THE FORMATION AND STRUCTURE OF MYELIN SHEATHS IN THE CENTRAL NERVOUS SYSTEM

The development and structure of myelin sheaths have been studied in the optic nerves of rats and of Xenopus laevis tadpoles. Both potassium permanganate- and osmium-fixed material was examined with the electron microscope. In the first stage of myelinogenesis the nerve fibre is surrounded by a cell process which envelops it and forms a mesaxon. The mesaxon then elongates into a loose spiral from which the cytoplasm is later excluded, so that compact myelin is formed. This process is similar to myelinogenesis in the peripheral nervous system, although in central fibres the cytoplasm on the outside of the myelin is confined in a tongue-like process to a fraction of the circumference, leaving the remainder of the sheath uncovered, so that contacts are possible between adjacent myelin sheaths. The structure of nodes in the central nervous system has been described and it is suggested that the oligodendrocytes may be the myelin-forming cells.     

HISTOCHEMICAL DEMONSTRATION OF ACID RIBONUCLEASE IN THE CENTRAL NERVOUS SYSTEM

Abstract— The staining pattern of acid ribonuclease activity in the nervous tissues of rats has been studied with different fixatives and incubation media. Certain modifications in the method adopted (V orbrodt , 1961) produced a definite improvement in the localization as well as distinct subcellular distribution at the pHs studied. The modifications introduced consisted of preincubation of tissue sections with prostatic acid phosphatase, omission of acid phosphatase from the standard incubation medium and its addition at a later stage of incubation. This avoids much of the diffused cytoplasmic and filamentous background staining at pH 5.2–5.8, eliminates the irregular nuclear staining at pH 4.5 and replaces the strong nuclear positivity at pH 5.2 by a fine granular dispersion.
A study of the enzyme activity of subcellular fractions at the different pHs used was performed to confirm the histochemical localization.     

DHPOSITION OF LIPIDS IN THE DEVELOPING CENTRAL AND PERIPHERAL NERVOUS SYSTEMS OF THE CHICKEN

Abstract— The lipid composition of chick brain and sciatic nerve was determined during development. It was confirmed that the addition of CaCl₂ to solvents during the extraction of lipids from brain results in much higher yields of diphosphoinositides particularly from unmyelinated embryo brain. Unlike the earlier report for rat brain, the recovery of triphosphoinositides was also Substantially increased. The amount of CaCl₂, required to achieve optimal recoveries decreased with increasing age and addition of more than this optimal amount depressed the yields of polyphosphoinositides, particularly triphosphoinositides. CaCl₂, addition did not improve the yield of diphosphoinositides from sciatic nerve of any age but drastically reduced recovery of triphosphoinositidcs. Differenccs in the effect of CaCl₂ were not the result of variation in the tissue concentrations of calcium or magnesium.
The lipid composition of sciatic nerve closely reflected that of the myelin. Both polyphosphoinositides were absent initially and their accumulation paralleled that of cerebrosides and sulfatides. The concentration of diphosphoinositides remained constant after the period of most active myelination while triphosphoinositides and the galactolipids continued to increase suggesting maturational changes in the myelin composition. The pattern of deposition in chick brain was similar except for the much greater contribution of non-myelin structures. Both polyphosphoinositides were present in equimolar amounts in pre-myelination embryonic tissue. The concentration of diphosphoinositides increased during active myelination only while triphosphoinositides continued to increase thereafter.     

EFFECT OF ZUCLOMIPHENE ON CHOLESTEROL FORMATION IN THE DEVELOPING CENTRAL NERVOUS SYSTEM OF THE RAT

Abstract— The effect of zuclomiphene, a hypocholesterolemic agent, on developing rat CNS cholesterol biosynthesis was examined. Sterol content and composition was studied in relation to age in four regions of the CNS, cerebrum, brain stem, spinal cord and cerebellum. Sterol content of all four regions was slightly lower in drug-treated animals than in controls. Brain stem and spinal cord were more susceptible to the effects of zuclomiphene than were cerebrum and cerebellum. Drug treatment resulted in the accumulation of desmosterol and zymosterol (5 x -cholesta-8,24-dien-3β-ol) in all CNS regions. After 15 days of drug treatment, desmosterol constituted more than 50% of the total sterol in the four examined regions. Six to 9% of the total sterol was zymosterol.
Examination by electron microscopy indicated only minimal morphological changes. Occasionally, neuronal membranous cytoplasmic inclusion bodies were evident.     

PYRIDOXINE DEFICIENCY AND DEVELOPMENT OF THE CENTRAL NERVOUS SYSTEM IN THE RAT

Pyridoxine (vitamin B₆) deficiency was produced in rats during the period of development of the central nervous system. The levels of pyridoxal phosphate and y-amino-butyric acid in whole brains of these rats were determined, together with the activities of glutamate decarboxylase (EC 4.1.1.15) and γ-aminobutyrate aminotransferase (EC 2.6.1.19). The lowered contents of pyridoxal phosphate and γ-aminobutyrate in the brains confirmed the existence of pyridoxine deficiency. The activity of the glutamate decarboxylase holo-enzyme was decreased, whereas the activity of the apoenzyme was increased. However, there appeared to be no difference in the activity of γ-aminobutyrate aminotransferase. Concomitantly, some electrophysiological parameters, such as EEG and auditory evoked potentials, were analysed. The EEG of pyridoxine-deficient animals showed spike activity, presumably indicative of the existence of seizures in many of the deficient rats. Evoked potentials presented abnormalities in their latency, wave form and response to repetitive stimuli, but the extent to which they were affected depended upon the intensity of the deficiency.     

FORMATION OF MYELIN IN THE CENTRAL NERVOUS SYSTEM OF MICE AND RATS, AS STUDIED WITH THE ELECTRON MICROSCOPE

Sections of brain and spinal cord of mice and rats at 1, 3, 5, and 17 days after birth were examined with the electron microscope. In the early stages of myelinization only two to four lamellae surround the axon. These laminae are formed from the plasma membraces of glial processes, and in particular of oligodendroglial processes. In a later stage of myelinization a larger number of flattened glial processes surround the axon with enclosed cytoplasm trapped within some of the membranes. Multiple extensions of the membranes of the flattened glial processes to the lamellae of the myelinated sheath are evident at this stage, with a variable number of membranes within the sheath at various positions along the fiber. Newly formed myelinated sheaths are sometimes larger than their enclosed fibers, extending as a projection of sheath which does not surround axoplasm. Loci are present in which the myelinated sheath is incomplete or interrupted.     

ERGOTHIONEINE IN THE CENTRAL NERVOUS SYSTEM

Abstract— Further investigations have been made into ergothioneine in the brains of several mammalian species, and the distribution of ergothioneine in the brain of the ox is described. It has not been possible to confirm many of the findings of earlier workers and the results do not appear to support their conclusion that ergothioneine is identical with the cerebellar factor.     

THE EFFECTS OF 4-HYDROXYBUTYRIC ACID ON THE BIOSYNTHESIS OF AMINO ACIDS IN THE CENTRAL NERVOUS SYSTEM

—The effect of 4-hydroxybutyrate (GHB) on cerebral glucose metabolism has been studied. GHB increases the glucose level, decreases the lactate concentration and diminishes the incorporation of glucose carbon into glutamic acid, glutamine, aspartic acid and GABA in the brain of the rat in a state of general anaesthesia. The data reported here suggest that GHB interferes in the metabolism of glucose in brain.     

BIPHASIC MYELINATION AND THE FATTY ACID COMPOSITION OF CEREBROSIDES AND CHOLESTEROL ESTERS IN THE DEVELOPING CENTRAL NERVOUS SYSTEM OF THE DOMESTIC PIG

Abstract— The chemical composition of four parts of the CNS (cerebrum, cerebellum, brain stem and spinal cord) was determined in 107 pigs at 11 stages of fetal and postnatal development and also in 6 adults. In cerebrum, cerebellum and brain stem, but not in spinal cord, the rate of increase in weight and the rates of change in lipid content slowed down for a period of about 10 days before and after birth. Cholesterol esters and desmosterol were only found in progressively decreasing amounts during the fetal stages of development and together with DNA these were exceptions to the general increases in the tissue concentrations and total amounts of other components during the period studied.
The onset of myelination, as measured by calculated daily increases in tissue contents of cerebroside took place between 70 and 80 days conceptual age and there were two peaks of activity, the first occurring 2 weeks before and the second 3 weeks after birth. Unlike the rate curve for total spinal cord weight the biphasic accumulation of DNA was not synchronous with myelin lipid accretion and the earlier prenatal DNA peak probably denotes proliferation of oligodendrocytes. The two phases of myelination are discussed in relation to an observed generalized pause in development immediately before and after birth.
Fatty acid analysis of cerebrosides indicated that, in spinal cord, chain elongation and desaturation are associated with myelination and continue with increasing activity until maturity. Consequently there was a progressive decrease in the proportion of saturated fatty acids. The fatty acid components of cholesterol esters in the developing pig were shown to be similar to those found during development in the CNS of other species but different from those found in demyelinating conditions.     

SYNAPSES IN THE CENTRAL NERVOUS SYSTEM

A number of different synapses have been described in the medulla, cerebellar cortex, and cerebral cortex of the rat. All of these possess the same fundamental fine structure as follows: 1. Close apposition of the limiting membranes of presynaptic and postsynaptic cells without any protoplasmic continuity across the synapse. The two apposed membranes are separated by a cleft about 200 A wide, and display localized regions of thickening and increased density. 2. The presynaptic expansion of the axon, the end-foot or bouton terminal, contains a collection of mitochondria and clusters of small vesicles about 200 to 650 A in diameter. Although the significance of these structures in the physiology of the synapse is still unknown, two suggestions are made: that the mitochondria, by means of the relation between their enzymatic activity and ion transport, participate in the electrical phenomena about the synapse; and that the small synaptic vesicles provide the morphological representation of the prejunctional, subcellular units of neurohumoral discharge at the synapse demanded by physiological evidence.     

BIOSYNTHESIS OF PSYCHOSINE AND LEVELS OF CEREBROSIDES IN THE CENTRAL AND PERIPHERAL NERVOUS SYSTEMS OF QUAKING MICE

Abstract— The levels of cerebrosides in neural tissues of adult mice were determined by densitometry of cerebroside spots on charred thin-layer chromatograms of washed total lipid extracts. Values for brain, spinal cord and peripheral nerves were 9·2, 33·0 and 36·9 mg/g of tissue, respectively. In adult Quaking mice these values were 6·4, 24 and 35 % of normal, respectively. Normal levels in brain, spinal cord and peripheral nerve of 21-day-old mice were 3·10, 13·5 and 17·8 mg/g, respectively. In 21-day-old Quaking mice the levels were reduced to 16,21 and 57% of normal, respectively. Biosynthesis of psychosine (galacto-sylsphingosine) by homogenates of Quaking brain, spinal cord and peripheral nerve, respectively, was 18, 24 and 42% of the normal rates at 21 days after birth and 16, 66 and 60% of the normal rates at 94 days. Our results suggest a quantitative relationship between the rate of formation of psychosine in vitro and the rate of accumulation of cerebrosides. in vivo. Biosynthesis of lactosylceramide was not reduced in homogenates of brain and spinal cord from Quaking mice. Cerebroside levels in normal and Quaking spinal cord and in normal brain increased 2- to 3-fold after 21 days of age, but in Quaking brain there was little or no increase.     

ELECTROPHORETIC CHARACTERIZATION OF BASIC PROTEINS IN ACID EXTRACTS OF CENTRAL NERVOUS SYSTEM TISSUE

A technique has been outlined for identification of myelin basic proteins in mixtures of CNS proteins. Myelin basic proteins can be recognized easily by high cathodic mobility at low pH, a unique electrophoretic pattern exhibited at high pH and a characteristic colour when complexed with Amido black. The major protein extracted at pH 3·0 from either brain or spinal cord is myelin basic protein. In the low pH electrophoretic pattern of these extracts it is the most conspicuous component and the component migrating farthest cathodically; it does not appear in comparable electrophoretic patterns of liver extracts. Guinea pig myelin basic protein appears as a single dense blue-green band in low pH electrophoretic patterns, in contrast to the other proteins which are stained greyish-blue or greyish-purple by Amido black. The pattern of rat myelin basic protein is similar except that it consists of a pair of dense blue-green bands. A third characteristic which facilitates the identification of myelin basic proteins in mixtures is a considerable cathodic mobility and electrophoretic heterogeneity at pH 10·6. Most other basic CNS proteins barely penetrate the gel at this pH. We have also examined in detail the behaviour of two other components of pH 3·0 extracts which migrate close to myelin basic protein at low pH. Both are present in pH 3·0 extracts of liver and brain but not of spinal cord, and both stain grey instead of blue-green, a characteristic which readily distinguishes them from myelin basic protein. Neither of these components affects the characteristic pattern of microheterogeneity observed in high pH electrophoretograms of myelin basic proteins. One of these components has been purified and tentatively identified as lysine-rich histone F1.