Fig. 3

Overexpression of mutant PDIA3Q481K in mice impacts motoneurons and neuromuscular junctions. (A) Representative photomicrographs of immunohistochemical staining against choline acetyltransferase (ChAT) for detection of motoneurons in the lumbar spinal cord of mutant PDIA3Q481K transgenic mice and non-transgenic littermates at 18 months of age. Scale bar, 250 μm. The graphs show the relative quantification of motoneurons in a segment spanning 1 mm of tissue block from caudal to rostral, with the sciatic nerve marking the start point, in male and female mice pooled in the analysis (left) or in animals grouped by sex as indicated. Data are shown as mean ± s.e.m. and statistical analysis was performed using two-tailed Student's t-test. Differences with p < 0.05 were considered statistically significant. Each symbol in the bar graph corresponds to an independent biological replicate. The number of motoneurons corresponds to the average of cell count in five independent transversal sections per animal. (B) Scheme for measuring compound muscle action potential (CMAP) in the skeletal muscles of the hindlimbs. Red arrow, recording electrode. Red dashed arrow, ground electrode. Blue arrow, stimulating electrode. Blue dashed arrow, ground electrode. (C) CMAP in gastrocnemius of mutant PDIA3Q481K transgenic mice and non-transgenic littermates at 18 months of age. Data from male and female mice were plotted and analyzed separately as indicated. Data are shown as mean ± s.e.m. and statistical analysis was performed using one-way ANOVA with Tukey's multiple comparisons test. Differences with p < 0.05 were considered statistically significant. If not indicated, comparisons were non-significant. Each symbol in the bar graphs corresponds to an independent biological replicate. (D) Same as C, in tibialis anterior. F values and degrees of freedom, F (DFn, DFd): genotype, F (3, 35) = 3.519; p = 0.025. (E) Representative photomicrographs of fluorescent α-BTX staining of muscle endplates in diaphragm of mutant PDIA3Q481K transgenic mice and non-transgenic littermates at 12 months of age. Scale bar, 50 μm. The middle panels show various types of morphologies of neuromuscular junction present at adult stage. The graph shows quantification of frequency of the different morphologies. Data are shown as mean ± s.e.m. and statistical analysis performed using two-way ANOVA with Sidak's multiple comparisons test. Differences with p < 0.05 were considered statistically significant. If not indicated, comparisons were non-significant. F values and degrees of freedom, F (DFn, DFd): interaction, F (3, 24) = 3.875 and p = 0.0216; morphologies, F (3, 24) = 286.3 and p < 0.0001; genotype, F (1, 24) = 1.707e-005 and p = 0.9967. Each symbol in the bar graph corresponds to an independent biological replicate. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)