Figure 3: GABAAR and NKCC1 expression in the spinal dorsal horn.
From: Presynaptic GABAergic inhibition regulated by BDNF contributes to neuropathic pain induction
(a–f) Co-localization of α1-GABAAR (green) with calcitonin gene-related peptide (CGRP)-positive terminals (red) in parasagittal sections of lumbar spinal cord of control and 2 d.p.i. mice. Arrows mark representative double-labelled terminals. (g) Quantification of the α1-GABAAR fluorescence intensity in the CGRP-positive areas in the dorsal horn of control and 2 d.p.i. mice (control: n=8 sections (2 mice); 2 d.p.i.: n=31 sections (3 animals); unpaired t-test, P<0.01). (h) Western blotting showing the specific band of NKCC1 in control and 2 d.p.i. spinal cord. GAPDH was used as housekeeping gene. (i) Quantification of the bands intensities relative to GAPDH expression (n=6 ipsilateral spinal cord (L4–L5) from injured animals and 6 halves spinal cord (L4–L5) from control animals. Three western blotting were performed in total; unpaired t-test, P>0.05). (j,k) Representative staining of NKCC1 (red) in control and 2 d.p.i. spinal cord dorsal horn. Nuclei (blue) were stained with DAPI. (l) Quantification of immunofluorescence signals for NKCC1 (n=3 sections of each spinal cord from three control and three 2 d.p.i. animals; unpaired t-test, P>0.05). Intraperitoneal administration of NKCC1 inhibitor bumetanide or saline 2 days (m), 14 days (n) or 21 days (o) after nerve injury, n=6 mice per group (bumetanide effect: one-way analysis of variance (ANOVA) with post-hoc Dunnett’s test; 2 d.p.i., P=0.001; 14 d.p.i., P=0.0001; 21 d.p.i., P>0.05. Ipsilateral+saline versus ipsilateral+bumetanide, two-way ANOVA with post-hoc Bonferroni’s test, 2 d.p.i., P=0.0001; 14 d.p.i., P<0.05; 21 d.p.i., P>0.05.). Scale bars: (a–f), 2 μm; (j,k), 100 μm (scale bar only shown in f and k). *P<0.05; **P<0.01, versus control (g), or versus before injection (m,n); +P<0.05; ++P<0.01; +++P<0.001 versus ipsilateral+saline (m,n). Error bars indicate s.e.m.
