ELECTRON MICROSCOPE AND LOW-ANGLE X-RAY DIFFRACTION STUDIES OF THE NERVE MYELIN SHEATH

1. A close correlation has been obtained between high resolution electron microscopy and low-angle x-ray diffraction studies of the myelin sheath of frog and rat peripheral and central nerves. Extensive studies were performed by application of both techniques to the same specimens, prepared for examination by OsO₄ or KMnO₄ fixation, and embedding either in methacrylate or in gelatin employing a new procedure. Controlled physical and chemical modifications of the myelin sheath prior to fixation were also investigated. 2. A correspondence was established between the layer spacings observed in electron micrographs and the fundamental radial repeating unit indicated by the low-angle x-ray diffraction patterns. The variations in relative intensities of the low-angle x-ray reflections could be related to the radial density distributions seen in the electron micrographs. 3. An analysis of the preparation procedures revealed that OsO₄ fixation introduces a greater shrinkage of the layer spacings and more pronounced changes in the density distribution within the layers than KMnO₄ fixation. The effects of methacrylate and gelatin embedding are described, and their relative merits considered in relation to the preservation of myelin structure by OsO₄ fixation. 4. The experimental modifications introduced by freezing and thawing of fresh whole nerve are described, particularly the enhancement of the intermediate lines and the dissociation of the layer components in the myelin sheath. A characteristic collapsing of the radial period of the sheath is observed after subjecting fresh nerve trunks to prolonged and intense ultracentrifugation. 5. Controlled extraction of fresh nerve with acetone at 0°C., which preferentially removes cholesterol, produces characteristic, differentiated modifications of the myelin sheath structure. Electron microscopy reveals several types of modifications within a single preparation, including both expanded and collapsed layer systems, and internal rearrangements of the layer components. Alcohol extraction leads to a more extensive structural breakdown, but in certain areas collapsed layer systems can still be observed. The components of the lipide extracts could be identified by means of x-ray diffraction. These modifications emphasize the importance of cholesterol in the myelin structure, and disclose a resistance of the dense osmiophilic lines to lipide solvents. 6. The significance of these structures is discussed in relation to present concepts of the molecular organization of myelin. The available evidence is consistent with the suggestion that the primary site of osmium deposition is at the lipoprotein interfaces and that the light bands probably represent regions occupied by lipide chains. The electron microscope and x-ray diffraction data also indicate the possibility of a regular organization within the plane of the layers, probably involving units of 60 to 80 A. The myelin sheath is regarded as a favourable cell membrane model for detailed analysis by combined application of x-ray diffraction and electron microscopy.     

ELECTRON MICROSCOPE AND X-RAY DIFFRACTION STUDIES OF THE EFFECTS OF DEHYDRATION ON THE STRUCTURE OF NERVE MYELIN : II. Optic Nerve

The dehydration of rat optic nerve has been studied by allowing specimens to become partially or fully dried before fixation and preparation for electron microscopy. A correlation is established between electron micrographs of the myelin sheath and corresponding small-angle x-ray diffraction patterns. The modifications of the optic nerve myelin layers during drying were very similar to those described in more detail for the myelin of frog sciatic nerve. The most striking difference was that the system of fine layers characteristic of the fully dried myelin was much more extensive in the case of the optic nerve, and the layer thickness was significantly greater than the corresponding layer in the frog sciatic nerve preparation. The significance of these correlations is discussed.     

PERMANGANATE FIXATION OF THE GOLGI COMPLEX AND OTHER CYTOPLASMIC STRUCTURES OF MAMMALIAN TESTES

Observations on the fine structure of KMnO₄-fixed testes of small mammals (guinea pig, rat, and mouse) reveal certain morphological differences between the spermatogenic and Sertoli cells which have not been demonstrated in the same tissue fixed with OsO₄. Aggregates of minute circular profiles, much smaller than the spherical Golgi vesicles, are described in close association with the Golgi complex of developing spermatids. Groups of dense flattened vesicles, individually surrounded by a membrane of different dimensions than that which bounds most of the other cell organelles, appear dispersed within the cytoplasm of some spermatogenic cells. Flattened vesicles of greater density than those belonging to the Golgi complex are reported confined to the inner Golgi zone of developing guinea pig spermatids between the Golgi cisternae and the head cap. The profiles of endoplasmic reticulum within spermatocytes appear shorter, wider, and more tortuous than those of Sertoli cells. Minute cytoplasmic particles approximately 300 A in diameter and of high electron opacity appear randomly disposed in some Sertoli cells. Groups of irregular-shaped ovoid bodies within the developing spermatids are described as resembling portions of cytoplasm from closely adjacent spermatids. Interpretation is presented regarding the fine structure of KMnO₄-fixed testes in view of what has already been reported for mammalian testes fixed in OsO₄.     

An Electron Microscope Study of Myelin Figures

In the electron microscope, thin sections of OsO₄-fixed myelin figures from the phospholipide fraction of human brain show a pattern of parallel dark lines with a repeating period of about 40 A. It is shown that the dark lines probably represent the reaction product of OsO₄ with double bonds in the fatty acid chains, thereby marking the central portion of one bimolecular lamella. The addition of globin results in dense lines 25 to 50 A wide that cover the surface of the myelin figures. When such a figure consists of only two bimolecular leaflets of lipide covered with globin, the structure shows striking similarity to the image of cell membranes in fixed tissue sections. A hypothetical schema is given of the molecular structure of the figure, and the distribution of OsO₄ in it.     

The Structure of Myelin Sheaths in the Central Nervous System of Xenopus laevis (Daudin)

The structure of myelinated nerve fibres has been studied in the spinal cord and optic nerve of the tadpoles of Xenopus laevis. Potassium permanganate-fixed material was examined with the electron microscope. The myelin sheath itself is made up of spirally arranged lamellae in which the intraperiod and dense lines alternate. Inside the myelin sheath an inner cytoplasmic process surrounds the axon and where the external surfaces of its bounding membrane come together an internal mesaxon is formed. The intraperiod line begins within the mesaxon and the dense line usually begins in the same region by apposition of the cytoplasmic surfaces of the membrane. The width of each lamella is 140 A. The outer line in the sheath is the dense line and this terminates in a tongue where the cytoplasmic surfaces of the myelin-forming glial cell separate. Thus, central myelin in Xenopus tadpoles is arranged in the same way as peripheral myelin, the only difference being that in central fibres, cytoplasm on the outside of the sheath is confined to that present in the tongue. For this reason adjacent central sheaths come into apposition without any intervening material being present. When this occurs an intraperiod line is formed between them.     

AN ELECTRON MICROSCOPE STUDY OF THE FINE STRUCTURE OF FEATHER KERATIN

Thin sections of the rachis of regenerating follicles of pigmented fowl feathers and of mature non-pigmented seagull feather rachis, embedded in methacrylate and Araldite respectively, were studied in the electron microscope. The late stages of development of keratin fibrils were examined in OsO₄-fixed follicle material, and after poststaining with lead hydroxide the keratin aggregates were found to be composed of fine microfibrils approximately 30 A in diameter apparently embedded in a matrix material which had absorbed the lead stain. The centre-to-centre separation of the microfibrils was of the order of 35 A. After bulk treatment by reduction with thioglycollic acid, OsO₄ staining, and poststaining with lead hydroxide, a similar microfibrillar fine structure was observed in mature rachis. Only after lead staining could the microfibrils be delineated, and their diameter and separation were similar to that found in the keratin of the follicle. It is suggested that feather keratin resembles α-keratins in consisting of microfibrils embedded in an amorphous protein matrix. However, in comparison with α-keratins, the microfibrils are much smaller in diameter, their arrangement is less orderly, and on the basis of the reactions towards the electron staining procedures, the cystine content of the matrix appears to be not greatly different from that of the microfibrils. The significance of a microfibrillar constitution of feather keratin is discussed in relation to current structural models for this fibrous protein deduced from x-ray diffraction studies. The boundaries between the component cells of feather rachis are desmosomal in character and similar to those of related keratinous structures and a number of different types of cells; the melanin granules are dissimilar to those of mammalian epidermis in their apparent lack of melanin-protein lamellae.     

ULTRASTRUCTURE OF THE SPOON TYPE SYNAPTIC ENDINGS IN THE NUCLEUS VESTIBULARIS TANGENTIALIS OF THE CHICK

The fine structure of the "spoon" type synaptic endings of the chick tangential nucleus was studied with the electron microscope. These endings often measure ~18 µ in length by ~3–4 µ in width. The axoplasm of the endings contains very few synaptic vesicles, a large number of neurofilaments oriented parallel to the long axis of the nerve fiber, and microtubules and numerous mitochondria. The synaptic membrane complex shows areas of localized occlusion of the synaptic cleft with the formation of an external compound membrane. It has not been decided whether these areas have a disc shape; their length measures between 0.04 and 0.47 µ. The five-layer pattern characteristic of an external compound membrane is shown in specimens fixed with formalin—OsO₄, glutaraldehyde—acrolein—OsO₄, and acrolein KMnO₄ but it does not appear in the glutaraldehyde-OsO₄-fixed specimens. The over-all thickness of the external compound membrane varies depending upon the fixative used. The synaptic clefts in the regions between the external compound membrane discs are widened and measure ~300 A. A condensation of dense material occurs in pre- and postsynaptic cytoplasms all along the synaptic membrane complex. The morphological relationships described in the spoon endings are suggestive of electrical transmission.     

Fine Structure of Succinate-Swollen Rhizobium trifolii 0403

Transmission electron micrographs of glutaraldehyde- OsO₄-fixed Rhizobium trifolii 0403 before and after cells were treated with 16.6 mM succinate showed that treated cells increased in mass by increasing cytoplasmic volume. The morphology of succinate-treated cells was identical to that of bacteroids, and the appearances of the envelope and periplasmic space were similar. The primary difference was in inclusion number and type.     

ELECTRON MICROSCOPIC RADIOAUTOGRAPHY OF THIN SECTIONS: THE GOLGI ZONE AS A SITE OF PROTEIN CONCENTRATION IN PANCREATIC ACINAR CELLS

Electron microscopic radioautographs of guinea pig pancreatic exocrine cells were obtained by covering thin sections (~ 600 A) of OsO₄-fixed, methacrylate-embedded tissue with thin layers of Ilford K-5 nuclear research emulsion. After an exposure of 13 days at 4°C., the preparations were photographically processed, stained with uranyl acetate, and examined in an electron microscope. The label used was leucine-H³ injected intravenously 20 minutes before collection of the specimens. Conventional radioautographs of thicker sections (0.4 micron) were also examined in a phase contrast microscope. The advantages obtained from electron microscopic radioautography are: the higher radioautographic resolution (of the order of 0.3 micron) due to the thinness of the emulsion and the specimen, and a high optical resolution permitting a clear identification of the labeled structure. In the guinea pig pancreas this technique demonstrated that, at the time studied, newly synthesized proteins were concentrated in the structures of the Golgi complex and especially in large vacuoles partially filled with a dense material. The vacuoles are probably a precursor to the secretion granules (zymogen granules) in which the label becomes segregated at a later time. These observations demonstrate directly the role of the Golgi complex in the secretion process. They also illustrate the possibilities of this method for radioautography at the intracellular level.     

THE EFFECT OF VARIOUS FIXATION SCHEDULES ON THE SCANNING ELECTRON MICROSCOPIC IMAGE OF TROPAEOLUM MAJUS

Fresh, unfixed shoot tips of Tropaeolum provided excellent quality scanning electron micrographs if observations were brief and magnifications were relatively low. For extended observations or for high-magnification observations, fixation by OsO₄ vapor or Zirkle-Erliki's fluid provided the best scanning electron micrographs. Brief exposures to microscope vacuum do not appear to produce any apparent morphological changes in living shoot tips of Tropaeolum except in regions adjacent to a cut surface.     

Morphological changes of myelin sheaths in rats intracranially injected with apotransferrin

Previous findings from our laboratories indicate that the intracranial injection of apotransferrin (aTf) in neonatal rats produces an accelerated oligodendrocyte maturation and an enhanced production and deposition of myelin membranes in the brain. To evaluate the anatomical distribution and the morphological characteristics of the myelin in these rats, we analyzed the optic nerves, cerebellum, and selected areas of brain sections from aTf-treated and control rats by both light and electron microscopy. Microscopic identification of myelin using a specific staining procedure, showed that in aTf-injected rats, in coincidence with previous biochemical studies, there was an increased deposition of myelin in selected areas of the nervous system. Qualitative and quantitative analysis of electron micrographs from areas showing increased myelinaton, such as the optic nerves and the corpus callosum, showed that among other changes, the intracranial treatment with aTf produces ultrastructural evidences of myelin decompaction, consisting of an enlargement in the distance between adjacent major dense lines, a decreased density of the intraperiod line, and an increased electron density of the major dense line, accompanied by a significant increase in its width. The intracranial administration of aTf induces an increased deposition of myelin by oligodeudroglial cells (OLGc), and these myelin membranes, in spite of the changes in composition and in morphology, appear to function normally. Apotransferrin can be considered as a differentiation factor that could be used to stimulate remyelination in cases in which myelin has been destroyed by various pathological processes.     

A STRUCTURAL ANALYSIS OF THE MYELIN SHEATH IN THE CENTRAL NERVOUS SYSTEM

The cerebral white matter of rats subjected to a variety of noxious experimental conditions was examined in the electron microscope. Several unusual configurations of the myelin sheath are identified in addition to the usual configuration. These variations include the presence of (a) formed organelles within the inner and outer loops, (b) isolated islands of cytoplasm in unfused portions of the major dense lines, (c) apparently unconnected cell processes between the sheath and the axon, and (d) concentric, double myelin sheaths. A generalized model of the myelin sheath based on a hypothetical unrolling of the sheath is described. It consists of a shovel-shaped myelin sheet surrounded by a continuous thickened rim of cytoplasm. Most of the unusual myelin configurations are explained as simple variations on this basic theme. With the help of this model, an explanation of the formation of the myelin sheath is offered. This explanation involves the concept that myelin formation can occur at all cytoplasmic areas adjacent to the myelin proper and that adjacent myelin lamellae can move in relation to each other.     

FURTHER OBSERVATIONS ON THE STRUCTURE OF MYELIN SHEATHS IN THE CENTRAL NERVOUS SYSTEM

Direct evidence has been presented to confirm the existence of a spiral in the myelin sheaths of the central nervous system. An account of some of the variations in structure of central myelin sheaths has been given and it has been shown that the radial component of myelin sheaths has the form of a series of rod-like thickenings of the intraperiod line. These thickenings extend along the intraperiod line in a direction parallel to the length of the axon. The relative position of the internal mesaxon and external tongue of cytoplasm has been determined in a number of transverse sections of sheaths from the optic nerves of adult mice, adult rats, and young rats. In about 75 per cent of the mature sheaths examined, these two structures were found within the same quadrant of the sheath, so that the cytoplasm of the external tongue process tends to lie directly outside that associated with the internal mesaxon. The frequency with which the internal mesaxon and external tongue lie within the same quadrant of the sheath increases both with the age of the animal and with the number of lamellae present within a sheath. The possible significance of these findings is discussed.     

Electron Microscopy of Retinal Photoreceptors : The Use of Chromation Following Formaldehyde Fixation as a Complementary Technique to Osmium Tetroxide Fixation

The fine structure of the cone and rod outer segments of the toad was studied under the electron microscope after fixation in osmium tetroxide and fixation in formaldehyde followed by chromation. In the OsO₄-fixed specimens, the rod outer segment appears to be built of a stack of lobulated flattened sacs, each of which is made of two membranes of about 40 A separated by an innerspace of about 30 A. The distance between the rod sacs is about 50 A. The sacs in the cone outer segment are originated by the folding of a continuous membrane. The thickness of the membranes and width of the spaces between the cone sacs is the same as in rod, but the sac innerspace is slightly narrower in the cone (~ 20 A). After fixation in formaldehyde and chromation, two different dense lines (l₁ and l₂) separated by spaces of less density appear. One of the lines, l₁, has a thickness of 70 A and is less dense than the other, l₂, which is 30 A thick. The correlation of the patterns obtained with both fixatives is considered and two possible interpretations are given. The possibility that l₂ is related to a soluble phospholipid component is discussed. It is suggested that the outer segments have a paracrystallin organization similar to that found in myelin.     

LOCALIZATION OF THE SITES OF γ-AMINOBUTYRIC ACID (GABA) UPTAKE IN LOBSTER NERVE-MUSCLE PREPARATIONS

The principal sites of γ-aminobutyric acid (GABA) uptake in lobster nerve-muscle preparations have been determined with radioautographic techniques after binding of the amino acid to proteins by aldehyde fixation. Semiquantitative studies showed that about 30% of the radioactive GABA taken into the tissue was bound to protein by fixation. Both light and electron micrographs showed dense accumulations of label over Schwann and connective tissue cell cytoplasm; muscle was lightly labeled, but axons and terminals were almost devoid of label. The possible role of Schwann and connective tissue cells in the inactivation of GABA released from inhibitory axons is discussed.     

THE FINE STRUCTURE OF NERVE CELL BODIES AND THEIR MYELIN SHEATHS IN THE EIGHTH NERVE GANGLION OF THE GOLDFISH

The eighth cranial nerve ganglion consists of bipolar nerve cell bodies each occupying part of an internodal segment. The perikaryal sheaths range from a single layer of Schwann cell cytoplasm on the smallest cells to typical thick compact myelin on the largest. On most perikarya, the sheath displays an intermediate form, consisting of multiple layers of Schwann cell cytoplasm (loose myelin), or of loose and compact myelin continuous with each other. Internodes beyond the one containing the cell body bear only compact myelin. In loose myelin the thickness of each layer of Schwann cell cytoplasm is about 100 A. It may be much greater (~ 3000 A) particularly in the outermost layers of the sheath, or the cytoplasm may thin and even disappear with formation of a major dense line. The cytoplasmic layers are separated from each other by a light zone, 40 to 200 A wide, which in its broader portions may contain an intermediate line. Desmosomes sometimes occur between lamellae. In addition to the usual organelles, the perikaryal cytoplasm contains granular and membranous inclusions. Large cells covered by compact myelin have a consistently higher concentration of neurofilaments, and some of the largest cells, in addition, show a reduced concentration of ribosomes. The functional significance and possible origins of perikaryal myelin sheaths are discussed.     

ULTRASTRUCTURAL STUDY OF REMYELINATION IN AN EXPERIMENTAL LESION IN ADULT CAT SPINAL CORD

This report presents ultrastructural observations on the cytological events that attend myelin formation occurring in the wake of demyelination in adult cat spinal cord. Lesions were induced in subpial cord by cerebrospinal fluid (c.s.f.) exchange (1, 2). Tissue from eleven cats at nine intervals from 19 to 460 days was fixed in situ by replacing c.s.f. with buffered OsO₄ and embedded in Araldite. After demyelination, axons are embraced by sheet-like glial processes. An occasional myelin sheath is first seen at 19 days; by 64 days, all axons are at least thinly myelinated. The cytoplasm of the myelin-forming cells, unlike that of either oligodendrocyte or fibrous astrocyte in normal cord, is dense with closely packed organelles and fine fibrils. Many of the myelinogenic cells become scarring astrocytes and at 460 days the lesion teems with their fibril-filled processes. Oligodendrocytes appear in the lesion after remyelination is under way. Phagocytes disappear gradually. A myelin sheath is formed by spiral wrapping of a sheet-like glial process around an axon. Where the first turn of the spiral is completed, a mesaxon is formed. As cytoplasm is lost from the process, the plasma membrane comes together along its outer and cytoplasmic surfaces to form compact myelin. Only a small amount of cytoplasm is retained; it is confined to the paramesaxonal region and, on the sheath exterior, to a longitudinal ridge which appears in profile as a small loop. This outer loop has the same rotational orientation as the inner mesaxon. These vestiges of spiral membrane wrapping are also found in normal adult and new-born cat cord. Nodes are present in all stages of remyelination and in normal adult cat and kitten cord. These observations suggest that myelin is reformed in the lesion in the same way it is first formed during normal development. The mechanism of myelin formation is basically similar to that proposed for peripheral nerve and amphibian and mammalian optic nerve; it does not agree with present views on the mechanism of myelinogenesis in mammalian brain and cord. This is the first demonstration of remyelination in adult mammalian central nervous tissue.     

PRESERVATION OF MYELIN LAMELLAR STRUCTURE IN THE ABSENCE OF LIPID : A Correlated Chemical and Morphological Study

The fine structure of myelin was studied in glutaraldehyde-fixed rat sciatic nerves depleted of lipid by acetone, chloroform:methanol (2:1 v/v), and chloroform:methanol:concentrated HCl (200:100:1, v/v/v). One portion of each of these nerves, plus the extracts, was saponified and analyzed by gas-liquid chromatography for fatty acids. The remainder of each nerve was stained in osmium tetroxide in CCl₄ (5g/100cc) and was embedded in Epon 812. Thin sections, examined in the electron microscope, revealed the preservation of myelin lamellar structure with a 170 A periodicity in nerves depleted of 98% of their lipids. Preservation of myelin lamellar structure depended on glutaraldehyde fixation and the introduction of osmium tetroxide in a nonpolar vehicle (CCl₄) after the lipids had been extracted. It is concluded that the periodic lamellar structure in electron micrographs of myelin depleted of lipid results from the complexing of osmium tetroxide, plus uranyl and lead stains, with protein.     

Structural alterations in nerve fibers produced by hypotonic and hypertonic solutions

In sections of KMnO(4)-fixed, developing mouse sciatic nerves, the central gap of mesaxons in myelinating fibers is normally closed with close apposition of the outside approximately 20 A dense strata of the two approximately 75 A Schwann cell membranes. The two combined outside strata make the intraperiod line bisecting each myelin lamella. The approximately 150 A mesaxon is elaborated spirally around the axon in either a right hand or left hand spiral, and its inside (cytoplasmic) approximately 20 A strata in apposition form the major dense lines of myelin. In hypotonic solutions the lamellae of adult frog sciatic myelinated fibers split apart along the outside membrane strata apposed at the intraperiod line throughout the spiral. Under similar conditions the inside (cytoplasmic) strata of the membranes, in apposition at the major dense lines, do not separate. The approximately 150 A membranous structure resulting from this is called an "internal compound membrane." The double membranes of normal and control frog sciatic unmyelinated fibers have a central gap approximately 100 to 150 A wide. After soaking in 4 to 10 times normal strength Ringer solution or 10 N sucrose-Ringer solution, this gap closes and a membranous structure approximately 150 A wide resembling developing mouse mesaxons results. This is designated by the term "external compound membrane." The latter membranes resemble internal compound membranes, but their central dense zones, each consisting of two apposed outside membrane strata, are less dense.     

SOME ELECTRON MICROSCOPICAL OBSERVATIONS ON LIQUID-CRYSTALLINE PHASES IN LIPID-WATER SYSTEMS

OsO₄ fixation preserves some liquid-crystalline phases of soaps and phospholipids to an extent that it is possible to observe their structure in electron micrographs of thin sections. Good agreement exists between the structure observed directly and that deduced from x-ray diffraction studies of the same systems.