Figure 6.
Effects of cholesterol enrichment on HCN channel function in nociceptor DRG neurons from naïve and SNI animals. (A) Representative protocol and membrane potential recordings using hyperpolarizing current injection (−90 pA) to activate HCN channels, leading to membrane depolarization. The ratio of the recovered potential (Vrec.) relative to the total hyperpolarization (Vpeak) reflects the percent of recovery in membrane potential mediated by HCN activation. (B) Time course of changes in the percent recovery (Vrec./Vpeak) after WSC treatment for naïve nociceptor DRG neurons (n = 5 patches), compared with untreated controls (n = 6 patches), mean ± SEM; red trace shows a single-exponential fit. (C) Time course of the change in the percent recovery for ipsilateral SNI DRG neurons (n = 4 patches with WSC treatment, mean ± SEM) using the same protocol as in panels A and B, compared with the no-treatment controls using ipsilateral neurons (n = 4 patches). (D–F) Time course of the change in the V1/2 and slope factor (Vs) derived from the G-V relationship (D, P = 2e−5, 4e−4, and 0.02 at 5, 10, and 15 min time points for V1/2), the current amplitude (E, P = 6e−4, 2e−4, and 7e−4 at 5, 10, and 15 min time points), and τ1 of channel activation (F, P = 6e−4, 1e−3, and 0.01 at 5, 10, and 15 min time points) after 0.5 mg/ml WSC application for naïve small DRG neurons; n.s., no statistical significance for the slope factor over time after WSC treatments, n = 7–10 patches. (G–I) Summary time course of the change in the V1/2 and slope factor (Vs) (G, P = 1e−4, 9e−4, and 4e−3 at 5, 10, and 15 min time points for V1/2, P = 0.004, 9e−5, and 0.014 at 5, 10, and 15 min time points for the slope), the current amplitude (H, P = 1e−4, 4e−5, and 1e−4 at 5, 10, and 15 min time points), and τ1 of channel activation (I, P = 0.03, 0.0017, and 0.026 at 5, 10, and 15 min time points) after 0.5 mg/ml WSC application for SNI small DRG neurons. Compared with naïve neurons (D), the slope factor in panel G showed significant changes in SNI neurons following WSC treatment. Data shown are mean ± SEM, n = 4–7 patches, *P < 0.05, **P < 0.01, two-sided paired t test. Refer to the image caption for details. The image contains multiple graphs depicting the effects of cholesterol enrichment via water-soluble cholesterol (W S C) on H C N channel function in dorsal root ganglion (D R G) neurons from naive and S N I (spared nerve injury) animals. Graph A shows representative membrane potential recordings using hyperpolarizing current injection; the ratio of the recovered potential (V rec) relative to the total hyperpolarization (V peak) reflects the percent recovery mediated by H C N activation, which decreases after W S C treatment. Graph B illustrates the time course of decreased percent recovery in naive D R G neurons after W S C treatment (tau equals 127 s) compared to untreated controls, while graph C shows a similar time course for S N I D R G neurons (tau equals 186 s). Graph D presents the time course of changes in the half-maximal activation voltage (V 1/2) and slope factor for naive DRG neurons, showing a significant hyperpolarizing shift in V 1/2. Graphs E and F show a decrease in normalized maximal current and an increase in the time constant (tau 1) of channel activation, respectively, for naive neurons following W S C application. Graph G summarizes the significant changes in V 1/2 and slope factor for S N I D R G neurons. Finally, the two graphs labeled H show the decrease in normalized maximal current and the increase in tau 1 activation for S N I D R G neurons over 15 minutes. Data are shown as mean plus or minus s.e.m., with statistical significance indicated by asterisks and n.s. for non-significant results. All values are approximate.

Effects of cholesterol enrichment on HCN channel function in nociceptor DRG neurons from naïve and SNI animals. (A) Representative protocol and membrane potential recordings using hyperpolarizing current injection (−90 pA) to activate HCN channels, leading to membrane depolarization. The ratio of the recovered potential (Vrec.) relative to the total hyperpolarization (Vpeak) reflects the percent of recovery in membrane potential mediated by HCN activation. (B) Time course of changes in the percent recovery (Vrec./Vpeak) after WSC treatment for naïve nociceptor DRG neurons (n = 5 patches), compared with untreated controls (n = 6 patches), mean ± SEM; red trace shows a single-exponential fit. (C) Time course of the change in the percent recovery for ipsilateral SNI DRG neurons (n = 4 patches with WSC treatment, mean ± SEM) using the same protocol as in panels A and B, compared with the no-treatment controls using ipsilateral neurons (n = 4 patches). (D–F) Time course of the change in the V1/2 and slope factor (Vs) derived from the G-V relationship (D, P = 2e−5, 4e−4, and 0.02 at 5, 10, and 15 min time points for V1/2), the current amplitude (E, P = 6e−4, 2e−4, and 7e−4 at 5, 10, and 15 min time points), and τ1 of channel activation (F, P = 6e−4, 1e−3, and 0.01 at 5, 10, and 15 min time points) after 0.5 mg/ml WSC application for naïve small DRG neurons; n.s., no statistical significance for the slope factor over time after WSC treatments, n = 7–10 patches. (G–I) Summary time course of the change in the V1/2 and slope factor (Vs) (G, P = 1e−4, 9e−4, and 4e−3 at 5, 10, and 15 min time points for V1/2, P = 0.004, 9e−5, and 0.014 at 5, 10, and 15 min time points for the slope), the current amplitude (H, P = 1e−4, 4e−5, and 1e−4 at 5, 10, and 15 min time points), and τ1 of channel activation (I, P = 0.03, 0.0017, and 0.026 at 5, 10, and 15 min time points) after 0.5 mg/ml WSC application for SNI small DRG neurons. Compared with naïve neurons (D), the slope factor in panel G showed significant changes in SNI neurons following WSC treatment. Data shown are mean ± SEM, n = 4–7 patches, *P < 0.05, **P < 0.01, two-sided paired t test.

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