Figure 5.

Descending 5-HT from RVM is upregulated after CCI-ION and nerve injury induced central terminal sensitization in Vc is blocked by depletion of descending 5-HT in the RVM. (A) Schematic diagram of descending RVM innervations into Vc. RVM (yellow) nerve fibers (red) innervate into dorsal lamina layers of Vc (blue). (B) Vc slices from mice 14 days after CCI-ION were doubly stained with anti-GFP (for GCaMP3; green) and anti-5-HT (red) antibodies. Scale bar: 50 μm. (C) Quantification of 5-HT levels in (B) and Figure S4B are expressed as the mean density. 5-HT level in ipsilateral Vc (Ipsi.; n=13, 48.40±1.35 arbitrary unit (a.u.)) after nerve injured is significantly higher than contralateral side (cont.; n=8, 34.33±1.55). Local gene transfer of Tph-2 shRNA into the RVM attenuated 5-HT in Vc (n=21, 17.18±1.59) compared with that by scrambled shRNA treatment (S.; n=10, 45.26±1.27) and background (n=21, 14.71±0.87). (D) Representative GCaMP3 imaging of Vc slices. Fourteen days after CCI-ION, central terminals in V3 of ipsilateral Vc slices, from mice, whose RVM was treated 3 days earlier with either Tph-2 (lower panels) or scrambled shRNA (upper panels), were activated by capsaicin (1 μM) or low KCl (20 mM). Scale bar: 50 μm. (E and F) Time course of the amplitude of the Ca²⁺ transient that was evoked by capsaicin (1 μM) or low KCl application at V3 in Vc. Capsaicin was bath applied from 0 to 60 sec during the time course. Ipsi./s. shRNA, ipsilateral scrambled shRNA (n=15); cont./s. shRNA, contralateral scrambled shRNA (n=15); ipsi./Tph shRNA, ipsilateral Tph-2 shRNA (n=17); cont./Tph shRNA, contralateral Tph-2 shRNA (n=17). (G) The effects of molecular depletion of descending 5-HT on mechanical hyperalgesia induced by CCI-ION at the V2 (injured) or V3 (uninjured) region. Behavioral measures were done before shRNAi (at 10 d after CCI-ION) and at 4 day after shRNAi (14 day after CCI-ION) with intra-RVM gene transfer of Tph-2 shRNA (n=8) or scrambled shRNA (n=7) *p<0.05; **p<0.01; ***p<0.001.