Figure 5. Loss of CA3 neurons in Scyl2-deficient mice is the result of apoptotic cell death. A, B, Caspase-3 activation in degenerating CA3 neurons. Immunohistochemical staining of brain sagittal sections obtained from postnatal day (P) 4.5, P6.5, and P8.5 NesCre+;Scyl2+/fl and NesCre+;Scyl2fl/fl mice, using an anticleaved caspase-3 antibody. Black arrowheads indicate cleaved caspase-3-positive cells. White arrowheads point to cleaved caspase-3-positive neuronal processes. B, The histogram represents the quantification of cleaved caspase-3-positive cells in the CA3 area of hippocampi obtained from P4.5, P6.5, and P8.5 NesCre+;Scyl2+/fl and NesCre+;Scyl2fl/fl mice. Values are mean ± SEM of 15 images from 3 different mice per genotype and age group. p values, determined by the two-tailed Student's t test, are indicated on the graph. Scale bar, 100 μm. C, D, CA3 degenerating neurons are positive for TUNEL staining. TUNEL staining of brain sagittal sections obtained from P4.5, P6.5, and P8.5 NesCre+;Scyl2+/fl and NesCre+;Scyl2fl/fl mice. D, Graph represents the quantification of TUNEL-positive cells in the CA3 area of hippocampi obtained from P4.5, P6.5, and P8.5 NesCre+;Scyl2+/fl and NesCre+;Scyl2fl/fl mice. Arrows point to TUNEL-positive cells within the CA3 area. Values are mean ± SEM of 15 images from 3 different mice per genotype and age group. p values, determined by the two-tailed Student's t test, are indicated on the graph. Scale bar, 100 μm. E, Generation of NesCre+;Scyl2fl/fl;Baxfl/fl mice. Western blot analysis of brain extracts obtained from NesCre+;Scyl2+/fl;Bax+/+, NesCre+;Scyl2fl/fl;Bax+/+, NesCre+;Scyl2+/fl;Baxfl/fl, and NesCre+;Scyl2fl/fl;Baxfl/fl mice, using antibodies against SCYL2, BAX, and β-actin. Note the complete absence of SCYL2 in the brains from NesCre+;Scyl2fl/fl;Bax+/+ and NesCre+;Scyl2fl/fl;Baxfl/fl mice and BAX expression in the brains from NesCre+;Scyl2+/fl;Baxfl/fl and NesCre+;Scyl2fl/fl;Baxfl/fl mice. F, Targeted deletion of Bax rescues the loss of CA3 neurons caused by Scyl2 deficiency. Representative micrographs of sagittal sections of hippocampi obtained from 5-week-old NesCre+;Scyl2+/fl;Bax+/+, NesCre+;Scyl2fl/fl;Bax+/+, NesCre+;Scyl2+/fl;Baxfl/fl, and NesCre+;Scyl2fl/fl;Baxfl/fl mice. Note the absence of CA3 pyramidal neurons in NesCre+;Scyl2fl/fl;Bax+/+ mice compared with mice also deficient for Bax (NesCre+;Scyl2fl/fl;Baxfl/fl). Scale bar, 200 μm. G, Cellularity in the CA3 area of NesCre+;Scyl2+/fl;Bax+/+, NesCre+;Scyl2fl/fl;Bax+/+, NesCre+;Scyl2+/fl;Baxfl/fl, and NesCre+;Scyl2fl/fl;Baxfl/fl mice. Values are mean ± SEM of 20 images from 4 different mice per genotype. p values, determined by the two-tailed Student's t test, are indicated on the graph.