Thinner myelin sheath in R-Ras1^−/−, R-Ras2^−/−, and R-Ras1^−/−;R-Ras2^−/− adult (P90) mutant mice. A, Transverse sections of the optic nerve from adult (P90) control, R-Ras1^−/−, R-Ras2^−/−, and R-Ras1^−/−;R-Ras2^−/− mice analyzed by electron microscopy showing a significant decrease of myelin in single and double mutants compared with controls. B, Higher-magnification images showing no differences in myelin structure, compaction, and periodicity in mutant mice compared with controls. However, R-Ras1^−/−, R-Ras2^−/−, and, more dramatically, R-Ras1^−/−;R-Ras2^−/−, show less wrapping of myelinated axons compared with controls. C, Scatter plots showing the morphometric quantification of g-ratios (y-axis) of individual fibers relative to the respective axon diameters (x-axis) from optic nerves. Significant increase in g-ratios were found in R-Ras1^−/− (0.87 ± 0.04, ***p = 0.000012, n = 924 axons analyzed), R-Ras2^−/− (0.8 ± 0.08, ***p = 0.000011, n = 1014 axons analyzed), and R-Ras1^−/−;R-Ras2^−/− (0.89 ± 0.06, ***p = 0.000072, n = 1037 axons analyzed) mice compared with controls (0.75 ± 0.09, n = 764 axons analyzed). D, Graph showing the percentage of myelinated axons present in the optic nerve of R-Ras1^−/− = 102 ± 2.7%, R-Ras2^−/− = 72.1 ± 5.2%, and R-Ras1^−/−;R-Ras2^−/− = 23.9 ± 2.5% compared with the control = 100 ± 2.8%. Significant differences were observed in the R-Ras2^−/− (***p < 0.0001) and the double-mutant mice (***p < 0.0001). E, Scatter plots showing the variation of myelin thickness with respect to the diameter of axons in single and double mutants compared with the control (control = 251 ± 60 nm, n = 764 axons analyzed; R-Ras1^−/− = 105 ± 53 nm, ***p < 0.0001, n = 924 axons analyzed; R-Ras2^−/− = 159 ± 75 nm, ***p < 0.0001, n = 1014 axons analyzed; R-Ras1^−/−;R-Ras2^−/− = 73 ± 40 nm, ***p < 0.0001, n = 1037 axons analyzed). F, Graph showing the percentage of axons classified according to their diameters in control and double-mutant mice. Compared with the control, R-Ras1^−/−;R-Ras2^−/− mice showed a significant increase in the smallest axons, measuring <600 nm (control = 30%, R-Ras1^−/−;R-Ras2^−/− = 42.6% of axons, ***p < 0.0001), and a significant decrease in axons, measuring >1000 nm (control = 30.3%, R-Ras1^−/−;R-Ras2^−/− = 21.5% of axons, ***p = 0.0002). All samples were obtained from adult mice (P90) (n = 3). Scale bars: A, 500 nm; B, 250 nm.