Figure 4.

Chronic VPA administration delays motoneuronal death, maintains high levels of CBP protein, and upregulates CREB/CBP-dependent transcription in vivo. WT and G86R mice were chronically injected with VEH (0.09% NaCl) or VPA (250 mg · kg⁻¹ · d⁻¹) from 60 d of age until score 2 (105 d; G86R-D105) or score 0 (end stage; G86R-ES). A, Nonadjacent spinal cord sections from WT (n = 5) or transgenic mice were immunostained using an anti-ChAT antibody followed by a FITC-conjugated antibody. ChAT-positive MNs with an area >600 μm² are represented as means ± SEM per section. The counting was performed on G86R-D105 and G86R-ES; white and gray histograms correspond to untreated and VPA-treated animals, respectively. *p < 0.05 and ***p < 0.0001 compared with WT conditions; ^#p < 0.05 compared with G86R plus VEH condition. B, Representative semithin section of an L4 ventral root from a WT mouse or a G86R-D105. Scale bars, 50 μm. C, D, Representative pictures (C) and quantification (D) of myelinated fibers occupancy in the different groups of mice (a, WT; b, G86R-D105 plus VEH; c, G86R-D105 plus VPA; d, G86R-ES plus VEH; e, G86R-ES plus VPA). Scale bar, 20 μm. D, The percentage of occupancy of myelinated axons within the L4 VR is represented for each group. The data represent means ± SEM. The number of each animal tested is noted within the histogram. Statistical analysis were performed with ANOVA followed by the post hoc Newman–Keuls multiple-comparisons test (Prism). All situations are compared with WT, and in each group, VPA treatment is compared with VEH. *p < 0.05; **p < 0.001. E, Representative pictures of CBP protein immunostaining in lumbar spinal cord from WT and G86R mice (score 0) chronically injected with VEH or VPA (250 mg · kg⁻¹ · d⁻¹). Arrowheads depict lumbar motor neurons. Scale bars, 40 μm. F, Expression of bcl-2 and smn were analyzed by semiquantitative RT-PCR 24 h after injection in spinal cord extracts from 105-d-old WT or G86R (score 2) that received or not an acute injection of VEH, VPA (250 mg/kg), TSA (2 mg/kg), or NaBu (640 mg/kg). Agarose gels were scanned, and specific bands were quantified. The results are represented relative to the expression levels of the house-keeping 18S gene mRNA levels. ^#p < 0.05 and ^##p < 0.001 compared with the untreated G86R condition.