Fig. 1: Neuronal activity and persistent inflammatory nociception trigger the nuclear export of HDAC4 and histone 3 acetylation in spinal cord neurons.

a Representative images of cultured primary spinal cord neurons (DIV10) immunostained for endogenous HDAC4 (green) and the neuronal marker NeuN (red); Hoechst (blue) marks the nuclei. Scale bar is 20 µm. b Quantification of the relative fluorescence intensity of the nuclear HDACs signal in spinal cord neurons normalized to respective controls. Each point represents the mean value derived from one independent experiment. Ca. 40 cells were analyzed per condition and experiment (HDAC1 n = 4 F_(3,8) = 1.120; HDAC3 n = 4 F_(3,12) = 0.9239; HDAC4 n = 6 F_(3,20) = 8.676; HDAC5 n = 4 F_(3,12) = 3.210; HDAC7 n = 6 F_(3,20) = 2.726; HDAC9 n = 5 F_(3,20) = 0.7916; HDAC10 n = 4 F_(3,8) = 2.502; HDAC11 n = 4 F_(3,12) = 0.6623). c Quantification of the relative fluorescence intensity of the nuclear signal of acetylated histone H3-Lys9 (AcH3) in spinal cord neurons, treated as indicated. Ca. 30 cells were analyzed per sample (n = 4 F_(3,12) = 5.854). d Representative images of primary cultured spinal cord neurons (DIV10) immunostained for AcH3 (green). Cells were treated with Bic (50 µM) for the indicated times. Nuclei were labelled with Hoechst (blue) and NeuN (red) as a neuronal marker. Scale bar is 40 µm. e Representative images of spinal cord dorsal horn sections of lumbar spinal segments L3–5 of mice 24 h after intraplantar CFA or saline injection, immunostained for HDAC4 or acetylated histone H3-Lys9 (AcH3) shown in green; NeuN (red). Nuclei were labeled with Hoechst (blue) and scale bar is 40 µm. Higher magnifications of the upper laminae are shown in the right panels with a scale bar of 40 µm. f Quantifications of the average nuclear fluorescence intensities from immunolabeled HDACs and AcH3 in neurons of the spinal cord dorsal horn, normalized to values obtained from saline-injected mice and integrated over a time course series (n = 4 mice per condition and timepoint). Timepoints used were 0.5, 2, 6, and 24 h. Statistically significant differences were determined by one-way ANOVA followed by Dunnett’s post hoc test (b, c) or two-tailed Student’s t-test (f). ***p < 0.001; **p < 0.01; *p < 0.05. In bar graphs, each point represents a value derived from one independent culture or mouse. Graphs represent mean ± SEM. See also Supplementary Figs. 1 and 2.