Fig. 6. Layer 5 (L5) activation in the S1 hindlimb cortex (S1HL) is antinociceptive.

a ChR2-EYFP-expression (green) in S1HL of a L5-ChR2 mouse showing fluorescence in L5 neurons. S1HL cortex (right panel) with L5 neurons depth-registered relative to S1HL layer borders (dashed lines, estimated based on soma sizes and densities using DAPI signals, blue). A representative example from n = 19 mice. b, c Representative S1HL cortex silicon-probe recording from an L5-ChR2 mouse demonstrating optogenetic control of the L5 pathway. b L5 units were identified based on their short-latency, low-jitter response to 10 ms light pulses (1201 mW/mm²). Each marker shows unit depth vs. mean ± SD latency to the first evoked spike (n = 32 out of 203 units recorded at a depth of L5, data from n = 2 mice). c Example raster plot of one unit from b in response to 5 s laser stimulation (1201 mW/mm²). d Optogenetically-evoked pain-like behaviors (i.e., paw shaking and withdrawal) were absent in the case of L5 stimulation (red, n = 6). L6-CT (blue, n = 11, replotted from Fig. 1f). Behavioral responses were considered in the 5 s optogenetic stimulation period, paw lifting (solid line), and limb shaking (dashed line). See also Supplementary video 2. e Within-animal comparison of paw withdrawal probabilities in response to graded von Frey stimulation of the left hindpaw at baseline (black, laser off) and during laser stimulation (red, laser on, 5 s continuous pulse) in the contralateral S1HL of L5-ChR2 mice (n = 6, p < 0.001). L5-EGFP control animals in Supplementary Fig. 4a. f Same as in (e) but after animals were injected with Complete Freund’s adjuvant (CFA) in the left hindpaw to induce paw inflammation (n = 12). Comparison between pre- and post-CFA in Supplementary Fig. 5. g Comparison of filament forces that evoked paw withdrawal in 60% of the stimulation trials as a function of L5 activation without (Naive; n = 6 mice, p = 0.0004) and with inflammation (CFA; n = 12 mice, p = 0.03). Higher withdrawal thresholds indicate decreased sensitivity. h Conditioned place preference (CPP) test. Aggregated positional tracking heatmaps from L5-EGFP (n = 6) and L5-ChR2 (n = 5) mice across all respective baseline (no stimulation) and conditioning (20 Hz laser stimulation of S1HL cortex) sessions. Percentages show relative time spent in the laser-paired chamber (blue outlines). Animals were injected with CFA (see Methods) one day before the first baseline session. i Population analysis of total time spent in the laser-paired chamber at baseline (Laser off, black/gray) and during stimulation (Laser on, red 20 Hz laser stimulation in S1HL cortex) of L5-EGFP (n = 6) and L5-ChR2 (n = 5) mice. j Average chamber preference indices (PI) for L5-ChR2 (n = 5) and L5-EGFP (n = 6) mice. A PI of 1 indicates a full preference, while a PI of −1 indicates full avoidance of the laser-paired chamber. PIs were significantly different between groups during laser stimulation, but not at baseline. * and # represent p < 0.05; 6e–j: Two-way repeated measures ANOVA with post hoc Bonferroni test. Exact F and p values are in Supplementary Table 1. Data were shown as mean ± SEM. Source data for b–j are provided as a Source Data file.