Fig 2: Mechanical hypersensitivity after PNI was ameliorated by AAV-CRISPR-Cas9-mediated a2d-1 knockdown in SDH neurons. (A) Schematic of an AAV vector containing a neuronal SaCas9 expression cassette, and vectors containing the sgRNA targeting Cacna2d1 or control sgRNA fused with a neuronal mCherry expression cassette. (B) Representative images showing expression of mCherry (red) and SaCas9 (green) in the SDH. (C) Representative images showing a2d-1 expression (green) in mice injected with the Cas9 expression vector and sgControl (upper) or sgCacna2d1 (lower) containing vector, respectively. (D) Summary of the a2d-1 immunofluorescent intensity of mCherry-positive neurons in sgCacna2d1 and sgControl mice (n = 4, 3 sections from 4 animals, and data are presented as mean ± SEM of averaged data from individual animals, two-tailed unpaired t-test, t = 3.12, df = 6, *p < 0.05). (E) Summary data of the paw withdrawal threshold of naïve mice [n = 10 (sgControl), n = 9 (sgCacna2d1), two-tailed unpaired t-test, t = 0.391, df = 17, p = 0.701]. (F) Summary data of the latency for mice to lick their hindpaws or jump after being placed on a 52°C hot plate (n = 4 each, two-tailed unpaired t-test, t = 0.692, df = 6, p = 0.515). (G) Summary of the duration for mice to lick their formalin-injected hindpaw for 0–5 min (first phase, left, n = 7 each, two-tailed unpaired t-test, t = 0.984, df = 12, p = 0.344) and for 10–60 min (second phase, right, n = 7 each, two-tailed unpaired t-test, t = 0.663, df = 12, p = 0.520) after formalin injection. (H) Paw withdrawal threshold ipsilateral to the injured side of sgControl or sgCacna2d1 mice before (pre) and after PNI. [n = 10 (sgControl), n = 9 (sgCacna2d1), two-way repeated measurements ANOVA post-hoc Bonferroni’s test, Group, F(1,17) = 46.1, p < 0.0001, Time, F(7,119) = 20.2, p < 0.0001, Interaction F(7,119) = 5.91, p < 0.0001, **p < 0.01, ***p < 0.001]. (I) Same as (H) but contralateral to the injured side [n = 10 (sgControl), n = 9 (sgCacna2d1), two-way repeated measurements ANOVA post hoc Bonferroni’s test, Group, F(1,17) = 0.00552, p = 0.942, Time, F(7,119) = 1.07, p = 0.386, Interaction F(7,119) = 0.788, p = 0.599]. (J) Paw withdrawal threshold ipsilateral to the injured side of sgControl or sgCacna2d1 mice before (pre) and after sham operation [n = 6 each, two-way repeated measurements ANOVA post-hoc Bonferroni’s test, Group, F(1,10) = 0205, p = 0.660, Time, F(7,70) = 0.155, p = 0.993, Interaction F(7,70) = 0.293, p = 0.955]. (K) Same as (J) but contralateral to the operated side [n = 6 each, two-way repeated measurements ANOVA post-hoc Bonferroni’s test, Group, F(1,17) = 0.894, p = 0.367, Time, F(7,70) = 1.83, p = 0.988, Interaction F(7,70) = 0.356, p = 0.924]. Data are mean ± SEM.

Fig 3: a2d-1 mRNA was selectively expressed in excitatory neurons in the SDH. (A,B) Representative images of RNA scope in situ hybridization for mRNAs of Cacna2d1 (encoding a2d-1 subunit, red) and Slc17a6 (Vglut2, gray) in the SDH of naïve mice. Low magnification images (A) and high magnification images (B). (C) Summary of coexpression of Cacna2d1 and Slc17a6. n = 3, 3 sections from 3 animals. (D) Representative images of Cacna2d1 (red) and Slc32a1 (Vgat, gray) mRNAs. (E) Summary of coexpression of Cacna2d1 and Slc32a1. n = 3, 3 sections from 3 animals. (F) Representative images of Cacna2d2 (encoding a2d-2 subunit, red) and Slc17a6 (gray) mRNAs. (G) Summary of coexpression of Cacna2d2 and Slc17a6. n = 3 sections from 3 animal. (H) Representative images of Cacna2d2 (red) and Slc32a1 (gray) mRNAs. (I) Summary of coexpression of Cacna2d2 and Slc32a1. n = 3, 2–3 sections from 3 animals. (J) Representative images of Cacna2d3 (encoding a2d-3 subunit, red) and Slc17a6 (gray) mRNAs. (K) Summary of coexpression of Cacna2d3 and Slc17a6. n = 3, 2–3 sections from 3 animals. (L) Representative images of Cacna2d3 (red) and Slc32a1 (gray) mRNAs. (M) Summary of coexpression of Cacna2d3 and Slc32a1. n = 3, 2–3 sections from 3 animals. Data are mean ± SEM of averaged data from individual animals.