Figure 4. Sorted immature neurons survive in vitro and reach functional maturity.

Immature neurons were sorted from P1 (FSC^low SSC^low) population and plated in various culture conditions to find supportive factors that can increase their survival and functional maturity. (A): Comparison of the survival effects of different factors on the sorted immature neurons 6 days after plating (mean±SEM; n = 4 independent experiments, * P = 0.0205, one-way ANOVA on log transformed data). (B): Representative micrographs from different conditions on day 6 after plating. BMP4 treated condition showed the greatest number of process bearing β III-tubulin IR cells/field of view. (C): Depolarizing current injection-induced repetitive action potentials from a RMP of differentiating neurons cultured in astrocyte conditioned medium plus BMP-4 (20 ng/ml). (D): Effect of various culture conditions on action potential (AP) occurrence (n≥5). APs were evoked with depolarizing current injections, and cells were divided into three groups; cells with APs from a RMP, with APs only from a hyperpolarized holding potential (HP) of −60 mV and without APs. (E): I–V relationship of voltage-gated, tetrodotoxin-sensitive Na⁺ channel current of differentiating neurons under various culture conditions (n≥4). Series resistance (Rs) compensation was not applied. A voltage error of ∼10 mV was estimated for the maximum Na⁺ channel current amplitude given Rs of 2–5 MΩ. Scale bar = 50 µm. Abbreviations: RMP = resting membrane potential, BMP-4 = Bone morphogenetic protein-4.