Journal: The Journal of Neuroscience
Article Title: TREK2 Expressed Selectively in IB4-Binding C-Fiber Nociceptors Hyperpolarizes Their Membrane Potentials and Limits Spontaneous Pain
Figure Lengend Snippet: TREK2 knockdown in vivo decreased SFL 1 d after CFA. Ipsilateral SFL in rats 1 d after an intradermal plantar CFA injection in one hind foot in the same region as an intradermal plantar injection of either TREK2 siRNA or scrambled siRNA given 3 d earlier. Numbers in B and C (superscripts) and D and E (beside data points) relate to rats from which tissue in the lanes of A was obtained; these numbers are included to enable comparison of TREK2 protein expression with other data from the same rats. A , TREK2 Western blot of skin tissue, at the site of scr (1, 2) or siRNA (3, 4) injection 4 d previously, and 1 d after either saline (1, 3) or CFA injection (2, 4) at the same site. Top, The lower TREK2 band (MW ∼50 kDa, the band that we and others find in DRG tissues) was almost eliminated by siRNA, not scr, treatment, regardless of whether rats were injected with CFA or saline. The upper band was not reduced after siRNA. The MWs of these bands are consistent with them being two of the three known rat TREK2 isoforms (see Results and Discussion). Bottom, α-Tubulin loading controls. B , TREK2 knockdown by TREK2 siRNA in nerve fibers and epidermis (Ep) in plantar skin. Left, after scr siRNA injection. Right, after TREK2 siRNA injection. Both treatments were followed by CFA injection. Tissue was taken 4 d after scr/siRNA treatment (1 d after CFA). Triple fluorescence staining for β-tubulin III (blue, to stain nerve fibers), IB4 binding (green), and TREK2 (red). White dashed lines indicate junctions between epidermis (above) and dermis (below). In the merged images, colocalization of all three markers shows as white or very pale (bottom left image). After siRNA, red TREK2 staining in epidermis and in groups of nerve fibers (shown as blue and/or green) was reduced, showing substantial knockdown in vivo of TREK2 near the siRNA injection site. Scale bar, 25 μm. C , TREK2 knockdown by local TREK2 siRNA in the nerve innervating plantar skin. This knockdown, seen 5–10 mm from the siRNA injection site, occurred whether rats were subsequently treated with saline or CFA. There was decreased fiber TREK2 immunostaining (red) in TREK2 siRNA, not scr siRNA, treated rats, so that IB4 + fibers (green) showed little/no TREK2 (red). Scale bar, 50 μm. D , E , SFL after CFA increased after TREK2 siRNA compared with scr control. Ipsilateral SFLd ( D ) or SFLn ( E ) after siRNA C compared with scr treatment did not increase before saline injection (day 3, columns 1 and 2) or after saline injection (day 4, columns 3 and 4), or before CFA injection (day 3, columns 5 and 6). However, after CFA, it caused increased SFLd ( D ) and SFLn ( E ) in both scr ( p
Article Snippet: Three days after transfection, the HEK cell cultures expressing the different K2P channels were washed with PBS, fixed with 4% paraformaldehyde in 4.2% sucrose (in PBS, pH 7.2) for 10 min at room temperature, rinsed with PBS, and processed for TREK2 immunocytochemistry with the rabbit anti-TREK2 antibody from Alomone Labs as described above.
Techniques: In Vivo, Injection, Expressing, Western Blot, Fluorescence, Staining, Binding Assay, Immunostaining