Figure 4. Bidirectional modulation of expression of Rac1 in spinal dorsal horn neurons in vivo and its structural and functional impact.

(a) Western blotting of spinal lysates for validation of spinal knockdown and overexpression of Rac1 via AAV-mediated shRNA delivery or Rac1 cDNA delivery in comparison with corresponding controls. Lower panel represents quantitative summary of Rac1 expression normalized to corresponding Tubulin control. (b,c) Typical examples of traces derived from Golgi-impregnated spinal neurons (b) and their quantitative summary (c) in mice with spinal AAV-mediated knockdown or overexpression of Rac1, using mice spinally expressing a non-targeting shRNA or EGFP as controls (Scale bar, 50 μm; 15–18 neurons from three animals per treatment group). (d) Attenuation of phase II nocifensive behaviour, but not of phase I, in the formalin test by spinal knockdown of Rac1 (n=7–8 per group). (e) Spinal neuron-specific knockdown of Rac1 led to a reduction of mechanical hypersensitivity induced by intraplantar CFA injection as compared with mice expressing control RNA (n=6 per group). (f) Overexpression of Rac1 in neurons of the spinal dorsal horn led to enhanced basal sensitivity to mechanical nociceptive stimuli (n=6–8 mice). (g) Functional impact of overexpression of Rac1 or EGFP (control) in the spinal dorsal horn of SDH-Kal-7^−/− mice on sensitivity to mechanical nociceptive stimuli and CFA-induced hypersensitivity (n=6 mice per group). (h) Quantification of changes in spine density on overexpression of Rac1 or EGFP in spinal neurons lacking Kal-7 at 24 h post-CFA (25–30 neurons were analysed from from animals each per treatment group). *P<0.05 as compared with control group; ^†P<0.05 as compared with basal condition; two-way ANOVA, Bonferroni post hoc test. Error bars represent s.e.m.