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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.

Sequences of 15 minute X-ray scattering spectra were recorded with rat sciatic and optic nerves, superfused with tetracaine-containing Ringer solutions. The spectra were analysed using the algorithm advocated in this series of papers. The main results, as a function of the time of exposure to tetracaine, were: the mean value of the repeat distance increases; its variance decreases; the average number of membrane pairs per coherent domain decreases; the fraction of isolated membrane pairs increases. Eventually, the spectra were observed to give way to the continuous intensity curve of a single, isolated membrane pair. At all stages of the experiment the continuous intensity curves were found to differ from one type of nerve to the other, and to be invariant, for each type of nerve, with respect to the tetracaine treatment. The X-ray scattering study clearly identified the nature of the structural differences between the two types of myelin sheaths: in that of native sciatic nerves, packing disorder preferentially affects the cytoplasmic space of the membrane pair, and tetracaine disrupts the packing in that space; in the myelin of optic nerves it is the external space that is preferentially affected by packing disorder and disrupted by tetracaine. The time-course of the structure parameters showed that, at any stage of the experiment, tetracaine acts preferentially on the more highly disordered regions of the structure and totally disrupts them. These results corroborate earlier conclusions reported in the previous papers of this series. An electron microscope study was also performed on tetracaine-treated nerves: the results, in close agreement with those of the X-ray scattering study, neatly confirm the conclusions given above. In a more general way, the remarkable agreement between the results of the analysis of the X-ray scattering spectra and the electron microscope observations strongly supports the validity of the physical model used in this series of papers and the correctness of the mathematical treatment that we advocate. Finally, the relations between this work and the work of others are discussed. It must be stressed that the present work bears on the toxic rather than on the anaesthetic effects of tetracaine.

An X-ray scattering study was performed on optic nerves dissected from rats aged from ten days to one year. The spectra were analysed using the procedure described in the previous papers of this series. Each experiment yields the values of a variety of parameters: the average D and the variance sigma D of the repeat distance, the average number mean value of N of motifs per crystallite, the fraction alpha loose of myelin that does not belong to the compact sheaths, the sets [idiff(h/D)] and [imotif(k/2D)] that suffice to define, respectively, the spurious scattering and the continuous intensity curve of the elementary membrane pair. A surprising result is that, in the native optic, as previously found in the swollen sciatic nerves, the stacking disorder affects the external space, whereas in native sciatic nerves the disorder affects the cytoplasmic space. An analysis of the evolution of the structure parameters as a function of the age of the animal and a comparison with the results previously obtained with rat sciatic nerves led to the following conclusions: the structure of the elementary membrane pair is constant throughout myelinogenesis; mean value of N is much smaller in optic than in sciatic nerves; mean value of N and the degree of myelination increase with age in the two types of nerve; D is smaller in optic than in sciatic nerves; in optic nerves, D decreases slightly with age, but in sciatic nerves it increases; sigma D is strongly age-dependent in optic nerves, but almost age-independent in sciatic nerves. In contrast to sciatic, the structure of optic nerve myelin was found to be almost insensitive to hypertonic solutions. Finally, a pair of electron density profiles was selected, quite similar to those selected previously in sciatic nerves, 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.

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.