The Role Of Potassium Channels In The Differentiation And Development Of Hippocampal Neural Stem Cells

Neural stem cells (NSCs) are the cells which can be self-renewing and have the potential to form main kinds of neural cell types in central neural system (CNS). NSCs not only are essential to cell replacement therapy and transplantation in clinic, but also provide a unique and useful model for the researches of neurogenesis and epigenesis. Thus, the functional properties of NSCs and NSC-derived neurons, especially the developmental properties of ion channels which play an important role in neural electric signal system is becoming the hot spot. However, little is know about the changes of voltage-gated potassium channels in developmental neurogensis. Therefore, in this experiment, we investigated the electrophysiological properties of Kv channels in undifferentiated cNSCs and NSC-derived neurons at various time points after plating from 0 (day of plating) to >18d of newborn SD rat hippocampus in vitro, using patch-clamp techniques, in order to provide important clue for the clinic utilization of cNSCs and the elucidation of neurogenesis mechanism. ObjectiveStuding the developmental electrophysiological properties of Kv channels in undifferentiated cNSCs and NSC-derived neurons at various time points of newborn SD rat hippocampus in vitro, in order to provide important clue for the clinicutilization of cNSCs and the elucidation of NSCs neurogenesis mechanism.MethodsNSCs were isolated from the hippocampus of both sexes newborn rats with serum-free incubation containing basic fibroblast growth factor (bFGF), brain-derived neurotrophic factor (BDNF) or other growth factors and single-cell cloning technique in vitro. RA and fetal bovine serum (FBS) was added to induce NSC differentiation. Immunochemistry technique was employed to identify cNSCs and NSCs-derived progeny. The date was recorded using cell-attached, whole-cell and inside-out configuration of patch-clamp techniques. Results(1) The floating neural spheres from the hippocampus of newborn rats, from several cells to hundreds of round and healthy cells, were formed in serum free medium after 6~8 days in vitro and had the potential to self-renew and showed the expression of Nestin antigen. The Nestin+ cells were able to differentiate into neural cells which connected or crisscrossed with each other and expressed Tuj 1 and GFAP respectively under induce condition of RA and FBS.(2) Compared with the control group, EPO supplementation group displayed an average twofold to fourfold increase in Nestin positive cells over a wide range of plating densities during proliferation, however showed a striking decrease in Nestin positive cells during differentiation, following an early appearance of Tujl or GFAP positive cells. The percentage of Tujl positive cells increased significantly to 57% in EPO supplementation group versus 47% in control group, but decreased grossly to 28% in anti-EPO supplementation group. EPO increased the Tujl positive cells yield in a dose-depending manner at same plating densities.(3) Three types of outward Kv channels were observed in undifferentiated cNSCs by whole-cell patch-clamp techniques. Type I was characterized with a slowlyactivation with depolarization for about 20~30ms to a sustained outward current, hardly any inactivation was found throughout the recording process. External 5mM TEA blocked the steady state currents by as much as 56.8±5.9% (n=17) of the control at +40mV. This current was similar to the Idr (Delayed rectifier outward K+ currents) in other type cells. Type II was activated rapidly to a maximal level, then inactivated spontaneously, and was sensitive to 5mM 4-AP, identifying this current component as Ia (Transient A type K+ currents). The majority of undifferentiated cNSCs (42 vs 48 cells, i.e., 87.5 %) exclusively possessed Idr whereas lacking of the IA, other cNSCs (6 vs.48 cells, i.e., 12.5 %) possessed both IAand Idr. Typically, type III could be inhibited by Iberiotoxin (IbTX). The most fundamental response that can be measured was the fraction of channels that were open as a function of [Ca2+]i. The Po (open probability) of the channels increased as the membrane potential was depolarized or the [Ca2+]i level increased, which means the channel displayed sensitivity to both voltage and [Ca2+];. In excised inside-out recordings performed in symmetrical 140mmol/L K+ conditions, the conductance of single channel was approximately 184±17.6 pS (n=8) and the reverse potential was about 0 mV. Thus, on the basis of these findings, this channel was identified as BKca (Large-conductance Ca2+- activated potassium channels).(4) The extracellular application of EPO enhanced the currents density of BKca channels greatly in cNSCs under whole-cell configuration of patch-clamp techniques. The bath apphcation of 10U/L EPO induced an increase in Po (open probability) of BKca channel under cell-attached configuration of patch-clamp techniques. Meanwhile, application of 10U/L EPO via the patch pipette result in a more rapid and marked increase in Po of BKca channel. Moreover, the increase in BKca channel activity was sustained in the continued presence of EPO for up to 30 min. The calcium sensitivity of the BKca channels was also increased in both bath and pipetteapplication of EPO. Application of EPO under above situation induced an increase in Po of single channel through the increased open time constant without changing the current amplitude, closed time constant, unitary conductance and voltage dependence of BKca channel. The effect of EPO on the BKca channels could be inhibited and reversed by the application of MAPK inhibiter PD98059.(5) The passive membrane properties of undifferentiated cNSCs were measured using whole-cell patch-clamp techniques. The resting membrane potential (RMP) of the undifferentiated cNSCs varied range from -33.2 mV to -50.1mV and averaged -37.82±4.01 mV (n=59), which was more positive than mature hippocampus neurons. The input resistance (Rin) and r was 908±198.02 MQ. (n=35) and 7.16 ± 1.29 (n=32) respectively. The Cm was 15.84± 3.23pF (n=26).Compared with undifferentiated cNSCs, NSC-derived neurons had a more negative RMP, a higher Cm, a lower Rin and r at all comparable time points. Our data demonstrated that, RMPs were the most positive in the undifferentiated cNSCs and decreased progressively in 0-18d DIV. Statistically significant differences were observed in RMP between each group before 18d DIV. The developmental changes in Rin and r followed a similar pattern to that of RMP. The cells exhibited the highest Rin and t in DIV 0~6d and the values decreased progressively. Compared with undifferentiated cNSCs, dramatic increases in Cm were found after cells differentiation (DIV 0~6d). The cells showed a marked increase trend in Cm in 0-18d DIV. However, no significant differences were found in the RMP, Cm, Rin and r beyond 18d DIV.(6) The current amplitude and density of IDr increased in NSC-derived neurons DIV 0~6d whereas remained constant in DIV>6d. The activation process of IDR was also altered in DIV0~6d whereas remained constant in DIV>6d. The positive shift in steady-state activation curve of Idr revealed an increase of V1/2, however the slopefactors k remained unchanged. The inactivation properties of Idr were not altered before and after differentiation.(7) The current amplitude and density of U remained constant after differentiation. The negative shift in steady-state activation curve revealed a decrease in V1/2, but the slope factors k remained unchanged. The inactivation properties of IA were not altered before and after differentiation. Conclusion(1) The hippocampus of both new-born and adult SD rats contains NSCs which can be self-renewing and have the potential to differentiate into neurons and glia. EPO promoted proliferation of cNSCs originated from newborn rat hippocampus and increased the neuron differentiation in a dose-depending manner.(2) Three types of outward Kv channels, including Ia, Idr and BKca, are present in undifferentiated cNSCs. The BKca channels in cNSCs of newborn rat hippocampus had high Ca2+ sensitivity and voltage dependence. These channels coupled the membrane excitability to intracellular Ca2+ concentration and were believed to play an important role in intracellular signaling.(3) EPO enhanced the activities of BKca channels. It seems reasonable to assume that BKca channels play an important role in the regulation of EPO during cNSCs proliferation and differentiation process. The effects of EPO on BKca channels could be inhibited and reversed by PD98059, which indicated the participation of MAPK ERK1/ERK2 in the EPO action. It is also possible that phosphorylation and dephosphorylation modulation of BKca channels interact to play an important role in the functional profile and BKca channels is in a mixed phosphorylation state.(4) The passive membrane properties of undifferentiated cNSCs derive from newborn SD rat hippocampus shared similar and special properties with heterogeneous groups of NSCs. The variances between them are probably due to thedifference in species, region of CNS, methods of isolation, culture condition and the time cells spent in culture.(5) The development of passive membrane properties ceased at about DIV 18d and was thought to be independent of length of time in culture but dependent of the culture condition.(6) The electrophysiological characteristics of both Ia and Idr were altered in DIV 0~6d, suggesting the essential role of Kv channels in neurogenesis and early stage of differentiation / development process is very important for the functional mature.