Figure 5

SPC-01 generates functional neurons. (a) Preincubation with Cd²⁺ (100 μM) together with Ni²⁺ (50 μM), for 5 minutes significantly reduced the [Ca²⁺]_i responses induced by K⁺ in all cells tested by 93% ± 9.2% (n = 5), indicating the involvement of voltage-activated Ca²⁺ channels in depolarization-induced Ca²⁺ entry. (b) The specific L-type Ca²⁺ channel blocker nicardipine (1 μM) reduced the [Ca²⁺]_i responses by 28% ± 7% (n = 5; P = 0.002). The trace in (c) shows the [Ca²⁺]_i responses observed in a selected SPC-01-derived neuron induced by 50 mM K⁺, preincubated for 2 minutes with Ω -GVIA (800 nM) and then stimulated with K⁺. After a washout of 10 minutes, the same cells were subjected to K⁺ to observe recovery. Similarly, the effect of the P/Q-Ca²⁺ channel blocker, Ω -Aga-IVA, 300 nM was tested before and during stimulation with high K⁺(d). After washout of the toxin, the [Ca²⁺]_i response was shown to be reversible. The histogram (e) summarizes the results presented in (c) and (d). The resting [Ca²⁺]_i level in these cells is indicated as basal. The results are expressed as mean ± SEM; n = 4 (Ω-GVIA) and n = 5 (Ω-Aga-IVA). Asterisks indicate the statistical significance (P > 0.05) versus control K⁺ stimulus.