(A) Timeline and schematic for electrophysiological recordings in freely moving rats. Each trial of peripheral stimulation lasted until paw withdrawal or in cases of no withdrawal (non-noxious stimulus or NS) a total of 5 s. (B) Histology showing the location of ACC tetrode recordings. (C) Raster plots and peristimulus time histograms (PSTHs) before and after NS, LS and HS stimulations. Time zero denotes the onset of stimulus. Y-axis shows z-scored firing rates. To calculate the z-scored firing rate, we used the following equation: (FR – mean of FRb) / SD of FRb, where FR indicates firing rate and FRb indicates baseline firing rate prior to NS, LS or HS (see Materials and methods section). (D, E) A subset of neurons was identified among pain responsive neurons that showed higher firing rates after HS stimulation compared with LS (see Materials and methods for definition of pain responsive neurons). n = 14 out of a total of 88 neurons; for D, p<0.0001, paired Student’s t test. (F) Population-decoding analysis using a SVM classifier demonstrated decoding accuracy to distinguish between HS and NS (85%) or HS and LS (76%), n = 9. See Materials and methods. (G) Raster plots and PSTHs before and after NS, LS and HS stimulations in rats 10 days after CFA injections in the opposite paws. (H, I) A subset of neurons was found among pain responsive neurons that showed higher firing rates after HS stimulation compared with LS in the chronic pain condition. n = 10 out of a total of 93 neurons; for H, p<0.0001, paired Student’s t test. (J) Population-decoding analysis demonstrated decoding accuracy to distinguish between HS and NS (81%) or HS and LS (67%) in rats after chronic pain, n = 15. (K) A robust linear regression model was used to fit the peak z-scored firing rates and to calculate slope of the fit for all ACC neurons that demonstrated higher firing rates at HS compared with LS. Slope = 1.31 ± 0.09, R2 = 0.5937, n = 50 neurons. After chronic pain, neurons with higher firing rates at HS than LS showed a flatter tuning curve between LS and HS responses (see Materials and methods). Slope = 1.07 ± 0.07, R2 = 0.6904, n = 53 neurons. The two slope parameters are statistically different (p<0.05, unpaired Student’s t-test). (L) Decoding analysis showed that after chronic pain, there was a decrease in decoding accuracy to distinguish between HS and LS, n = 9 for pre-CFA, 15 for post-CFA; p<0.05, unpaired Student’s t test.