Malnutrition and myelin structure: an X-ray scattering study of rat sciatic and optic nerves

Taking advantage of the fast and accurate X-ray scattering techniques recently developed in our laboratory, we tackled the study of the structural alterations induced in myelin by malnutrition. Our work was performed on sciatic and optic nerves dissected from rats fed with either a normal or a low-protein caloric diet, as a function of age (from birth to 60 days). By way of electrophysiological controls we also measured (on the sciatic nerves) the height and velocity of the compound action potential. Malnutrition was found to decrease the amount of myelin and to impair the packing order of the membranes in the sheaths.     

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.     

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.     

Structural and electrophysiological effects of local anesthetics and of low temperature on myelinated nerves: implication of the lipid chains in nerve excitability

X-ray scattering and electrophysiological experiments performed on toad sciatic nerves as a function of the exposure to either low temperature or tetracaine yielded the following results: (i) the main structural effect is to thicken the individual membranes, thus to stiffen the acyl chains and increase the repeat distance of the one-dimensional lattice, phenomena that are typical of lipid-containing systems with disordered chains; (ii) the electrophysiological effect is to decrease the amplitude and velocity of the compound action potential; (iii) the structural and physiological effects of the two agents are practically identical. Since the structural and the electrophysiological parameters have different origins in the nerves (the structure regards the myelin sheath, the electrical signals originate at the nodes of Ranvier) it is inferred that tetracaine and low temperature exert similar effects on the membranes of both the myelin sheath and the nodes of Ranvier. Also, since local anesthetics act by inhibiting the Na+ channels, these observations suggest that the acyl chain conformation modulates the channel function and thus the generation of action potential.     

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.     

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.     

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.     

Order-disorder phenomena in myelinated nerve sheaths. II. The structure of myelin in native and swollen rat sciatic nerves and in the course of myelinogenesis

The algorithm described in the accompanying paper was applied to X-ray scattering experiments performed with rat sciatic nerves, either as a function of the age of the animal (4 to 30 days), or with adult nerves swollen in non-isotonic media. The results were all consistent with the model of disorder used in the theoretical treatment. The algorithm leads, in one step, from the data to the numerical values of the parameters, avoiding all intermediate manipulation. For each experiment a variety of parameters was determined: the average D and the variance sigma 2D of the repeat distance, the average number [N] of motifs per crystallite, the set [idiff(h/D)], which defines the diffuse scattering, the fraction alphaloose of myelin that does not belong to the compact sheaths, and the set [imotif (k/2D)], which suffices to define the continuous intensity curve of the motif imotif(s). Note the remarkable wealth of information, especially by contrast with conventional analyses which, as a rule, only yield the values of D and of the set [imotif(h/D)] (insufficient to determine the function imotif(s]. The function imotif(s) and the parameters D and sigma D (and thus the local structure of the myelin sheaths) were shown to be almost invariant in the course of myelinogenesis; what varies is mainly the total amount of myelin in the nerve and the number of membranes per sheath. Swelling agents have a dramatic influence on the X-ray scattering spectra, but in spite of the conspicuous variation of D, sigma D and [N] the structure of the motif is invariant. The structure of the motif was shown to be quite different in the native and in the swollen samples; the stacking disorder appears to involve mainly the cytoplasmic space in native myelin, the external space in swollen nerves. The very notion of electron density profile, when disorder is present, is discussed. Two criteria were proposed to select the "best" signs of the reflections: two sets came out at almost the same rank, one corresponding to Caspar & Kirschner's the other to Worthington & McIntosh's proposals, neither of which can be ruled out according to the criteria used in this work.     

Physical structure of the excitable membrane of unmyelinated axons: X-ray scattering study and electrophysiological properties of pike olfactory nerve

The aim of this work was to elicit correlations between physical structure and physiological functions in excitable membranes. Freshly dissected pike olfactory nerves were studied by synchrotron radiation X-ray scattering experiments and their physiological properties were tested by electrophysiological techniques. The scattering spectra contained a sharply oriented equatorial component (i.e. normal to the nerve axis), and an isotropic background. After background subtraction, the equatorial component displayed a weak and fairly sharp spectrum of oriented microtubules, and a strong and diffuse band of almost the same shape and position as the band computed for an isolated myelin membrane. We ascribed this spectrum to the axonal membranes. Under the action of temperature and of two local anesthetics, the spectrum underwent a contraction (or expansion) in the s-direction, equivalent to the structure undergoing an expansion (or contraction) in the direction perpendicular to the plane of the membrane. The main observations were: (i) with increasing temperature, membrane thickness decreased with a thermal expansion coefficient equal to -0.97(+/-0.19) 10(-3) degrees C(-1). The polarity and amplitude of this coefficient are typical of lipid-containing systems with the hydrocarbon chains in a disordered conformation. The amplitude and propagation velocity of the compound action potentials were drastically and reversibly reduced by lowering the temperature from 20 degrees C to 5 degrees C. (ii) Exposing the nerve to two local anesthetics (tetracaine and dibucaine) had the effect of decreasing membrane thickness. Action potentials were fully inhibited by these anesthetics. (iii) Upon depolarization, induced by replacing NaCl with KCl in the outer medium, approximately 25 % of the membranes were found to associate by apposing their outer faces. Electrophysiological activity was reversibly impaired by the KCl treatment. (iv) No detectable structural effect was observed upon exposing the nerves to tetrodotoxin or veratridine. Electrophysiological activity was fully impaired by tetrodotoxin and partially impaired by veratridine. The main conclusions of this work are that axonal membranes yield highly informative X-ray scattering spectra, and that these spectra are sensitive to the functional state of the nerve. These results pave the way to further studies of more direct physiological significance.     

THE EFFECTS OF UNDERNUTRITION ON THE PROTEINS OF OPTIC AND SCIATIC NERVES DURING DEVELOPMENT

Abstract— Polyacrylamide gel electrophoresis has been used to assess the appearance of some optic and sciatic nerve proteins in normal developing rats and in undernourished rats. Of the myelin proteins, the'Wolfgram'proteolipid is already present about the time myelination begins. The basic myelin proteins appear later, first in sciatic and then in optic nerve. A non-myelin basic protein, assumed to be a histone, is present at high levels in both nerves before myelination begins. There is no apparent effect of undernutrition on the appearance and amount of myelin proteins at 12, 16 and 22 days of age. The'histone'protein is reduced in optic and sciatic nerves at times corresponding roughly to the transition periods from cellular proliferation to myelin formation. The possibilities are discussed that myelin basic proteins are synthesized as compact myelin formation occurs, and that there may be retarded cellular proliferation in nerves of undernourished rats.     

Divalent cations cooperatively stabilize close membrane contacts in myelin.

Intact nerve myelin compacts to a dehydrated structure of closely apposed membranes when exposed to isotonic solutions at least 10 mM in calcium or tetracaine. The repeat period of the membrane pair in the compacted structure measured by X-ray diffraction is about 126 A in both central and peripheral mammalian nerve myelins whereas the normal periods are about 158 and 178 A, respectively. The electron density profile of compacted myelin shows an asymmetric membrane unit with thickness similar to that of the symmetric bilayer of flocculated myelin lipids. The centrosymmetrically averaged myelin membrane profile is similar to that of the lipid bilayer except at the surface where residual protein is concentrated. Dispersions of extracted total myelin lipids flocculate under similar conditions to those causing myelin compaction, indicating that similar forces act in both processes. Compaction is always accompanied by lateral segregation of intramembrane particles out of the close-packed domains. Lateral displacement of intramembrane proteins form compacted domains can be driven by the attraction of the lipid surfaces for each other. Rates of compaction vary with compacting reagent, concentration, tissue, and temperature, and probably reflect the permeability of the tissue. Extensive compaction by calcium or tetracaine leads to disruption and vesiculation of the spirally wrapped myelin membranes.     

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.     

Divalent cations cooperatively stabilize close membrane contacts in myelin

Intact nerve myelin compacts to a dehydrated structure of closely apposed membranes when exposed to isotonic solutions at least 10 mM in calcium or tetracaine. The repeat period of the membrane pair in the compacted structure measured by X-ray diffraction is about 126 Å in both central and peripheral mammalian nerve myelins whereas the normal periods are about 158 and 178 Å, respectively. The electron density profile of compacted myelin shows an asymmetric membrane unit with thickness similar to that of the symmetric bilayer of flocculated myelin lipids. The centrosymmetrically averaged myelin membrane profile is similar to that of the lipid bilayer except at the surface where residual protein is concentrated. Dispersions of extracted total myelin lipids flocculate under similar conditions to those causing myelin compaction, indicating that similar forces act in both processes. Compaction is always accompanied by lateral segregation of intramembrane particles out of the close-packed domains. Lateral displacement of intramembrane proteins from compacted domains can be driven by the attraction of the lipid surfaces for each other. Rates of compaction vary with compacting reagent, concentration, tissue, and temperature, and probably reflect the permeability of the tissue. Extensive compaction by calcium or tetracaine leads to disruption and vesiculation of the spirally wrapped myelin membranes.     

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.     

The non-synchronous synthesis of myelin components during early stages of myelination in the rat optic nerve.

—Analysis of lipids and proteins in whole optic nerves during initial stages of myelination indicate a sequential deposition of myelin components. During this period sulfatide levels increase from 4 nmol/pair of nerves at 9 days to 29 nmol/pair of nerves at 21 days. Cerebrosides are not detected until the 12th day and attain a level of 46 nmol/pair of nerves in the 21-day old rat. Plasma-logen values increase from 2.1 to 31 nmol/pair of nerves and cholesterol values from 12 to 158 nmol/pair of nerves between 9 and 21 days. High molecular weight proteins are present in samples from 9-day old rats analyzed by discontinuous sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Under identical conditions myelin basic proteins are first observed in the 10 day sample, and the proteolipid protein is first detected in the 12 day sample. The 2′, 3′-cyclic nucleotide 3′-phosphohydrolase activity increases from 1.13 to 5.50 μmol/min per mg protein between 9 and 21 days.     

Evidence for the Association of 2',3'-Cyclic-Nucleotide 3'-Phosphodiesterase with Myelin-Related Membranes in Peripheral Nervous System

Abstract: In PNS, the specific activity of 2′,3′-cyclic nucleotide 3′-phospho–diesterase (CNP) in myelin was not enriched over the starting homogenate. Nevertheless, most of the total activity was recovered in myelin. In myelin-deficient mutants, low CNP activities were measured in sciatic nerves. CNP specific activities were similar in myelinated and non-myelinated nerves but in non-nervous tissues, they were significantly lower than in nervous tissue. There was no indication for the presence of an isoenzyme of CNP in peripheral nerves. These results indicate that CNP is present in PNS myelin and preferentially localized in Schwann cell plasma membranes.     

Myelin Abnormalities in the Optic and Sciatic Nerves in Mice With GM1-Gangliosidosis

GM1-gangliosidosis is a glycosphingolipid lysosomal storage disease involving accumulation of GM1 and its asialo form (GA1) primarily in the brain. Thin-layer chromatography and X-ray diffraction were used to analyze the lipid content/composition and the myelin structure of the optic and sciatic nerves from 7- and 10-month old β-galactosidase (β-gal) +/? and β-gal −/− mice, a model of GM1gangliosidosis. Optic nerve weight was lower in the β-gal −/− mice than in unaffected β-gal +/? mice, but no difference was seen in sciatic nerve weight. The levels of GM1 and GA1 were significantly increased in both the optic nerve and sciatic nerve of the β-gal −/− mice. The content of myelin-enriched cerebrosides, sulfatides, and plasmalogen ethanolamines was significantly lower in optic nerve of β-gal −/− mice than in β-gal +/? mice; however, cholesteryl esters were enriched in the β-gal −/− mice. No major abnormalities in these lipids were detected in the sciatic nerve of the β-gal −/− mice. The abnormalities in GM1 and myelin lipids in optic nerve of β-gal −/− mice correlated with a reduction in the relative amount of myelin and periodicity in fresh nerve. By contrast, the relative amount of myelin and periodicity in the sciatic nerves from control and β-gal −/− mice were indistinguishable, suggesting minimal pathological involvement in sciatic nerve. Our results indicate that the greater neurochemical pathology observed in the optic nerve than in the sciatic nerve of β-gal −/− mice is likely due to the greater glycolipid storage in optic nerve.     

Neuron-Like Differentiation of PC12 Cells Treated With Media Conditioned by Either Sciatic Nerves,Optic Nerves,or Schwann Cells

In previous works we reported the finding of neurotrophic activity in a serum-free Dulbeccos modified Eagles medium conditioned by rat sciatic nerves, previously maintained in culture for 11 days. This medium produces rapid neuron-like differentiation of cultured PC12 cells, as revealed by an increase in the size of the cell body and by the extension of short and/or long neurites by most of the cells. Neuregulin present in the conditioned medium was demonstrated to play a key role in the observed differentiation.In the present work, taking into consideration those latter results, the neurotrophic activity of conditioned media prepared with sciatic and optic nerves cultured during days 1–4 and 9–12 were studied.Evaluation of the trophic activities of those media revealed an opposite timing in the activities of sciatic and optic nerves conditioned media. The activity of the sciatic nerve was not observed in the 1–4-day period, increasing then up to the 9–12-day period. On the contrary, the optic nerve conditioned medium was active in the 1–4-day period, decreasing down to the 9–12-day period.These results led us to explore the contribution of the different cellular constituents of those nerves to their neurotrophic properties. As a first step in that direction we also investigated the neurotrophic activity of media conditioned during 12 days by cultured Schwann cells isolated from rat sciatic nerves. The Schwann cell conditioned media did produce a rapid differentiation of the PC12 cells similar to that caused by the sciatic nerve conditioned medium, though of a lower magnitude.Variations in the trophic activities of the conditioned media used in the present work is discussed taking into consideration the production of trophic and inhibitory factors by the peripheral and central glial cells. The role played by the optic nerve glia and myelin is being investigated at present.     

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.     

Carboxypeptidase M in Brain and Peripheral Nerves

Carboxypeptidase M (CPM), a plasma membrane-bound enzyme, cleaves C-terminal basic amino acids with a neutral pH optimum. We studied its distribution in human, baboon, and dog brain and in dog peripheral nerves. Areas were dissected, homogenized, centrifuged, and assayed for activity with dansyl-Ala-Arg. The corpus callosum and the pyramidal and optic tract were especially rich in CPM, whereas basal ganglia and cortex had low activity. The identity of the basic carboxypeptidase activity with CPM was shown by similarities in subcellular localization, membrane attachment, substrate hydrolysis, inhibition by a specific basic carboxypeptidase inhibitor, and cross-reaction with anti-human CPM antiserum. This antiserum immunoprecipitated an average of 85% of the activity in human and baboon brain and approximately 66% in dog brain. CPM co-purified with myelin extracted from the brain. Consistent with results obtained in placenta and cultured kidney cells, CPM in the brain appears to be membrane-bound via a phosphatidylinositol glycan anchor. In the peripheral nerves, the specific activity in dog sciatic nerve and in vagus was high (98 and 149 nmol/h/mg of protein, respectively). In immunohistochemical studies, glia in the brain, which appear to be oligodendrocytes or astrocytes, and the outer aspects of myelin sheaths and Schwann cells in sciatic and vagus nerves were stained. We conclude that in some areas of the CNS and the PNS, CPM is closely associated with myelin and myelin-forming cells. Northern blot analysis revealed the presence of mRNA coding for CPM in the brain, showing that the enzyme is indeed synthesized there.