Optogenetic Activation of Non-Nociceptive Aβ Fibers Induces Neuropathic Pain-Like Sensory and Emotional Behaviors after Nerve Injury in Rats

Immunohistochemical characterization of ChR2-expressing DRG neurons. A, Immunolabeling of ChR2⁺ neurons (Venus; green) with either NF200, TrkC, CGRP, TrkA, TRPV1, or IB4 (red) in the L4 DRG contralateral and ipsilateral to PNI on day 14. B, Percentage of colocalization of each marker in ChR2⁺ DRG neurons [n = 4 rats (two to three slices per rat)]. Size-frequency histogram (inset) illustrating the distribution of the cross-sectional areas of ChR2⁺ NF200⁺ DRG neurons in the L4 DRG ipsilateral (371 cells) and contralateral (316 cells) to PNI on day 14. Values represent mean ± SEM. Scale bar: 100 μm.

Light illumination activates primary afferents with conduction velocity in a range of Aβ fibers. A, Schematic diagram of whole-cell patch-clamp recording in SDH Lamina II neurons using sagittal spinal cord slices with the L4 dorsal root of W-TChR2V4 rats. A suction electrode was used to electrically stimulate the dorsal root. A monopolar stimulating electrode for calculating conduction velocity was placed at the proximal point. Optical fiber was placed between two electrodes. B–D, Representative averaged traces of EPSCs evoked by light (left) and electrical (right) stimulation recorded from Lamina II SDH neurons. The averaged traces of EPSCs recorded from the same neuron evoked by suction electrode (distal, black line) and monopolar electrode placed in proximal dorsal root (proximal, red line). E, Conduction velocity (m/s) of primary afferent fibers responded and non-responded by light illumination. Calculated conduction velocity of fibers with monosynaptic input to a recorded neuron was indicated by a single circle [red, responded neurons of naive rats, n = 17; blue, non-responded neurons of naive rats, n = 24; orange, responded neurons of PNI rats (day 14), n = 5]. F, Effects of light illumination on monosynaptic Aβ fiber-mediated EPSCs evoked by electrical stimulation (monopolar electrode) of the dorsal root in Lamina II neurons. The dorsal root was electrically stimulated before (Pre) and at 20, 70, 120, and 170 ms after light illumination for 100 ms. Similar results were seen in each of two experiments. Blue regions, light illumination to the dorsal roots. Values represent mean ± SEM.

Induction of c-Fos and pERK in SDH neurons by light after PNI. A, Number of c-Fos⁺ neurons in the superficial L4 SDH of W-TChR2V4 rats with or without light stimulation at day 14 post-PNI [n = 4 rats (two to four slices per rat); *p < 0.05, one-way ANOVA with post hoc Tukey’s test]. B, C, Immunofluorescence (B) and number (C) of pERK⁺ neurons in the superficial L4 SDH of W-TChR2V4 rats with or without light stimulation at day 14 post-PNI. WM, white matter; GM, gray matter [n = 4–5 rats (three slices per rat); **p < 0.01, *p < 0.05, one-way ANOVA with post hoc Tukey’s test]. Values represent mean ± SEM. Scale bar: 100 µm.

Light illumination excites Lamina I SDH neurons after PNI. A, Schematic diagram of whole-cell patch-clamp recording in Lamina I neurons using sagittal spinal cord slices with the L4 dorsal root taken from W-TChR2V4 rats with or without PNI (day 14). B, Representative traces of EPSCs (voltage-clamp mode; left) and action potentials (current-clamp mode; left) in Lamina I neurons at the L4 in spinal slices taken from W-TChR2V4 rats (naive and PNI). Blue regions, light illumination to the dorsal roots. C, Amplitude of light-evoked EPSCs in Lamina I neurons (naive and PNI; n = 10 cells; **p < 0.01, unpaired t test with Welch’s correction). D, E, Confocal images (D) and firing pattern (E) of recorded Lamina I neurons in W-TChR2V4 rats with or without PNI. Values represent mean ± SEM. Scale bar: 100 µm.

Activation of PBN and CeA by illuminating the hindpaw after PNI. A, B, Immunofluorescence (left) and number (right) of c-Fos⁺ neurons in the PBN (A) and CeA (B) of W-TChR2V4 rats [naive and PNI (day 14)] with or without light stimulation [n = 3–4 rats (three slices per rat); **p < 0.01, *p < 0.05, one-way ANOVA with post hoc Tukey’s test). Values represent mean ± SEM. Scale bar: 200 µm.