Different NaV subtypes produce equivalent excitability at different days in vitro (DIV).
(A) Representative responses of small DRG neurons to current injection at rheobase and 3x rheobase when tested on DIV0 (blue) or DIV4-7 (red) before (dark) and after (pale) bath application of 100 nM TTX. At DIV0, TTX did not alter spiking pattern (ξ2=0.25, p=0.617, McNemar test) or significantly reduce firing rate (F1,72=1.527, p=0.24, two-way repeated measure (RM) ANOVA; n=13). At DIV4-7, TTX significantly altered spiking pattern, converting all but one neuron to transient spiking (ξ2=20.05, p<0.0001), and it significantly reduced firing rate (F1,132=43.157, p<0.001, n=23). Only neurons with repetitive spiking at baseline are included in the firing rate plot. (B) At DIV0, TTX did not affect rheobase (Z24=1.129, p=0.265, Wilcoxon rank test) but did reduce spike height (T24=3.092, p=0.005, paired t-test). At DIV4-7, TTX increased rheobase (Z28=4.681, p<0.001, Wilcoxon rank test) and dramatically reduced spike height (T28=20.333, p<0.001, paired t-test). Notably, neurons at DIV0 and DIV4-7 did not differ in their baseline rheobase (U=316, p=0.425, Mann-Whitney test) or spike height (T52=0.322, p=0.749, t-test). (C) Neurons at DIV0 and DIV4-7 differed in their total capacitance (T52=6.728, p<0.001, t-test) and leak conductance density (U=216, p=0.011, Mann-Whitney test) but not in their resting membrane potential (T52=1.668, p=0.101, t-test). (D) Sample voltage clamp recordings with command voltage stepped from -85 mV to +15 mV in 5 mV increments, before and after TTX. Sodium current was not significantly reduced by TTX at DIV0 (F1,72=3.585, p=0.107, two-way RM ANOVA; n=7 neurons) but was completely abolished by TTX at DIV4-7 (F1,108=33.526, p<0.001; n=10 neurons). *, p<0.05; **, p<0.01; Student-Newman-Keuls post-hoc tests in A and D.