Highly synchronized cortical circuit dynamics mediate spontaneous pain in mice

Cortical neural dynamics mediate information processing for the cerebral cortex, which is implicated in fundamental biological processes such as vision and olfaction, in addition to neurological and psychiatric diseases. Spontaneous pain is a key feature of human neuropathic pain. Whether spontaneous pain pushes the cortical network into an aberrant state and, if so, whether it can be brought back to a “normal” operating range to ameliorate pain are unknown. Using a clinically relevant mouse model of neuropathic pain with spontaneous pain–like behavior, we report that orofacial spontaneous pain activated a specific area within the primary somatosensory cortex (S1), displaying synchronized neural dynamics revealed by intravital two-photon calcium imaging. This synchronization was underpinned by local GABAergic interneuron hypoactivity. Pain-induced cortical synchronization could be attenuated by manipulating local S1 networks or clinically effective pain therapies. Specifically, both chemogenetic inhibition of pain-related c-Fos–expressing neurons and selective activation of GABAergic interneurons significantly attenuated S1 synchronization. Clinically effective pain therapies including carbamazepine and nerve root decompression could also dampen S1 synchronization. More important, restoring a “normal” range of neural dynamics through attenuation of pain-induced S1 synchronization alleviated pain-like behavior. These results suggest that spontaneous pain pushed the S1 regional network into a synchronized state, whereas reversal of this synchronization alleviated pain.

with brain removed to show impingement of trigeminal nerve root on the right side. Lower panel: top-down view with brain removed and trigeminal nerve root lifted to show the Surgiform remained in situ at day 28 post FLIT surgery. Scale bar represents 1 mm. (C to J) Behavioral testing for the FLIT model. Mice underwent Sham and FLIT surgery followed by behavioral testing at indicated time points. Equal numbers of male and female mice were used for each group (n = 18, Mean ± SEM.). (C) Facial grooming counts (in 10 minutes) at indicated time points. Two-way ANOVA indicates significant difference present between the groups, post-hoc Bonferroni test indicates P values of FLIT vs. sham, **P < 0.01; ***P < 0.001. (D) Representative pictures of mouse eyes between groups. Asymmetric eye grimacing was present only in the FLIT group. Scar bar represents 5 mm. (E) Body weight of mice were examined at indicated time points. Two-way ANOVA indicates significant difference present between the groups, posthoc Bonferroni test indicates the P value of FLIT vs. IoN-CCI *P < 0.05; **P < 0.01. (F) Top: A representative picture of wood chewing assay at baseline (day 0) and day 7 post surgery. Scale bar represents 1 inch. Bottom: Wood chewing assay to measure wood weight changes for animals that underwent Sham or FLIT surgery. FLIT mice exhibited significant less wood chewing activity compared with sham mice at days 7, 14, 21, and 28. Two-way ANOVA indicates significant difference between the groups, post-hoc Bonferroni test was carried out to determine the P value of FLIT vs. sham group, * P < 0.05; ** P < 0.01; *** P < 0.001. (G) Left panel: Representative pictures of incisors taken for the same mouse to demonstrate incisors overgrowth in the FLIT model. Scale bar represents 2 mm. Right panel: Quantification of incisors length. Two-way ANOVA indicates significant difference present between the groups, post-hoc Bonferroni test was carried out to determine the P value of FLIT vs. sham group, **P <  Representative H&E staining of masseter muscle shown (40X). Scale bar represents 100 µm.
(C) Masseter muscles weight was examined at 28 days after injection. Unpaired t test with twotailed P value indicates significant difference present between the two groups (***P < 0.001).   representing specific cortical regions. Orange represents S1J, red represents S1ULp, and dark grey represents S1BF. Ten slices centered bregma 1.2 mm at 60 m in thickness were used to assess c-Fos expression in S1J; fifteen slices centered bregma 0.5 mm at 60 m in thickness were used to assess c-Fos expression in S1ULp; and fifteen slices between bregma -0.8 mm and -1.8 mm were used to assess c-Fos expression in S1BF. (C) Representative tangential slices of c-Fos staining of sham mice at 4X and 10X. Sequential slices from left to right represent coronal sections covering S1J (bregma 1.2mm), S1ULp (bregma 0.5mm), anterior S1BF (bregma -0.8mm) and posterior S1BF cortex (bregma -1.8mm). Slices between bregma -0.8 mm and -1.8 mm were co-stained with VGLUT2 (red) to visualize barrels. Lower panels represent boxed regions of corresponding upper panels. (D) Representative staining of c-Fos and VGLUT1 or GAD67 in S1ULp-S1J of FLIT group. (E) Percentage of VGLUT1+ and GAD67+ cells among c-Fos+ cells. Two-tailed unpaired t-tests were carried out to determine the difference of GLUT1 vs. GAD67. Data are presented as mean ± SEM. *** P < 0.001.  Figure S6. Chemogenetic manipulation of S1ULp-S1J but not S1BF suppresses pain-like behaviors. (A to C) Chemogenetic inhibition of c-Fos-induced Gi-expressing neurons leads to attenuated pain-like behavior. Behavioral testing was performed at indicated timepoints. At day 7, behavioral testing was performed prior to C21 administration to obtain pre-treatment baseline.

(A) Body weight. (B) Wood weight changes in 24 hours. (C) percentage of time eating solid
chow. Two-way ANOVA followed by Bonferroni post hoc test was carried out to determine the difference between the two groups. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01.
(D to L) TRAP2 mouse was injected with AAV-DIO-hSyn-Gi-mCherry or AAV-DIO-mCherry vector in S1BF at day -28 (n = 8 per group). FLIT surgery was performed for all mice at day 0 accompanied by tamoxifen administration. From day 7, C21 was intraperitoneally administrated twice daily. Mice were sacrificed at day 28 for brain slices. (D) Diagram of virus injection and flowchart of experiment timeline. (E) Upper panels: Representative tangential slices of S1J, S1ULp and S1BF demonstrating mCherry expression was primarily located in S1BF. Lower      Figure S10E, Day 0 Decompression is the same as Figure 6H pre decomp row, 2) Figure   S10E, Decompression Day 7 is the same as Figure 6H post decomp day 7, 3) quantification of data in Figure S10F and Figure 6I share a subset of data points.
Movie S1: Excessive facial grooming in FLIT model.

Movie S2:
Paroxysmal asymmetrical facial grimacing in FLIT model. Movie S3: Synchronized S1 neuronal activity in the same field of view of a FLIT mouse.