FIGURE 8.

Analyses of the effects of linopirdine under reduced extracellular Ca²⁺ conditions and assessment of SGNs apoptosis. A, upper panel shows SGNs in control culture with BDNF/NT3 growth factors. A denotes apical and B denotes basal SGNs. Consistent with the data in Fig. 7, the number of apoptotic SGNs increased when cultures were treated with 10 μm linopirdine (lino, 3rd and 4th rows) for 72 h. TUJ1, neuronal marker; Apop (apoptosis = TUNEL-positive; DAPI = nuclei stain). B, quantitative assessment of SGNs apoptosis histograms, comparing the relative number of TUNEL-positive SGNs under different experimental conditions. 10 μm lino = linopirdine, comparison of base-line data for apical and basal SGNs with linopirdine-treated conditions. Low bath Ca²⁺ concentration of ∼300 μm was achieved by adding 2 mm EGTA to the culture media. We reduced Ca²⁺ currents using a mixture of Ca²⁺ channel (Ca_v) blockers; 1 μm mibefradil to block T-type, rSNX-482 for R-type, 1 μm ω-conotoxin GVIA for N-type, 1 μm ω-agatoxin IVA for P/Q-type, and 10 μm nimodipine for L-type Ca²⁺ currents. Reduction of Ca²⁺ influx by decreasing bath Ca²⁺ or blocking Ca_v significantly reduced the effects of linopirdine. **, p < 0.01.