural stem cells cultured in vitro before and after differentiation.Method: Embryonic rat hippocampal neural stem cells(NSCs) were isolated and cultured in serum-free medium. Use nestin immunofluorescence labeling to identify if the cultured cells were neural stem cells. Passaged cells were plated on poly-llysine hydrochloride glass coverslips for 1 hour or differentiated for 1 to 3 days with differentiation serum-free medium. Useβ-III-tubulin and GFAP immunofluorescence labeling to identify the fate of these cells. Whole cell patch-clamp technique was used to record the voltage-gated ion channel currents in the neural like cells. To permit further visualization of the cell morphology, cells were routinely filled with Lucifer yellow (LY) during the recording, then colocalized withβ-III-tubulin to identify if the test cells were neuronal progenitors or neurons.Results: Cells aggregated could proliferate and form neurospheres which nestin immunoreactivity was within 3-5 days after primary culture. NSCs have been shown to differentiate into neurons, astrocytes in vitro. No inward Na+ current was detected in any of the undifferentiated NSCs.1 d after the induction of differentiating, cells exhibited voltage-gated sodium currents. The presence of two types of outward potassium currents, delayed recitifier potassium currents and transient potassium currents were also recorded before or after differentiation. Conclusion: NSCs from the embryonic rat brain could be isolated, cultured and differentiated. sodium current expression is a very early event following cell cycle exit in neurogenesis. The two types of outward potassium currents expressed either before or after differentztion.PART III MODULATION OF PASSIVE MEMBRANE PROPERTIES, VOLTAGE-GATED SODIUM CHANNELS AND VOLTAGE-GATED POTASSIUM CHANNELS BY BRAIN-DERIVED NEUROTROPHIC FACTOR DURING DIFFERENTIATIONObjective: To explore the brain derived neurotrophic factor (BDNF) modulation of passive membrane properties and the electrophysiological properties of voltage-gated sodium and potassium channels during differentiation.Method: Passaged cells were plated on poly-llysine hydrochloride glass coverslips with differentiation serum-free medium. Whole cell patch clamp recordings were used to monitor the passive membrane properties,sodium and potassium currents. Analyze the passive membrane properties,sodium current density, generated the activation curves, compared the mean half activation voltage (V1/2) and mean slope k at the early development stage (DIV 1-4) and the late development stage (DIV 8-14).Analyze the potassium current density. Different holding membrane potentials were used to identify the two current types: delayed recitifier potassium currents and transient potassium currents.generated the activation curves, compared the mean half activation voltage (V_1/2) and mean slope k at the early development stage (DIV 1-4) and the late development stage (DIV 8-14).Results: Compared with the time-match controls, Cells treated with BDNF were accompanied by a gradual increase in membrane capacitance, time constant and decrease in input resistance during the progress of development. Chronic stimulation of neuron-like cell cultures with BDNF evoked biphasic changes in sodium currents (I_dNa). During the early stage of differentiation chronic treatment with BDNF induced a significantly increase of sodium current density, but evoked opposite changes at the later stage, compared with the time-match control group.Chronic BDNF stimulation revealed an increase in total whole-cell potassium current density in BDNF-treated neurons at the early development stage and no significant difference at the later development stage compared with time-match controls. The delayed rectifier outward currents are observed in all neuron-like cells. The expression of I_K(A) increased significantly following induction of neural differentiation treated without BDNF, but the expression of I_K(A) decreased after the supplementation of BDNF. Current–conductance relationships indicated that there was negative shift in the V_1/2 of I_K(A) and I_K(DR) following chronic exposure of neuron-like cells to BDNF during the early stage of differentiation but no significant change of the mean slope during the two differentiate stages compared with time-match controls.Conclusion: significant changes in passive membrane properties accompany the dramatic morphological transition observed during the different differentiated stages of cells treated with BDNF. The early stage of differentiation might be a critical period, BDNF was involved in the regulation of the critical period by promoting the functional development of passive membrane. The difference of I_dNa between the early stage of differentiation and the late stage of differentiation stimulated by BDNF indicated that BDNF might play an important role by regulating the formation and/or activation of sodium channels. Our results strongly indicated that the early development stage (1-4days) was a critical period for the development of sodium channels and BDNF could play a protective role in the epileptic brain which reduced excitatory of the new born cells. BDNF might play an important role by regulating the formation and/or activation of potassium channels at the early development stage. There was a significant correlation between the supplementation of BDNF and the expression of I_K(A). I_K(A) might be suppressed by BDNF and might serve a developmental function during the period when it is present.The early development stage (DIV 1-4) might be a critical period of potassium channels during which BDNF might be involved in regulating potassium channels and served to limit excitability in neural cells.