Differential expression of slow and fast-repriming tetrodotoxin-sensitive sodium currents in dorsal root ganglion neurons

Figure 1. Recording of sodium current repriming in small DRG neurons of rats. (A) The voltage protocol used for recording of sodium current repriming. (B) Representative traces showing the repriming of sodium currents recorded in a small DRG neuron dissociated from adult Sprague–Dawley rats. (C) Comparison of time-dependent repriming of sodium current recorded in A. Note there was a selective increase in fast-activating and fast-inactivating currents after 3 ms repriming (the red trace). (D) Separation of TTX-S current from TTX-R current by subtracting maximally recovered TTX-R currents from total sodium currents.

Figure 2. Differential repriming of TTX-S currents in small DRG neurons of rats. (A–D) Representative traces showing the repriming of TTX-S sodium currents recorded from four small DRG neurons using the protocol presented in Figure 1. (E) The amplitude of each current was normalized to the current recorded at 258 ms in each neuron as shown in A–D. The current in A (red) and B (black) were best fitted by a single exponential function, while the current in C (blue) and D (green) were best fitted by a double exponential function. (F) The distribution of time constant for single-repriming TTX-S currents. (G) The distribution of combined single-repriming time constants and the faster time constants in neurons that had a slower time constant larger than 70 ms. The distribution pattern of fast and slow time constant domains is similar between (F,G). The two time constants fell in the 10–15 ms range were 10.1 and 11.2 ms. (H) DRG neurons were divided into three different groups: slow-repriming (SR), fast-repriming (FR), and double-repriming (DR) neurons based on their components of repriming TTX-S current, respectively.

Figure 3. Recording of sodium current repriming in small DRG neurons of mice. (A) Representative traces showing the repriming of sodium currents recorded in a small DRG neuron dissociated from adult C57BL/6 mice. The voltage protocol used for recording of sodium current repriming is the same as shown in Figure 1. (B) Comparison of time-dependent repriming of sodium current recorded in A. Note there was a selective increase in fast-activating and fast-inactivating currents after 3 ms repriming (the red trace). (C) Separation of TTX-S current from TTX-R current by subtracting maximally recovered TTX-R currents from total sodium currents. The box plots of the distribution of the fast (D) and the slow (E) time constants. (F) DRG neurons were divided into three different groups: slow-repriming (SR), fast-repriming (FR), and double-repriming (DR) neurons based on their components of repriming TTX-S currents, respectively.

Figure 4. Effects of ProTx-II on repriming of sodium currents in small DRG neurons. ProTx-II was pre-treated in culture plates for 45 min and kept in a recording chamber for 10–90 min before the repriming of sodium currents was recorded in small DRG neurons. Representative traces of sodium currents repriming in rat neurons were shown in control (A) and ProTx-II (B) groups. ProTx-II did not inhibit TTX-R currents in either rat or mouse neurons (see Results). However, it significantly inhibited SR currents in SR rat neurons (C) and the SR currents in DR mouse neurons (E). In addition, ProTx-II did not significantly decrease both SR and FR currents in DR rat neurons (D) or in DR mouse neurons (E). Data were presented as mean ± standard errors of mean. **, p < 0.01, Student t-test.

Figure 5. Recording of slow ramp currents in small DRG neurons of mice. Ramp sodium currents were activated by a slow ramp voltage protocol (−90 to +10 mV in 800 ms) in small DRG neurons dissociated from adult C57BL/6 mice. Five voltage ramps were applied to each neuron, and the five ramp currents were averaged. Representative traces of ramp currents were recorded from an FR neuron (A), a DR neuron with higher FR than SR currents (B), and a DR neuron with higher SR than FR currents (C). (D) The slow ramp currents were significantly different among the three groups of neurons (p < 0.01, one-way ANOVA). Data were presented as mean ± standard errors of mean. *, p < 0.01 (vs. FR neuron), post-hoc Tukey test; #, p < 0.05 (vs. DR neuron with higher FR than SR currents), post-hoc Tukey test.

Figure 6. Recording of fast and slow-repriming TTX-S currents in small DRG neurons of mice. Representative traces showing the dissection of repriming TTX-S currents recorded in a IB4+ (A) and a IB4− (B) small DRG neuron dissociated from adult C57BL/6 mice. The voltage protocol used for recording of sodium current repriming is the same as shown in Figure 1. Current density of total TTX-S (C), TTX-R (D), SR TTX-S currents (E), and FR TTX-S currents (F) in IB4+ (green bars) and IB4− (gray bars) neurons were compared among control (Con), paclitaxel (Pac), and high glucose (HG) groups. Paclitaxel (1 μM) or high glucose (30 mM compared to 10 mM in control) were pre-treated overnight in the culture medium. Data were presented as mean ± standard errors of mean. **, p < 0.01 (vs. IB4− neurons), student t test; #, p < 0.05 (vs. control), one-way ANOVA and post-hoc Tukey test.

Figure 7. Recording of membrane excitability in simulated small DRG neurons of mice. A cell model of a small mouse DRG neuron was constructed using NEURON simulation software. Mouse Nav1.6 and/or Nav1.7 channels were inserted into the model cell, along with TTX-R Nav1.8, transient and delayed rectifier potassium channels. Action potentials were triggered by a series of square (D) or ramp (H) current injection in model neurons with Nav1.7 but not Nav1.6 (A,E), half amount of Nav1.7 and Nav1.6 (B,F), and Nav1.6 but not Nav1.7 (C,G), respectively. The amount of Nav1.6 in Nav1.6 neurons was the same as the amount of Nav1.7 in Nav1.7 neurons.