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. 2022 Oct 24;11:e79917. doi: 10.7554/eLife.79917

Figure 2. Transcriptomic and functional expression of sodium channels in proprioceptors.

(A, B) Representative confocal images of cryoprotected adult dorsal root ganglia (DRG) sections (25 μm) with quantifications indicating the percentage of NaV+ and NaV- neurons in TdTomato+ proprioceptors. Sections were hybridized using RNAscope with probes targeting NaV1.6 or NaV1.7 (Scn8a and Scn9a, respectively, magenta) and stained with anti-DsRed (yellow). (A) Snc8a, n = 298, (B) Scn9a, n = 166. Scale bar set to 50 μm. White arrowheads indicate NaV+ neurons. (C–, D) Frequency distribution plots of integrated fluorescence density of NaV1.6 and NaV1.7 mRNA in TdTomato+ proprioceptors, respectively. (E) The average integrated density of Scn1a, Scn8a, and Scn9a RNAscope probe signal. Dots represent individual cells. Statistical significance was determined using a Kruskal–Wallis test with Dunn’s post-hoc comparisons. (F) Top: experimental workflow of serial pharmacological blockade of NaV channels expressed in proprioceptors. We first elicited a whole-cell sodium current in the absence of drug. We next bath applied 500 nM of ICA 121431 to block current carried by NaV1.1. Subsequently, we bath applied 9-anhydroustetrodoxin (AH-TTX) (300 nM) to block NaV1.6-mediated current, and PF-05089771 (25 nM) to block the NaV1.7-mediated current. Finally, tetrodotoxin (TTX) (300 nM) was used to block residual current and to confirm there was no contribution of TTX-resistant NaVs in proprioceptors. Bottom: representative current traces following application of NaV-selective inhibitors. All drugs were applied for 1 min. (G) Quantification of the average percentage of the whole-cell sodium current that was sensitive to the individual drugs used (n = 8). n = cells. ****p<0.0001.

Figure 2.

Figure 2—figure supplement 1. 0.1% DMSO vehicle does not change INa in proprioceptors.

Figure 2—figure supplement 1.

(A) Representative current trace before (black trace) and after (red trace) application of 0.1% DMSO to TdTomato+ proprioceptors during in vitro electrophysiological experiments. Quantification of current density (B) and current decay kinetics (C) before (normal external solution, black circles) and after DMSO application (red circles). A paired t-test was used to determine statistical significance (B), p=0.0855 and (C), p=0.3125, n = 5 cells.