Fig. 1.
Effect of MEX and growth factors on the expression of macroscopic KCa current in LMNs in vitro. A, Representative traces of outward currents evoked in E8 LMNs and after 72 hr in the presence of MEX (50 μg/ml). Outward currents were evoked in control and Ca2+-free salines (left traces) by 25 msec depolarizing pulses to +30 mV from a holding potential of −40 mV (shown at bottomleft). Net macroscopic KCa was obtained after digital subtraction of raw traces (right trace). B, Summary of the effects of MEX and trophic factors on the functional expression of KCa in cultured LMNs. Motoneurons were dissociated on E8 and maintained in culture for 72 hr in the presence of 50 μg/ml MEX or 10 ng/ml of the trophic factors BDNF, NT-3, NT-4, and GDNF. All of these factors are capable of promoting LMN survival in vitro. E8 represents control neurons examined 3 hr after dissociation. Note robust stimulation of KCa by MEX, GDNF, and BDNF. C, Time course of GDNF stimulation of KCa channel expression. Maximal expression of KCa channel occurred after 24 hr exposure to GDNF (10 ng/ml). The effect of GDNF on KCa channel expression is independent of electrical activity because TTX did not prevent the functional expression of KCa channels. Note that 24 hr incubation with NT-4 fails to increase KCa channel expression. In these experiments, LMNs were dissociated on E8 and maintained in culture for 3, 12, or 24 hr in the presence of GDNF as indicated. Control represents E8 LMNs examined 3 hr after dissociation. In this and subsequent figures, error bars indicate SEM, the number of cells recorded is given above each bar, andasterisks denote p < 0.05 from control as determined by one-way ANOVA followed by Tukey's honest significant difference test for unequal n.