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A sural nerve dissected from a recently dead patient displayed an unusual X-ray diffraction pattern, suggesting that in situ and at the time of the patient's death the myelin sheaths were in a swollen state. Diffraction patterns of the swollen type were also recorded from: (1) a sural nerve from the corpse of a neurologically healthy person after soaking the nerve with Ringer solution at pH 5.5; (2) sciatic nerves dissected from rat cadavers at increasing time after death. In all the cases the swollen patterns reversed to the native type upon superfusion with Ringer solution at pH 7.3. The postmortem effect is to decrease the pH of the fluids surrounding the nerves in the cadavers. Our experiments show that the early postmortem processes have the effect of acidifying PNS nerves and that as a consequence of acidification the myelin sheaths swell.

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.

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., (Copyright 1999 Academic Press.)

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.

We report the X-ray scattering study of sciatic and optic nerve myelin from shiverer, jimpy and quaking mice mutants and from the corresponding controls. These three mutations are known to affect dramatically central nervous system (CNS) myelin and to induce comparatively minor alterations in peripheral nervous system (PNS) myelin. Scattering experiments and data reduction were carried out using the techniques and algorithms developed in our laboratory and previously applied to several problems involving the structure of myelin. In sciatic nerve the fraction of myelin elementary pairs of membranes (total myelin) decreases in shiverer and quaking nerves (by approximately 30%) but not in jimpy nerves; in all three mutants the fraction of myelin membrane pairs that are not regularly stacked in the sheaths (loose myelin), the average number of membranes per sheath and the packing disorder are the same as in the control nerves; the repeat distance D and the membrane distance Dcyt across the cytoplasmic space increase in shiverer and decrease in jimpy; in quaking, D also decreases and the decrease is smaller than in jimpy and is not specific for Dcyt; small changes are also observed in the electron density profiles. As for the optic nerve the myelin content decreases dramatically in the three mutants; the very weak signal attests to a tiny amount of pairs of membranes structurally similar to normal CNS myelin. It is surprising that the structure of CNS myelin should be almost normal in the absence of the major structural components, namely myelin basic protein (MBP) for shiverer of proteolipid protein (PLP) for jimpy. The question arises whether the composition of the residual pairs of membranes, operationally identified as myelin in the X-ray scattering experiments, mirrors the composition determined by chemical means on the fraction of nerve tissue histologically identified as myelin, or whether in all circumstances it remains approximately the same.

We describe in this work X-ray scattering and electron microscope studies of rat sciatic and optic nerves as a function of temperature. The scattering experiments were analyzed as described in the previous papers of this series: a variety of parameters were determined, some of which characterize the lattice disorder, others the structure of the motif. The main results are the following. All the parameters determined by the X-ray scattering study vary with temperature and the temperature-dependence is specific for the type of nerve (sciatic or optic). Most of the disorder-related parameters display a minimum or a maximum in the vicinity of physiological temperature (38 degrees C in rat); this observation, strongly supported by the electron microscope study, shows that the degree of organization of myelin is highest near physiological temperature. The structure of the motif, as revealed by the electron density profile, is fairly different in the two types of nerves (in contrast with the assumption made by previous workers); the structure also varies with temperature and the temperature-induced alterations are nerve-type specific. In the two types of nerve the thickness of the lipid bilayer varies with temperature as expected for a lipid-containing system with hydrocarbon chains in the disordered conformation. In sciatic nerve the thickness of the (thinner) cytoplasmic polar layer, which is also the layer most affected by lattice disorder in this type of nerve, decreases dramatically with increasing temperature. In optic nerve, in which lattice disorder predominantly affects the extracellular layer, the thickness of both the cytoplasmic and the extracellular layer is barely affected by temperature.

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.

We have previously shown by X-ray diffraction techniques that the stability of the myelin lattice is decreased by repetitive stimulation of the nerve. We now show that in rat myelinated fibres this structural change is accompanied by the appearance of voltage-sensitive potassium channels which do not normally participate in the generation of the action potential. Since it has previously been shown that structures associated with potassium channels are found in the internodal and paranodal axolemma membrane but are normally concealed under the myelin sheath, we suggest that the acceleration of myelin swelling after repetitive propagation of the action potential may be due to the opening of paranodal axoglial junctions. To our knowledge this is the first demonstration that nerve activity may regulate the structural properties of the paranode.

Changes induced in the structure and electrical activity of myelin were recorded simultaneously from frog sciatic nerves treated with n-alkanes. The results suggest that the effect of n-alkanes seems to be two-fold: (a) there is an initial reversible phase, in which a significant modification of the X-ray diffraction patterns, concomitant with the continuous fall of the action potential, is observed; (b) there is a final phase which is irreversible. This occurs some time after the complete abolition of the electrical activity. At this stage, further changes of the X-ray diffraction patterns are detected, the most significant of them being in the n-pentane-treated myelin, and consist of an increase in the membrane bilayer thickness.

The x-ray diffraction pattern of myelin membranes from a frog sciatic nerve was recorded in vivo at 13 A resolution using a new experimental technique recently developed in our laboratory. The electron density profile was determined for the myelin membranes first in vivo and then, after elimination of the intraneural blood microcirculation, by transection of the nerve. The small structural differences observed between these conditions were statistically nonsignificant (at 95% confidence level). In addition, our results show that the stacking disorder that is present in the myelin membranes of freshly dissected frog sciatics is also characteristic of the myelin sheath in vivo.

Equatorial sections of the x-ray diffraction patterns of myelin from toad sciatic nerves recorded at 11 A resolution during long supramaximal repetitive stimulation (240 Hz for two hours) were used to calculate the relative electron density profile of the myelin membranes in this physiological state. The profile was compared to the one determined for the same nerve at rest. The difference between the stimulated profile and the control profile was found to be statistically non-significant. Therefore, when the sciatic nerve fibers are excited the structure of the internodal myelin membranes does not significantly change at 11 A resolution.