Figure 6.

Axonal morphometry and neurofilament subunit phosphorylation in optic axons of NFH-null mice. (A) Axon caliber distributions at optic nerve levels 50 and 700 μm from the eye in NFH^+/+ and NFH^−/− mice. The number of axons in each size class, expressed as a percentage of the total axonal population analyzed, is plotted as a function of axon cross-section area. A total of 9,864 axons from 5 wild-type and 5 NFH-null optic nerves were analyzed at each axonal level. (B) Regional axonal expansion (mean caliber [700 μm − 50 μm/50 μm] × 100) for the populations of axons in A. Mean regional neurofilament accumulation (neurofilament number [700 μm − 50 μm/50 μm] × 100) for 1,350 axons of caliber size representative of the whole fiber population. Lower regional differences in these 1.5-yr-old mice compared with 4-mo-old mice in Fig. 1 are due to aging-related increases in caliber and neurofilament number at the 50-μm level (Nixon, R.A., unpublished data). (C–E) Neurofilament subunit levels and phosphorylation in control and NFH-null mice. (C) Total NFM levels are not altered when detected with a phosphorylation-independent neurofilament antibody to NFM (RM04) generously provided by Dr. Virginia Lee (University of Pennsylvania, Philadelphia, PA. Representative examples of one-dimensional electroblots (n = 4) immunostained with SMI34 and SMI31 show no compensatory increase in levels of these phosphoepitopes in NFH^−/− mice. Two-dimensional immunoblots of optic nerves from NFH^+/+ (D) and NFH^−/− (E) mice stained with RT97 demonstrate the absence of NFH and presence of RT97-positive NFM in NFH^−/− mice. The identity and precise position of NFM in D (indicated by oval) were confirmed by reprobing the blots with RM04 (data not shown). The smaller quantity of NFH breakdown products in sciatic nerve samples allowed increases of RT97 on NFM in null mice to be appreciated (E, inset).