Repetitive propagation of action potentials destabilizes the structure of the myelin sheath. A dynamic x-ray diffraction study

Time courses of myelin lattice swelling in toad sciatic nerves preexposed to different treatments were determined by x-ray diffraction using a one-dimensional position-sensitive detector. In the nerves supramaximally stimulated for 1 h at 200 Hz, the subsequent process of myelin swelling occurred 45.0 +/- 7.3 min (n = 24) sooner than in resting controls. Sciatic nerves incubated for 1 h in a Ringer's solution deprived of divalent cations (Ca++ and Mg++) exhibited a kinetics of swelling similar to that shown by the stimulated nerves, that is, 52.5 +/- 14.2 min (n = 6) sooner than controls preincubated for the same time in normal Ringer's solution (with divalent cations). The fact that both pretreatments supramaximal stimulation and removal of divalent cations from the perfusion solution produced a similar effect; namely, a decrease of the myelin lattice stability against swelling in distilled water, suggests that the repetitive propagation of action potentials could modify the ionic composition at either the intraperiod channel or the paranodal axoglial junction complexes.     

Effects of ZnCl2 on membrane interactions in myelin of normal and shiverer mice

X-ray diffraction was used to record the effects of metal cations on the structure of peripheral nerve myelin. Acidic saline (pH 5.0) either with or without added metal cations caused myelin to swell by 10-20 A from its native period of 178 A. The X-ray patterns usually showed broad reflections, and higher orders were either weak or unobserved. With added ZnCl2, however, the swollen myelin gave diffraction patterns that retained sharp reflections to approx. 15 A spacing. Alkaline saline (pH 9.7) containing ZnCl2 produced a reduction of the myelin period by approx. 5 A which was at least twice as much as that produced by other metals. To examine the underlying chemical basis for these unique interactions of Zn2+ with myelin, we carried out parallel X-ray experiments on sciatic nerve from the shiverer mutant mouse, which lacks the major myelin basic proteins. Shiverer myelin responded like normal myelin to ZnCl2 in acidic saline; however, in alkaline saline shiverer myelin showed broadened X-ray reflections which indicated disordering of the regularity of the membrane arrays, and additional reflections were recorded which indicated lipid phase separation. This breakdown may come about by the binding of Zn2+ to negatively-charged lipids which could be more exposed due to the absence of myelin basic proteins. Electron density profiles were calculated on the assumption that, except for changes in their packing, the myelin membranes were minimally altered in structure. For both normal and shiverer myelins, treatments under acidic conditions resulted in swelling at the extracellular apposition and a slight narrowing of the cytoplasmic space. This swelling is likely due to adsorption of protons and divalent cations. Interaction between Zn2+ and myelin P0 glycoprotein could preserve an ordered arrangement of the apposed membrane surfaces. Alkaline saline containing ZnCl2 produced compaction at the cytoplasmic apposition in both normal and shiverer myelins possibly through interactions with a portion of P0 glycoprotein which extends into the cytoplasmic space between membranes.     

Myelin labeled with mercuric chloride. Asymmetric localization of phosphatidylethanolamine plasmalogen

We have recorded modified X-ray diffraction patterns to 15 Å spacing from sciatic nerves treated with mercuric chloride (HgCl₂) at concentrations of 0.5 to 32 mm in water or in saline. The observed changes in repeat period and in the intensities of the low-order reflections indicate closer packing of membranes at their cytoplasmic surfaces after treatments with HgCl₂. In addition, HgCl₂ at 0.25 mm or more prevents swelling in water at the extracellular boundaries. By comparing the distinctive diffraction patterns from nerves treated under different conditions with HgCl₂, we have interpreted the changes in intensities of the higher order X-ray reflections and have calculated electron density profiles of the modified membranes. The most striking difference between membrane profiles before and after treatment with HgCl₂ is the large increase in electron density in the region of the lipid headgroup peak in the cytoplasmic half of the bilayer. The magnitude and location of this increase suggests labeling of myelin lipid. To examine this possibility, we analyzed the lipids from mercury-treated sciatic nerves.Thin-layer chromatography of lipids extracted from nerves treated with HgCl₂ shows a marked decrease of phosphatidylethanolamine, which exists in myelin primarily as plasmalogen. At the same time, a new spot identified as lysophosphatidylethanolamine appears. An identical result was obtained by treating extracted lipids with HgCl₂, suggesting that the same sites of interaction are present in the intact membrane as in the dispersed lipids. Previous studies on plasmalogens indicate that mercury adds to the β-carbon of the α,β-unsaturated ether group to produce a lyso-lipid and an aldehyde with bound mercury (Norton, 1959). From a correlation of our X-ray structural analysis and the chemical studies, we conclude that phosphatidylethanolamine plasmalogen is preferentially localized in the cytoplasmic half of the myelin membrane bilayer.     

The action of local anesthetics on myelin structure and nerve conduction in toad sciatic nerve

X-ray scattering and electrophysiological experiments were performed on toad sciatic nerves in the presence of local anesthetics. In vitro experiments were performed on dissected nerves superfused with Ringer's solutions containing procaine, lidocaine, tetracaine, or dibucaine. In vivo experiments were performed on nerves dissected from animals anesthesized by targeted injections of tetracaine-containing solutions. In all cases the anesthetics were found to have the same effects on the x-ray scattering spectra: the intensity ratio of the even-order to the odd-order reflections increases and the lattice parameter increases. These changes are reversible upon removal of the anesthetic. The magnitude of the structural changes varies with the duration of the superfusion and with the nature and concentration of the anesthetic molecule. A striking quantitative correlation was observed between the structural effects and the potency of the anesthetic. Electron density profiles, which hardly showed any structural alteration of the unit membrane, clearly indicated that the anesthetics have the effect of moving the pairs of membranes apart by increasing the thickness of the cytoplasmic space. Electrophysiological measurements performed on the very samples used in the x-ray scattering experiments showed that the amplitude of the compound action potential is affected earlier than the structure of myelin (as revealed by the x-ray scattering experiments), whereas conduction velocity closely follows the structural alterations.     

X-ray diffraction study of myelin structure in immature and mutant mice

X-ray diffraction patterns were obtained from freshly dissected central and peripheral nerves of quaking, myelin synthesis deficiency ($\text{msd}$), and trembler mutants, as well as immature and adult normal mice. The patterns were compared with respect to strength of myelin diffraction, background scatter level, repeat period, and intensity and linewidth of Bragg reflections. The deficiency of myelin in optic nerves was found to be (in decreasing severity): quaking > immature > trembler ? normal adult; and in sciatic nerves: $\text{trembler > immature > quaking}\text{msd ?}\text{normal}$ adult. Repeat periods about 3 Å less than that for normal adult sciatic myelin were detected in corresponding nerves from immature, quaking, and trembler mice. In some trembler sciatic nerves a second phase having a 190–200 Å period and accounting for about 60% of the total ordered myelin was also evident. Comparison of electron density profiles of membrane units calculated from the repeat periods and diffracted intensities for sciatic myelins indicate structural differences at the molecular level. The main findings are: (1) quaking myelin shows a significant elevation of density in the external protein-water layer between membrane bilayers; (2) the membrane bilayer of immature myelin is ≈ 2 Å thinner than that for normal adult; (3) the membrane bilayer of the more compact phase in trembler myelin is ≈ 5 Å thinner than for normal; and (4) the difference in repeat periods for the two phases present in some of the trembler nerves can be accounted for predominantly by distinct membrane bilayer separations at the external boundary.     

Myelin Structure and Composition in Zebrafish

To establish a standard for genotype/phenotype studies on the myelin of zebrafish (Danio rerio), an organism increasingly popular as a model system for vertebrates, we have initiated a detailed characterization of the structure and biochemical composition of its myelinated central and peripheral nervous system (CNS; PNS) tissues. Myelin periods, determined by X-ray diffraction from whole, unfixed optic and lateral line nerves, were approximately 153 and approximately 162 Angstrom, respectively. In contrast with the lability of PNS myelin in higher vertebrates, zebrafish lateral line nerve myelin exhibited structural stability when exposed to substantial changes in pH and ionic strength. Neither optic nor lateral line nerves showed swelling at the cytoplasmic apposition in CaCl(2)-containing Ringer's solution, in contrast with nerves from other teleost and elasmobranch fishes. Zebrafish optic nerve showed greater stability against changes in NaCl and CaCl(2) than lateral line nerve. The nerves from zebrafish having mutations in the gene for myelin basic protein (mbpAla2Thr and mbpAsp25Val) showed similar myelin periods as the wildtype (WT), but gave approximately 20% less compact myelin. Analysis of proteins by SDS-PAGE and Western blotting identified in both CNS and PNS of WT zebrafish two orthologues of myelin P0 glycoprotein that have been characterized extensively in trout--intermediate protein 1 (24 kDa) and intermediate protein 2 (28 kDa). Treatment with endoglycosidase-F demonstrated a carbohydrate moiety of approximately 7 kDa, which is nearly threefold larger than for higher vertebrates. Thin-layer chromatography for lipids revealed a similar composition as for other teleosts. Taken together, these data will serve as a baseline for detecting changes in the structure and/or amount of myelin resulting from mutations in myelin-related genes or from exogenous, potentially cytotoxic compounds that could affect myelin formation or stability.     

Compaction and particle segregation in myelin membrane arrays

Compacted membrane arrays are formed in the nerve myelin sheath by lowering the water activity (through evaporation or immersion in hypertonic solutions of nonelectrolytes or monovalent salts) or by binding specific cations (Ca(++), La(+++), and tetracaine at concentrations above 5-10 mM). X-ray diffraction observations on intact, hydrated nerves treated to induce compaction provide a control to assess the significance of structural changes seen by freeze-fracture electron microscopy. Compaction inevitably leads to lateral segregation of particles away from the closely packed membrane arrays into contiguous normal, or slightly expanded, period arrays. In the particle-enriched layers, the E fracture face is more particle-dense than the P face, whereas no particles are found on either face in the compacted layers. Morphologically, compaction induced by the all-or-nothing, relatively irreversible action of specific cations cannot be distinguished from compaction to the same extent induced by the graded, reversible effects of nonelectrolytes. Compaction by sodium chloride resembles that by specific- cation binding in that the repeat period is independent of reagent concentration; but, like dehydration by nonelectrolytes, the extent of compaction is reversibly related to reagent concentration. Sodium chloride-compacted myelin can be distinguished morphologically by a lack of the elongated border particles at the boundary between smooth and particle-enriched membrane observed for other compacting treatments. Fracture faces in compacted arrays are not always smooth, but the unusual appearances can be duplicated in purified myelin lipid multilayers subjected to similar treatments, which indicates that the particle-free membrane fracture faces are uninterrupted lipid hydrocarbon layers. Correlation of x-ray diffraction and electron microscopy observations provides a direct basis for identifying the intramembrane particles with transmembrane protein. The transmembrane protein appears to play a significant role in maintaining the normal membrane separation; swelling of the particle-enriched arrays in myelin compacted by tetracaine at low ionic strength provides information about the charge distribution on the transmembrane protein. Swelling of the compacted arrays following irreversible particle segregation shows that the interaction properties of the particle-free membranes are similar to those of pure lipid multilayers. Compaction and the consequent particle segregation in lyelin results from conditions stabilizing close apposition of the lipid bilayers. Particle segregation in areas of close contact between other cell membranes may also be driven by interbilayer attractive forces.     

X-ray observations on a collagen fibril lattice structure in peripheral nerve

It has been known for more than two decades that peripheral nerve shows X-ray reflections other than those originating from the myelin sheath. These extra reflections are at small angles of diffraction and arise from a variation of electron density in the radial direction. X-ray diffraction studies since 1973 have identified these reflections as coming from a lattice structure of filaments, the filament axes are parallel to the nerve fibre axis. The present X-ray observations were obtained using a high-resolution X-ray camera and we conclude that these reflections arise from a collagen fibril lattice structure within peripheral nerve. Estimates of fibril radius and the separation distance between fibrils in intact rat, rabbit and frog sciatic nerves have been obtained using X-ray analysis.     

Shiverer and Normal Peripheral Myelin Compared: Basic Protein Localization, Membrane Interactions, and Lipid Composition

We have correlated membrane structure and interactions in shiverer sciatic nerve myelin with its biochemical composition. Analysis of x-ray diffraction data from shiverer myelin swollen in water substantiates our previous localization of an electron density deficit in the cytoplasmic half of the membrane. The density loss correlates with the absence of the major myelin basic proteins and indicates that in normal myelin, the basic protein is localized to the cytoplasmic apposition. As in normal peripheral myelin, hypotonic swelling in the shiverer membrane arrays occurs in the extracellular space between membranes; the cytoplasmic surfaces remain closely apposed notwithstanding the absence of basic protein from this region. Surprisingly, we found that the interaction at the extracellular apposition of shiverer membranes is altered. The extracellular space swells to a greater extent than normal when nerves are incubated in distilled water, treated at a reduced ionic strength of 0.06 in the range of pH 4-9, or treated at constant pH (4 or 7) in the range of ionic strengths 0.02-0.20. To examine the biochemical basis of this difference in swelling, we compared the lipid composition of shiverer and normal myelin. We find that sulfatides, hydroxycerebroside, and phosphatidylcholine are 20-30% higher than normal; nonhydroxycerebroside and sphingomyelin are 15-20% lower than normal; and ethanolamine phosphatides, phosphatidylserine, and cholesterol show little or no change. A higher concentration of negatively charged sulfatides at the extracellular surface likely contributes to an increased electrostatic repulsion and greater swelling in shiverer. The cytoplasmic surfaces of the apposed membranes of normal and shiverer myelins did not swell apart appreciably in the pH and ionic strength ranges expected to produce electrostatic repulsion. This stability, then, clearly does not depend on basic protein. We propose that P0 glycoprotein molecules form the stable link between apposed cytoplasmic membrane surfaces in peripheral myelin.     

Membrane Interactions Are Altered in Myelin Isolated from Central and Peripheral Nervous System Tissues

Isolated myelin has been used for determinations of membrane surface charge density and topographical mapping of components in the membrane. To determine how similar such myelin is to myelin of intact tissue, we have used x-ray diffraction to compare their intermembrane interactions. The interactions were monitored by measuring the myelin period in samples treated with distilled water, buffered saline at pH 4-9 and ionic strength 0.06-0.18, and saline containing HgCl2 or triethyl tin sulfate. Myelin was isolated from whole brains and sciatic nerves of mice by conventional methods involving sucrose gradient centrifugation and osmotic shock. Consistent with previous findings, electron microscopy showed that the multilamellar morphology, staining, and repeat periods of isolated myelin were essentially like those of intact myelin; however, the membrane stacks were less extensive than those in whole tissue. X-ray diffraction revealed that isolated CNS myelin was like intact myelin in showing reversible compaction in acidic media and in distilled water. However, unlike the myelin in whole tissue, isolated CNS myelin did not swell in hypotonic or alkaline media, or in the presence of HgCl2-saline or triethyl tin. The altered membrane interactions could result from an increase in adhesiveness of the apposed membrane surfaces. Reorganization of proteolipid protein and/or a reduction of surface charge could account for the change in surface properties of isolated CNS myelin. Isolated PNS myelin, like the membranes in whole tissue, showed both compaction and swelling; however, the membrane pairs were disordered in the swollen structure. This irregular membrane swelling could result from charge variation in the extracellular surfaces.     

Reversible changes in myelin structure and electrical activity during anesthesia in vivo

X-ray diffraction patterns have been recorded from sciatic nerve myelin by means of dynamic X-ray diffraction either from frogs, during the early stages of anesthesia in vivo induced by n-pentane inhalation, and from frog and rat sciatic nerves isolated immediately after the animal was anesthetized. This approach has enabled to resolve minor changes in myelin structure that occur during anesthesia which were found to be similar in frogs and mammals. The X-ray patterns show a reversible slight decrease in intensity of the even reflections during anesthesia. The electron density profiles from myelin of anesthetized and recovered nerves revealed that the unit membrane structure is practically identical in both circumstances. However, during anesthesia myelin membrane pairs move toward the cytoplasmic side becoming more closely packed by 1.6 A. Physiological activity was estimated during the recovery process: compound action potential recovered its maximal amplitude before myelin recovered its native structure. On the contrary, the conduction velocity seemed to be closely related to the structural recovery. This work provides evidence that early stages of anesthesia by n-pentane in vivo does not change membrane bilayer structure but perturbs the surface interactions between adjacent membrane pairs.     

The swelling property of central nervous system nerves using x-ray diffraction.

Low-angle X-ray diffraction patterns have been recorded from cattle and rabbit optic nerves swollen in glycerol solutions. The new X-ray data have a resolution of about 15 to 16 Å. Analysis of the low-angle X-ray data indicates that the myelin layers of optic nerves swell in units of four membranes, that is, two membrane pairs adhere together during the process of swelling. Fourier syntheses of glycerol-treated cattle optic nerves are described. Differences in structure between the normal and swollen membrane pairs are apparent.     

Membrane structure in isolated and intact myelins.

The biochemical composition of myelin and the topology of its constituent lipids and proteins are typically studied using membranes that have been isolated from whole, intact tissue using procedures involving hypotonic shock and sucrose density gradient centrifugation. To what extent, however, are the structure and intermembrane interactions of isolated myelin similar to those of intact myelin? We have previously reported that intact and isolated myelins do not always show identical myelin periods, indicating a difference in membrane-membrane interactions. The present study addresses the possibility that this is due to altered membrane structure. Because x-ray scattering from isolated myelin sometimes consists of overlapping Bragg reflections or is continuous, we developed nonlinear least squares procedures for analyzing the total intensity distribution after film scaling, background subtraction, and Lorentz correction. We calculated electron density profiles of isolated myelin for comparison with membrane profiles from intact myelin. The change in the width of the extracellular space and the relative invariance of the cytoplasmic space as a function of pH and ionic strength that we previously found for intact nerve was largely paralleled by isolated myelin. There were two exceptions: isolated CNS myelin was resistant to swelling under all conditions, and isolated PNS myelin in hypotonic saline showed indefinite swelling at the extracellular apposition. However, electron density profiles of isolated myelins, calculated to 30 A resolution, did not show any major change in structure compared with intact myelin that could account for the differences in interactions.     

The regulation of ATPase-ATPase interactions in sarcoplasmic reticulum membrane. I. The effects of Ca2+, ATP, and inorganic phosphate

Two-dimensional crystalline arrays of Ca2+-ATPase molecules develop after treatment of sarcoplasmic reticulum vesicles with Na3VO4 in calcium-free medium (Dux, L., and Martonosi, A. (1983) J. Biol. Chem. 258, 2599-2603). The formation of Ca2+-ATPase crystals is inhibited by Ca2+ (2 microM), or ATP (5 mM), but not by ADP, 5'-adenylylimidodiphosphate, or adenylylmethylenediphosphonate. ATPase crystals did not form at 37 degrees C and exposure of preformed crystals to 37 degrees C for 1 h caused the disappearance of crystal lattice. Inorganic orthophosphate (1 mM at pH 6.0) promoted the formation of a distinct crystal form of Ca2+-ATPase, which was different from that produced by Na3VO4. These observations indicate that Ca2+, ATP, inorganic phosphate, pH, and temperature influence the interactions between ATPase molecules in the sarcoplasmic reticulum membrane.     

The in vivo synthesis of myelin proteins in rabbit sciatic nerve

Rabbits were injected into the sciatic nerves with either ³⁵S-methionine, or ³H-fucose. After times ranging from 45 min to 15 days the nerves were removed and the total particulate material from the nerves fractionated to give seven subfractions with densities between 0.2 and 1.2 M sucrose. The patterns of radio-labelled proteins were examined by SDS-PAGE and quantitative fluorography. The results showed that the P₂ basic protein was metabolically far more active than either the major P₀ glycoprotein, or the basic protein BP. The P₂ protein also entered the myelin fractions more rapidly than either P₀, or BP components. The net synthesis of P₀ was slower than P₂ and BP and this intrinsic membrane protein remained associated with the denser membrane fractions (>0.7 M sucrose) for longer than the basic proteins prior to entering myelin. Newly synthesized high molecular weight proteins remained concentrated in the denser membrane fractions and turned over faster than the myelin proteins.

A low density myelin fraction (B) was detected in which both the P₂ protein and certain high molecular weight proteins became more rapidly labelled than in compact myelin. In this fraction the specific activity remained higher than that of compact myelin for up to five days after the injection of ³⁵S-methionine into the nerve.

The results indicate that the major PNS myelin proteins are incorporated into and turn over in the various compartments of the Schwann cell plasma membrane—myelin continuum at very different rates.     

X-ray diffraction of myelin membrane. I. Optimal conditions for obtaining unmodified small angle diffraction data from frog sciatic nerve

The X-ray diffraction pattern of myelin of frog sciatic nerve has been investigated, using a Kratky small angle slit camera to obtain the electron density distribution across the membrane. All major reflections observed were related to a fundamental repeat distance of 171 ± 2.8 A. There was no further increase in the number of reflections on varying the experimental conditions (varying pH, applying tension, immersion in various isotonic buffer solutions, etc.) or by varying the camera slit arrangement. The degree of disorder within the myelin sheath was examined by comparing the crystallite size to the half-width of the diffraction peak at half-height. The limiting of the diffraction spectra to five major reflections was determined not to be caused by disorder. It is concluded that the observed X-ray diffraction pattern is a consequence of the particular electron density distribution of the membrane. Therefore, the membrane cannot contain sharply distinct step-function regions of electron density, but approaches a modified cosine distribution.     

An X-ray study of the effect of enzymes on frog sciatic nerve myelin

Low-angle X-ray diffraction patterns have been recorded from frog sciatic nerve after digestion with trypsin and Pronase. Reproducible X-ray patterns were obtained by swelling the nerves in distilled water before treatment with enzymes. The X-ray patterns of enzyme-treated nerves are distinctly different from the X-ray pattern of normal (live) nerve. It would appear that the normal asymmetric nerve myelin membrane becomes symmetric about its center after treatment with enzymes as a result of proteolytic cleavage and a subsequent redistribution of protein components.     

Phenobarbital induces slow recovery from sodium inactivation at the nodal membrane

Voltage-clamp experiments were performed on single myelinated nerve fibres of Rana esculenta at 20 degrees C in Ringer's solution and in solutions containing phenobarbital-sodium ([PB] less than or equal to 5 mM). The reduction of the sodium current under phenobarbital could be explained by an increase in the resting sodium inactivation; h infinity (E) was shifted towards more negative membrane potentials. The recovery from sodium inactivation proceeded with two time constants. The fast process could be described with the same time constant as in Ringer's solution, whereas the slow process had a time constant approx. 40 times larger. The slow process was also potential-dependent and could be described by 1/(0.025 alpha h + beta h), where alpha h and beta h denoted the rate constants in Ringer's solution. With the measured blockage of sodium channels by phenobarbital, both the shift of h infinity (E) and the slow recovery from sodium inactivation could be explained.     

The isolation and characterization of a plasma membrane and a myelin fraction derived from oligodendroglia of calf brain.

—A method is described for the fractionation of bulk isolated oligodendroglial cells from calf brain to produce both a plasma membrane and an attached myelin fraction. The cells are homogenized in a sucrose solution containing Mg²⁺ and K+ at a pH of 6·5. Crude membrane fractions are obtained from this homogenate by discontinuous sucrose density gradient centrifugation. After being subjected to osmotic shock, these fractions are purified by continuous sucrose density gradient centrifugation. The plasma membrane fraction, which bands at 1·0 m -sucrose, was identified by its morphology and enzyme content. Electron microscopy showed it to be a homogeneous preparation of vesicles composed, for the most part, of smooth trilaminar membranes. Enzymatic analysis revealed the presence of high specific activities of Na+, K+-ATPase, 5′-nucleotidase and 2′,3′-cyclic AMPase. Lipid analysis showed a higher galactolipid and lower phospholipid content than has been reported for neuronal and synaptic membranes. The attached myelin fraction, which bands at 0·7 m -sucrose has the typical multilamellar appearance of myelin, but differs considerably from normal myelin in having high concentrations of plasma membrane marker enzymes, and a lipid composition intermediate between normal myelin and the plasma membrane fraction. The ganglioside content and protein patterns of these fractions have also been examined.