Article Figures & Data
Figures
- Figure 1.
Distribution and functionality of Arch-EGFP pumps in the peripheral sensory pathways of Nav1.8-Arch+ mice. Confocal micrographs showing the fluorescence of Arch-EGFP (green), CGRP immunostaining (blue), P2X3 labeling (red), and the merge. A, Arch-EGFP colocalizes with either P2X3 or CGRP in dorsal root ganglia neurons, validating its selective expression in nociceptors. B, Arch-EGFP fluorescence overlaps with CGRP and P2X3 labeling in laminae I and II of the dorsal horn of spinal cord. C, Arch-EGFP colocalizes with CGRP and P2X3 in free nerve endings in the lower and upper dermis of glabrous skin. Insets are at lower magnification (20×). Scale bars, 50 μm. D, Representative yellow (589 nm) light-induced outward photocurrent and membrane hyperpolarization in a DRG neuron. Magnification shows an inward deflection in the voltage-clamp trace (arrow), illustrating a proton-mediated ASIC-like current. This inward current did not translate into a depolarization in the current-clamp trace. E, Arch-mediated blockade of electrically induced action potentials (10 ms, 0.4 nA current injection) in DRG neurons. Optical stimulation was continuous (intensity, 0.83 mW/mm2). Vh = −60 mV in voltage-clamp configuration; and the resting membrane potential was −62.23 ± 2.92 mV in current-clamp configuration (n = 8–9 cells).
- Figure 2.
Analgesic effect of acute yellow light stimulation under normal and inflammatory conditions in Nav1.8-Arch+ mice, and under prolonged blue light stimulation in Nav1.8-ChR2+-Arch+ mice. A, Mechanical sensitivity of Nav1.8-Arch+ mice under normal conditions, using different yellow light (589 nm) intensities (n = 8–17 mice/condition; paired Student’s t test, No light vs 0.25 mW/mm2 measurements, p = 0.0707a; Table 1). B, C, Optical stimulation reduced mechanical allodynia under capsaicin- and zymosan- induced inflammation. Yellow light (0.25 mW/mm2) was applied to the hindpaw of Nav1.8-Arch+ mice simultaneously with the mechanical stimulus at each time point postinjection (n = 6–13 mice/condition). D, E, Simultaneous application of yellow and blue light to the hindpaw of Nav1.8-ChR2+-Arch+ mice prevented the development of blue light-induced mechanical (D) and thermal (E) hypersensitivity. Post-treatment measurements were taken without any optical stimulation (n = 6–13 mice/condition). Symbols represent the mean ± SEM of hindpaw withdrawal thresholds and latencies before (Pre) and after (Post) treatment. Data were analyzed using repeated-measures two-way ANOVA followed by Sidak post hoc test. Significance (*) is reported between Ipsi-Light and Ipsi-Control (B, C) and between Ipsi-Blue and Ipsi-Blue + Yellow (D, E). *p < 0.05, **p < 0.01, ****p < 0.0001. F, Representative yellow (589 nm) and blue (473 nm) light-induced photocurrents in DRG neurons from Nav1.8-ChR2+-Arch+ mice. Arch-mediated membrane hyperpolarization was sufficient to block ChR2-induced action potentials (50 ms pulses) in the same neuron (n = 4–5 cells). Light intensity was 0.78 mW/mm2 (blue) and 0.83 mW/mm2 (yellow).
- Figure 3.
Analgesic effect of prolonged yellow light stimulation under inflammatory conditions in Nav1.8-Arch+ mice. Orange bars represent the hour-long yellow light stimulations. A, Prolonged silencing of Nav1.8+ fibers led to poststimulation analgesia, reducing zymosan-mediated mechanical allodynia. Optical stimulation was performed right after, 2 h after, and 4 h after zymosan injection (n = 7–11 mice/condition). B, Prolonged yellow light stimulation reduced CFA-induced thermal hypersensitivity when applied 24 h after CFA injection but not right after (n = 5–7 mice/condition). C, Hour-long stimulation does not affect thermal sensitivity in naive Nav1.8-Arch+ mice (n = 6 mice). D, Representative traces showing consistent and reproducible Arch-mediated hyperpolarizations in cultured DRG neurons at 1 and 10 min during a sustained 3 s ON, 1 s OFF stimulation protocol (n = 3 cells). Symbols represent the mean ± SEM of hindpaw withdrawal thresholds and latencies before (Pre) and after (Post) treatment. Significance between Ipsi-Light and Ipsi-Control measured using repeated-measures two-way ANOVA followed by Sidak post hoc test: *p < 0.05, ***p < 0.001, ****p < 0.0001.
- Figure 4.
Analgesic effect of prolonged optical stimulation under neuropathic conditions in Nav1.8-Arch+ mice. Orange bars represent the hour-long optical stimulations. Prolonged yellow light application on the neuropathic hindpaw decreased SNI-induced mechanical allodynia, showing partial analgesia lasting up to 24 h poststimulation, at 3, 4, and 6 weeks after SNI surgery (n = 7–14 mice for SNI; n = 3–7 mice for sham). Symbols represent the mean ± SEM of hindpaw withdrawal thresholds before (Pre) and after (Post) treatment. Significance between SNI-Light and SNI-Iso, measured using repeated-measures two-way ANOVA followed by Sidak post hoc test: *p < 0.05. Significance between each Post time point and Pre in the SNI-Light group, measured using repeated-measures one-way ANOVA followed by Dunnett’s post hoc test: #p < 0.05, ##p < 0.01.
Tables
Data Test p a Figure 2A Force (g) No light vs 0.25 mW/mm2 Paired Student’s t test 0.0707 Data Source of variation Light Time Interaction Matching dfn, dfd F p dfn, dfd F p dfn, dfd F p dfn, dfd F p b Figure 2B Force (g) 1, 10 9.206 0.0126 2, 20 49.6 <0.0001 2, 20 4.775 0.0202 10, 20 2.371 0.0481 c Figure 2C Force (g) 1, 23 12.63 0.0017 5, 115 44.5 <0.0001 5, 115 7.556 <0.0001 23, 115 2.298 0.0021 d Figure 2D Force (g) 1, 16 8.039 0.0119 3, 48 7.704 0.0003 3, 48 3.194 0.0317 16, 48 2.353 0.0115 e Figure 2E Latency (s) 1, 17 6.192 0.0235 3, 51 6.753 0.0006 3, 51 2.111 0.1103 17, 51 1.418 0.1672 f Figure 3A, left Force (g) 1, 12 11.97 0.0047 5, 60 26.74 <0.0001 5, 60 2.285 0.0574 12, 60 2.566 0.0082 g Figure 3A, middle Force (g) 1, 13 5.769 0.032 5, 65 32.44 <0.0001 5, 65 4.424 0.0016 13, 65 1.016 0.4471 h Figure 3A, right Force (g) 1, 16 0.2823 0.6025 5, 80 68.17 <0.0001 5, 80 4.293 0.0016 16, 80 1.734 0.0567 i Figure 3B, right Latency (s) 1, 12 0.5795 0.4612 8, 96 63.15 <0.0001 8, 96 2.216 0.0327 12, 96 1.634 0.0947 j Figure 4, left 50% threshold (g) 1, 19 8.91 0.0076 5, 95 1.826 0.1151 5, 95 1.861 0.1085 19, 95 3.687 <0.0001 dfn, df numerator; dfd, df denominator.
Data Test p k Figure 2B At 30 min: Ipsi-Light vs Ipsi-Control Sidak 0.0018 At 60 min: Ipsi-Light vs Ipsi-Control Sidak 0.0424 l Figure 2C At 2 h: Ipsi-Light vs Ipsi-Control Sidak <0.0001 At 4 h: Ipsi-Light vs Ipsi-Control Sidak 0.0104 At 6 h: Ipsi-Light vs Ipsi-Control Sidak 0.0036 m Figure 2D At 1 h: Ipsi-Blue vs Ipsi-Blue + Yellow Sidak 0.0034 At 3 h: Ipsi-Blue vs Ipsi-Blue + Yellow Sidak 0.0215 n Figure 2E At 1 h: Ipsi-Blue vs Ipsi-Blue + Yellow Sidak 0.0106 o Figure 3A, left At 2 h: Ipsi-Light vs Ipsi-Control Sidak 0.0141 At 4 h: Ipsi-Light vs Ipsi-Control Sidak 0.0128 p Figure 3A, middle At 4 h: Ipsi-Light vs Ipsi-Control Sidak <0.0001 q Figure 3A, right At 6 h: Ipsi-Light vs Ipsi-Control Sidak 0.0007 r Figure 3B, right At 24 (+1) h: Ipsi-Light vs Ipsi-Control Sidak 0.0331 s Figure 4, left At 1 h: SNI-Iso vs SNI-Light Sidak 0.0221 At 2 h: SNI-Iso vs SNI-Light Sidak 0.0375 Data Time Matching dfn, dfd F p dfn, dfd F p t Figure 4, left 50% threshold (g) 3.54, 46.02 3.783 0.0123 13, 65 3.687 0.0002 u Figure 4, middle 50% threshold (g) 2.142, 27.85 3.304 0.0486 13, 52 3.288 0.0011 dfn, df numerator; dfd, df denominator.
Data Test p v Figure 4, left SNI-Light: Pre vs 1 h Post Dunnett’s 0.0093 SNI-Light: Pre vs 2 h Post Dunnett’s 0.0321 SNI-Light: Pre vs 2.5 h Post Dunnett’s 0.0181 SNI-Light: Pre vs 3 h Post Dunnett’s 0.0483 w Figure 4, middle SNI-Light: Pre vs 1 h Post Dunnett’s 0.0437
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