Internodal myelination during development quantitated using X-ray diffraction Deepika Agrawal, Rachel Hawk, Robin L. Avila, Hideyo Inouye, Daniel A. Kirschner * Biology Department, Boston College, 140 Commonwealth Ave (Higgins Hall), Chestnut Hill, MA 02467-3811, USA article info Article history: Received 8 May 2009 Received in revised form 19 June 2009 Accepted 28 June 2009 Available online 1 July 2009 Keywords: X-ray diffraction PNS development CNS development Membrane interactions Ultrastructure Mouse abstract Characterizing the formation, accretion, and stability of myelin during development, maturation, and senescence is important for better understanding critical periods in the function of the nervous system in normal growth and following environmental insult or genetic mutation. Although there are numerous studies on the ultrastructural, biochemical, and genetic aspects of myelin development and maturation, few have used X-ray diffraction (XRD), which can rapidly provide unique metrics about internodal myelin based on measurements from whole, unfixed tissue. Besides periodicity (the classic attribute of inter- nodal myelin measured by XRD), other parameters include: relative amount of myelin, membrane dimensions, and packing disorder. To provide a baseline for future experiments on myelin structural integrity, we used XRD to characterize internodal myelin as a function of age (from 5 to 495 days) in the mouse, a species increasingly used for developing transgenic models of human neurological diseases. As expected, the relative amount of myelin increased with age in both PNS and CNS, with the most rapid accumulation occurring in the youngest age group. Changes in rate of myelin accretion yielded three dis- tinct age brackets during which small but significant changes in structural parameters were detected: in PNS, myelin period increased, packing distortion decreased, width of extracellular apposition (EXT) decreased, and widths of cytoplasmic apposition (CYT) and lipid bilayer (LPG) increased; in CNS, myelin period decreased, packing distortion decreased, EXT and CYT decreased, and LPG increased. We propose that the data obtained here can serve as a basis for rapidly detecting abnormal pathologies during myelination. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction Nerve conduction in vertebrates is greatly speeded up by the ensheathment of its axons with myelin, an insulating, multilamel- lar membrane assembly (Lazzarini, 2004). Internodal myelin, which is the portion of the sheath between nodes of Ranvier, con- sists of membrane pairs that are closely apposed, adhering at their cytoplasmic and extracellular appositions. Compared to other membranes that are loci for enzymatic activities, internodal myelin has a high lipid-to-protein ratio (Norton and Poduslo, 1973). The proteins in compact myelin include P0, MBP, PMP-22, and P2 in PNS, and PLP and MBP in the CNS (Kursula, 2008; Simons and Trot- ter, 2007; Trapp and Kidd, 2004). The recent co-localization of MBP and ATP synthase in optic nerve myelin (Ravera et al., 2009) may indicate a more active physiological role for compact myelin. The protein composition is the predominant determinant of myelin’s periodicity, as the thickness of the membrane bilayer is fairly con- stant across species whereas the extent of close apposition be- tween bilayers—or membrane packing—varies considerably (Kirschner and Blaurock, 1992; Kirschner et al., 1989). Depending on animal species the periodicity ranges from 160 to 185 Å in the PNS, and 150 to 170 Å in the CNS. Both ultrastructural and biochemical approaches have been used to follow myelination. For example, electron microscopy re- veals that for PNS myelin the ratio of the number of myelin layers to the axon area rapidly increases during the first 75 days (Friede and Samorajski, 1967; Low, 1976). Based on biochemical metrics of brain weight, myelin yield, and accumulation of MBP and spe- cific myelin lipids, CNS myelination shows an initial rapid increase followed by a more gradual increase (Barbarese et al., 1978; Morell et al., 1972; Norton and Poduslo, 1973; Uzman and Rumley, 1958). The few XRD studies that have been undertaken to characterize myelin accretion and changes in myelin structure with age show that within the first few weeks there is an initial rapid accumula- tion of PNS and CNS myelin and small changes in their periods (Kirschner and Sidman, 1976; Mateu et al., 1990, 1991; Vargas et al., 2000). To quantitate more thoroughly the formation and development of internodal myelin and its growth trends, we used XRD with an electronic detector to analyze whole nerves in both the PNS and CNS as a function of age. The advantage of this technique is the rapidity of the measurements, the large sampling volume due to the use of intact nerves, and the fact that the tissue is unfixed 1047-8477/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2009.06.019 * Corresponding author. Fax: +1 617 552 2011. E-mail address: kirschnd@bc.edu (D.A. Kirschner). Journal of Structural Biology 168 (2009) 521–526 Contents lists available at ScienceDirect Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi