In vivo CRISPR/Cas9 KO HDAC6 mice affect NMJs but do not lead in behavioral disorders. 9-wk-old WT mice (WT-CTL) and KO HDAC6 mice (HDAC6^−/−) were treated with TubA or with vehicle-control (veh) for 31 consecutive days. (A) To evaluate the level of HDAC6 expression, Western blots were performed. (B) Levels of α-tubulin acetylation in WT-CTL and HDAC6^−/− mice in TA muscles were evaluated by Western blot analysis. (C) Quantification of acetylated tubulin protein level normalized to β-tubulin. (A and B) GAPDH was used as a loading control (n = number of mice used per condition; WT-CTL = 5 and HDAC6^−/− = 5). (D) NMJs of isolated TA fibers labeled with α-BTX–A488 (in gray). (E) Graphical summary of NMJ compactness (n = total number of NMJs counted on five mice for each condition; WT-CTL = 171, and HDAC6^−/− = 175). (F) Fragmentation index and distribution of number of fragments have been quantified (n = total number of NMJs counted on five mice for each condition; WT-CTL = 171 and HDAC6^−/− = 175). (G) Open-field behavior. Distance traveled and time spent in the center or in corners of the open-field chamber are shown on the y axis. (H) Beam break test was realized for 12 h. Motor habituation and activity are shown on the y axis. (I) Grip strength was measured on a grid measuring maximal forelimb and hind limb grip strength. (G–I)n = number of mice used per condition (WT-CTL = 5, and HDAC6^−/− = 5). (D) Scale bars: 25 µm. n.s, not significant. β-tub, β-tubulin; Mr(K), relative molecular weight in kiloDalton; Nb, number; ac-tub and ac-tubulin, acetylated tubulin. Quantifications show means ± SEM. *, P < 0.05; **, P < 0.01; Mann-Whitney U test.