Conditional Deletion of the L-Type Calcium Channel Cav1.2 in Oligodendrocyte Progenitor Cells Affects Postnatal Myelination in Mice

Black Gold II staining for myelin in the Cav1.2^KO brain. A, Black Gold II staining in the brains of control and Cav1.2^KO mice injected at P4, P10, and P30. Representative coronal sections of the lateral area of the corpus callosum (CC) and the striatum are shown. Scale bar, 360 μm. B, Black Gold II staining intensity was quantified in the CC, the cingulate cortex (cortex), and striatum. Values are expressed as mean ± SEM of four independent experiments. *p < 0.05, **p < 0.01 versus the respective controls.

Reduced myelin protein synthesis in the postnatal Cav1.2^KO brain. A, Representative coronal sections of the central area of the corpus callosum (CC) and the cingulate cortex (cortex) of control and Cav1.2^KO mice injected at P4 and P10 and immunostained with anti-MBP and anti-MOG antibodies. Scale bar, 180 μm. B, Myelin was quantified by analyzing fluorescence intensity of MBP and MOG in the CC and in the cingulate cortex (cortex). C, Control and Cav1.2^KO mice were injected at P10 and immunostained with anti-MBP and anti-PLP antibodies at P60. Myelin was quantified by analyzing fluorescence intensity of MBP and PLP in the CC, cingulate cortex (CX), and striatum (ST). Values are expressed as mean ± SEM of four independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus the respective controls.

Western blot analysis of myelin protein expression. Total proteins were collected from the corpus callosum (A), cortex (B), olfactory bulb (C), optic nerve (D), cerebellum (E), and spinal cord (F) of Cav1.2^KO animals to assess the expression of MBP, MOG, PLP, and CNP by Western blot. Representative Western blots are shown. GAPDH was used as the internal standard and data from four independent experiments are summarized based on the relative spot intensities and plotted as percentage of the P4 control. Because no statistical differences between experimental groups were detected, the quantitative bar graphs for D–F are not shown. Values are expressed as mean ± SEM of four independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus the respective controls.

Electron microscopy of the Cav1.2^KO corpus callosum. A, Electron micrographs of axons in the corpus callosum of control and Cav1.2^KO mice injected at P10. Scale bars, 8 μm top, 2 μm bottom. B, C, Scatter plot and mean g-ratio values in the corpus callosum of control and Cav1.2^KO mice. D, Percentage of myelinated axons in the corpus callosum of control and Cav1.2^KO mice. E, F, Mean axonal diameter and distribution of axonal size in control and Cav1.2^KO mice fibers. Values are expressed as mean ± SEM. Five animals per experimental group and 100 fibers per animal were analyzed. ***p < 0.001 versus control.

Decreased number of mature oligodendrocytes in the Cav1.2^KO mouse. A, Representative coronal sections of the central area of the corpus callosum (CC) and the cingulate cortex (cortex) of control and Cav1.2^KO mice injected at P4 and P10 and immunostained with anti-Olig2 and anti-CC1 antibodies. Scale bar, 180 μm. B, C, Number of Olig2-, CC1-, and Olig2/CC1-positive cells was quantified stereologically in the central area of the CC and in the cingulate cortex (cortex) of control and Cav1.2^KO mice. Values are expressed as mean ± SEM of four independent experiments. *p < 0.05, **p < 0.01 versus the respective controls.

OPC proliferation in the Cav1.2^KO mouse. A, Representative coronal sections of the central and lateral areas of the corpus callosum (CC) of control and Cav1.2^KO mice injected at P4 and P10 and immunostained with anti-Olig2, anti-Ki67, and anti-Sox2 antibodies. Scale bar, 180 μm. B, C, Number of Olig2/Ki67-, Olig2/Sox2-, GFAP-, and CD45-positive cells was quantified stereologically in the central and lateral areas of the CC and in the cingulate cortex (cortex) of control and Cav1.2^KO mice. Values are expressed as mean ± SEM of four independent experiments. *p < 0.05, **p < 0.01 versus the respective controls.

Reduced in situ L-VOCC activity in Cav1.2^KO oligodendrocytes. A, D, Fura-2 time-lapse series of control GFP-positive OPCs located in the somatosensory cortex and in the lateral area of the corpus callosum at P10. Scale bar, 40 μm. Arrowheads indicate GFP-positive cells that were selected for the analysis. An increased fura-2 fluorescence ratio is indicated by warmer colors and time is denoted in minutes in the lower right corner. B, F, L-VOCC activity was examined in cortical OPCs and in cortical neurons from control and Cav1.2^KO/GFP mice at P10. Note that each trace corresponds to a single cell and the horizontal bar indicates the time of high K⁺ addition. C, E, Cortical and callosal OPCs from control and Cav1.2^KO/GFP mice were also stimulated with high K⁺ (50 mm) in the presence of verapamil (5 μm) and nifedipine (5 μm). The graphs show the average amplitude calculated from the responding cells expressed as percentage of change of the emission intensities. Values are expressed as mean ± SEM of at least four independent experiments. ***p < 0.001 versus the respective controls.

Decreased maturation of Cav1.2^KO/GFP OPCs in the postnatal brain. A, Representative coronal sections of the cingulate cortex of control and Cav1.2^KO/GFP mice injected at P4 and immunostained with anti-Olig2 and anti-CC1 antibodies. Scale bar, 150 μm. B, C, Number of double-positive cells for Olig2, Olig1, NG2, and CC1 was quantified stereologically in the corpus callosum, cingulate cortex, and striatum. D, Morphological examination of GFP-positive cells in the same brain areas. E, Proportion of double-positive cells for Ki67 and caspase-3 was quantified stereologically in the corpus callosum, cingulate cortex, and striatum. Values are expressed as mean ± SEM of four independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus the respective controls.