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. 2021 Dec 27;145(10):3637–3653. doi: 10.1093/brain/awab482

Figure 5.

Figure 5

Rodent C-LTMRs that lack Nav1.7 show smaller sodium currents and hypo-excitability. (A) THCreERT2 (control) or THCreERT2Nav1.7flox/flox (THCreERT2Nav1.7-KO) mice received and intrathecal injection of AAV.Flex.eGFP to target C-LTMRs prior to tamoxifen administration. Subsequent tamoxifen injection initiated simultaneous eGFP expression and Nav1.7 ablation. (B) Virally targeted C-LTMRs were cultured, eGFP expression used to identify the population and voltage-clamp used to analyse sodium currents in both control and THCreERT2Nav1.7-KO mice. (C) Example traces of recorded total sodium currents in eGFP-positive C-LTMRs from THCreERT2 and THCreERT2Nav1.7-KO mice. (D) C-LTMRs lacking Nav1.7 had a significantly reduced sodium current density compared with control C-LTMRs (THCreERT2n = 12 cells, THCreERT2Nav1.7-KO n = 11 cells. Mann–Whitney U-test, U = 28, P < 0.018, *). (E) Example sodium current traces from THCreERT2 and THCreERT2Nav1.7-KO C-LTMRs to determine the sodium current-voltage (I/V) relationship. (F) Quantification of the I/V relationship displayed as I/V curves. C-LTMRs from THCreERT2Nav1.7-KO mice showed a significantly smaller I/V curve compared with C-LTMRs from THCreERT2 mice (THCreERT2: n = 12 cells, THCreERT2Nav1.7-KO: n = 11 cells. Two-way ANOVA, F(1,432) = 24.05, P < 0.0001, ***, with Sidak–Holm post hoc test, −25 pA, t(432) = 3.342, P = 0.021, *). (G) Single fibre recordings from the mouse skin-nerve (saphenous) preparation comparing recordings from Nav1.7-WT (grey) and THCreERT2:Nav1.7-KO (blue) mice. C-LTMR conduction velocities were normal and comparable between both WT and C-LTMRs lacking Nav1.7. [WT: n = 14 units, KO: n = 12 units, two-tailed Student’s unpaired t-test, t(24) = 0.103, P > 0.91, n.s.] (H) The mechanical thresholds of THCreERT2:Nav1.7-KO C-LTMRs were significantly higher than Nav1.7-WT control C-LTMRs [WT: n = 14 units, KO: n = 12 units, two-tailed Student’s unpaired t-test, t(24) = 4.070, P = 0.0004,***] (I) Example trace of evoked action potentials in response to a supra-threshold mechanical stimulus applied to a single Nav1.7-WT and THCreERT2:Nav1.7-KO C-LTMR receptive field. (J) The increasing force stimulus-response function showing that C-LTMRs lacking Nav1.7 were significantly hypo-excitable to supra-threshold stimuli compared with control C-LTMRs [WT: n = 14 units, KO: n = 12 units, two-way ANOVA, F(1,95) = 11.87, P = 0.0008, ***]. (K) The increasing velocity stimulus-response function of Nav1.7-WT and THCreERT2:Nav1.7-KO C-LTMRs. C-LTMRs lacking Nav1.7 were hypo-excitable with a significantly reduced firing frequency to dynamic stimuli [WT: n = 14 units, KO: n = 12 units, two-way ANOVA, F(1,96) = 6.212, P = 0.014, *]. (L) The 31–14°C cooling stimulus-response of Nav1.7-WT and THCreERT2:Nav1.7-KO C-LTMRs. C-LTMRs lacking Nav1.7 were hypo-excitable to cooling stimuli. The linear regression fitted slopes are significantly different between WT and KO mice [WT: n = 10 units, KO: n = 8 units, linear regression F-test, F(1,306) = 9.32, P = 0.0024, **]. (M) The 14–42°C warming stimulus-response of Nav1.7-WT and THCreERT2:Nav1.7-KO C-LTMRs were similar. The non-linear regression Gaussian fitted curves are not significantly different between WT and KO mice, both groups and share a common curve (green) [WT: n = 10 units, KO: n = 8 units, non-linear regression F-test, F(3,504) = 0.763, P = 0.515, n.s.]. (N) The 42–14°C cooling stimulus-response of Nav1.7-WT and THCreERT2:Nav1.7-KO C-LTMRs. C-LTMRs lacking Nav1.7 are hypo-excitable to cooling stimuli. The non-linear regression Gaussian fitted curves are significantly different between WT and KO mice [WT: n = 10 units, KO: n = 8 units, non-linear regression F-test, F(3,508) = 5.106, P = 0.0017, **]. All data represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.