Electron microscopy of myelin: Structure preservation by high-pressure freezing

Highlights

• Novel techniques improve visualization of myelin biogenesis, structure and pathology.

• High-pressure freezing preserves myelin structure in terrestrial and aquatic species.

• Electron microscopy is independent of sequence data, antibodies or transgenic lines.

• Model species of myelin evolution can be analyzed using high-pressure freezing and EM.

Abstract

Electron microscopic visualization of nervous tissue morphology is crucial when aiming to understand the biogenesis and structure of myelin in healthy and pathological conditions. However, accurate interpretation of electron micrographs requires excellent tissue preservation. In this short review we discuss the recent utilization of tissue fixation by high-pressure freezing and freeze-substitution, which now supplements aldehyde fixation in the preparation of samples for electron microscopy of myelin. Cryofixation has proven well suited to yield both, improved contrast and excellent preservation of structural detail of the axon/myelin-unit in healthy and mutant mice and can also be applied to other model organisms, including aquatic species.

This article is part of a Special Issue entitled SI: Myelin Evolution.

Introduction

Neurons obtain glycolysis products and thus metabolic support from glial cells in species ranging from mice (Nave and Werner, 2014; Magistretti and Allaman, 2015) to flies (Schirmeier et al., 2015; Volkenhoff et al., 2015). This has founded the concept that the metabolic symbiosis of neurons and glia may be principally conserved throughout the evolution of bilaterian nervous systems. In the murine central nervous system (CNS), lactate and/or pyruvate are shuttled to neurons from both astrocytes (Suzuki et al., 2011; Weber and Barros, 2015) and oligodendrocytes (Funfschilling et al., 2012; Lee et al., 2012). The latter cell type additionally provides many axons with multiple layers of ensheathing myelin membrane and thus insulation, thereby restricting action potentials to the nodes of Ranvier and accelerating nerve conduction. In the peripheral nervous system (PNS), axons benefit from the support by Schwann cells with respect to energy homeostasis (Beirowski et al., 2014; Viader et al., 2013), myelination, and other physiological and pathophysiological functions (Stolt and Wegner, 2015; Klein and Martini, 2015; Jessen et al., 2015; Salzer, 2015).

In the vertebrate lineage, myelin has evolutionarily emerged in ancestral placoderms over 400 million years ago (Werner, 2013; Zalc et al., 2008; Zalc, 2015), at approximately the same time as hinged jaws (Kuratani, 2012). The most ancient living myelinated vertebrates are thus cartilaginous fish (sharks and rays) (de Bellard, 2016). However, the ultrastructure and major constituents of myelin underwent further evolutionary changes (Yoshida and Colman, 1996; Inouye and Kirschner, 2015). Indeed, the phylogenetic relationships of myelin proteins can now be well calculated, facilitated by the availability of sequence data from a continuously increasing range of species (Möbius et al., 2008; Nawaz et al., 2013; Gould et al., 2005; Shypitsyna et al., 2011; Myllykoski et al., 2015; Li and Richardson, 2015). However, most functional and genetic analyses of myelination and axoglial interactions are performed in mice owing to the easy access to the required molecular tools. Only more recently, zebrafish became an equally relevant model species in myelin research (Brosamle and Halpern, 2002; Ackerman and Monk, 2015), which allows a spectrum of advantageous approaches (Czopka and Lyons, 2011) including unbiased genetic screening (Pogoda et al., 2006; Kazakova et al., 2006) and comparatively straightforward live-imaging of myelin biogenesis and adaptation in the developmentally transparent fish (Snaidero et al., 2014; Hines et al., 2015; Baraban et al., 2015; Mensch et al., 2015). On the other hand, the limited availability of antibodies against zebrafish proteins needs to be overcome by the generation of suitable transgenic reporter lines (Czopka, 2016; Preston and Macklin, 2015). Notably, the genetics of zebrafish is more complex compared to other vertebrate groups due to the whole-genome duplication at the root of teleost fish, which resulted in the existence of numerous duplicate genes in zebrafish including those encoding proteolipid protein (PLP), myelin basic protein (MBP), and reticulon 4 (RTN4/NOGO) (Nawaz et al., 2013; Schweitzer et al., 2006; Pinzon-Olejua et al., 2014).

Taken together, mice and zebrafish continue to be the most frequently used model species to investigate the structure, function, regulation and adaptation of myelin. Interestingly, this includes evolutionary aspects, not because these species would be particularly relevant models of myelin evolution but owing to the existence of molecular and genetic tools. For example, myelin proteome analysis (de Monasterio-Schrader et al., 2012; Jahn et al., 2009; Patzig et al., 2011; Werner et al., 2007) remains much more efficient in mice, humans and zebrafish compared to all other myelinated species owing to the accessibility of extensive sequence data required for the annotation of mass spectra. We thus find it unlikely that other relevant model species of evolutionary biology – such as cartilaginous fish, lungfish or marsupials-can soon catch up with mice or zebrafish with respect to molecular tools available for myelin-related research, notwithstanding that genome sequencing is now applied to a larger range of species.

Here, we briefly review the recent application to myelin research of nervous tissue fixation by high-pressure freezing and freeze substitution in the preparation of samples for transmission electron microscopy (Fig. 1). We highlight publications in which this novel visualization technique has proven valuable for investigating the biogenesis and pathology of myelin in mice. We then show that the technique can also be applied to aquatic model species including zebrafish (Danio rerio). However, the technique׳s independency of the availability of sequence data, antibodies or transgenic lines makes it readily applicable to other species. For illustration, we conclude this review by looking at the ventral nerve cord of an invertebrate species, the earthworm Lumbricus terrestris, in which giant axons are ensheathed with multiple membrane layers.