Neurofilament accumulation induced in synapses by leupeptin.

The hypothesis that the usual absence of neurofilaments in synaptic terminals is due to their degradation by the calcium-activated protease present in axoplasm was tested by injecting leupeptin, which inhibits the protease, into the optic tectum of goldfish kept at 15 degrees and at 25 degrees C. The resulting accumulation of neurofilaments in synaptic terminals provides in vivo evidence in support of the hypothesis. The significance of these results and the potential uses of this pharmacological tool are discussed.

Modifications of myelin basic protein in DM20 transgenic mice are similar to those in myelin basic protein from multiple sclerosis.

Transgenic mice containing different numbers of transgenes (2-70) of the myelin proteolipid protein DM20 were phenotypically normal up to 3 mo of age, after which the mice containing 70 copies of the transgene spontaneously demyelinated and died at 10-12 mo. Since we demonstrated that demyelination in multiple sclerosis involved specific chemical changes in myelin basic protein (MBP), we investigated the MBP in our transgenic line for similar changes. Both the total amount of MBP in brain and the MBP mRNA levels were unaffected at the different ages. All the isoforms (14-21 kD) of MBP were present, but the microheterogeneity (a posttranslational event) was changed resulting in a higher proportion of the less cationic components reminiscent of the changes in MBP found in multiple sclerosis. An increased amount of the citrullinated form of MBP was found by Western blot analysis. Immunogold labeling of cryosections of brain revealed a greater density of particles with the anticitrulline antibody at 10 mo and that the levels of peptidylarginine deiminase (which deiminates protein-bound arginine to citrulline) were increased. This stable transgenic line represents a useful animal model for the human disease multiple sclerosis.

Morphological and biochemical characterization of light and heavy myelin isolated from developing rat brain.

Myelin from developing rat brains was separated on a discontinuous sucrose gradient into subfractions of two different densities, i.e. light and heavy myelin. Electron photomicrographs showed that heavy myelin consisted primarily of large compacted multilamellar structures with a distinct intraperiod line characteristic of myelin in situ. Light myelin, on the other hand, was composed of small vesicles having a unilamellar structure. Similar to whole myelin, both membrane subfractions were highly enriched in 2',3'-cyclic nucleotide-3'-phosphohydrolase. The specific activity of the enzyme, however, showed no developmental trend. Both subfractions contained all the four major proteins characteristic of the whole myelin membrane. There were, however, quantitative differences in the relative distribution of these proteins between light and heavy myelin. Basic protein accounted for 55% and proteolipid protein for 46% of the total myelin proteins of light and heavy myelin, respectively. DM-20 (Agrawal, H.C., Burton, R. M., Fishman, M.A., Mitchell, R.F. and Prensky, A.L. (1972) J. Neurochem. 19, 2083-2089) exhibited a developmental "switch" between light and heavy myelin. Light myelin appeared to contain more DM-20 in 15- to 20-day-old rat brain, whereas the concentration of this protein was higher in heavy myelin at subsequent ages studied.

Neurofilamentous changes in goldfish (Carassius auratus L.) brain in relation to environmental temperature.

The presence of neurofilaments in the synaptic boutons in the brains of goldfish maintained at 5 degrees C for 177 days or more is reported. Neurofilaments are not found in boutons of control fish kept at 15degreesC. It is suggested that the neurofilaments may be equated with neurofibrillary rings seen by light microscopy. Neurofilaments have been implicated in axoplasmic transport and, therefore, irregularities in the occurrence or appearance of these filaments may reflect alterations in their functions. The unusual occurrence of neurofilaments in the boutons of cold temperature goldfish may be an expression of early degenerative changes or may represent a modification of axoplasmic transport. The suitability of goldfish as a model for the study of the functional effects of neurofibrillary/neurofilamentous accumulations is discussed.

The use of tetracycline hydrochloride as a rapid fluorescent stain for myelin membranes in vertebrates and invertebrates.

We report here the use of tetracycline as a fast and reliable histological stain of myelinated nerves. These results are significant in that they point to tetracyclines as potentially important probes to study the role of divalent and trivalent cations in myelinated nerves. The usefulness and specificity of tetracycline as a supravital probe for calcium and iron metabolism in myelinated nerves is currently under study.

Distribution of neurofilaments in myelinated axons of the optic nerve of goldfish (Carassius auratus L.).

Neurofilaments were counted in myelinated axons of the optic nerve of goldfish which were acclimated to 5 degrees and 25 degrees C. The number of neurofilaments increases markedly with increasing axonal size; axons of less than 0.1 micrometer 2 in area contain between 25 and 60 neurofilaments, while in the larger axons of area greater than 1.0 micrometer 2 there are approximately 190. The densities of the neurofilaments in the small axons are noticeably higher than in the larger ones (507 and 160, respectively). A variety of fixation procedures i.e. osmium tetroxide (OsO4) in phosphate buffer, glutaraldehyde (4%) in phosphate buffer or in ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid (EGTA) and piperazine-N-N'-bis-(2-ethanesulphonic acid) (PIPES) and post-fixed with OsO4 had no effect on the numbers of neurofilaments relative to the size of axon. The anaesthetic MS-222 (tricaine methanesulphonate) likewise had no effect on the numbers of neurofilaments. It is proposed that temperature acclimation alters the axon diameter concomitant with an alteration in the number of neurofilaments to fit the new diameter of the axons.

Isolated brain capillary endothelia: influence of various levels of essential fatty acids on the acyl group composition of glycerophospholipids.

On day seven of gestation. Wistar rats were assigned to a high essential fatty acid (EFA), low EFA, or a fat free diet. The same diets were continued during lactation. On weaning, the offspring were fed the same diets as their mother. Rats were killed at 222 days, brain capillary endothelia isolated, and total lipids extracted from the purified capillaries. The composition of the constituent fatty acids of ethanolamine glycerophospholipid (EGP), choline glycerophospholipid (CGP), and the alk-1-enyl EGP composition from each diet is reported. A decrease in dietary EFA led to reduced proportions of total saturated acyl groups in EGP with no change observed in the total saturated acyl groups from CGP, and an increase in monoenoic fatty acids, particularly 18:1n-9 for each phospholipid class. The proportions of 20:4n-6 in alk-1-enyl EGP were reduced in fat-free fed animals. In addition, the relationship between 20:3n-9 and 20:4n-6 fatty acids in brain capillary endothelia were markedly increased with a reduction in dietary fat. Low EFA and fat deficient animals showed a tendency to sequester 22:6n-3.

Lipid and fatty acyl composition of rat brain capillary endothelia isolated by a new technique.

A method is described for the isolation of pure capillary endothelia from rat brain and the phosphlipid composition of these cells is reported. This method is rapid and requires only a small amount of starting material. It involves: (a) tissue disruption by high speed homogenization, (b) separation of the capillary endothelia from other brain structures using sucrose gradients, and (c) a final purification using a glass bead column. Choline and ethanolamine phosphoglycerides were found to be the predominant lipid classes of these cells amounting to 31.9% and 24.4%, respectively, of total phospholipids. The choline phosphoglycerides consisted almost exclusively of 1,2-diacyl glycerophosphorylcholine, whereas the ethanolamine phosphoglycerides consisted of approximately equal amounts of 1,2-diacyl and 1-alk-1'-enyl-2-acyl glycerophosphorylethanolamine. The composition of the constituent fatty acids of both choline and ethanolamine phosphoglycerides and the alk-1-enyl composition of ethanolamine phosphoglycerides is reported. Saturated fatty acids accounted for 45% of the total fatty acids in choline phosphoglycerides and for 53% in ethanolamine phosphoglycerides. Arachidonic acid accounted for approximately 48% of the total fatty acids in alk-1-enyl ethanolamine phosphoglyceride.

Myelinating glia of earthworm giant axons: thermally induced intramembranous changes.

The median and lateral giant axons in the ventral nerve cord of the earthworm Lumbricus terrestris are ensheathed by extensive spiral glial cell wrappings which resemble vertebrate myelin. The other, smaller, axons are encompassed by attenuated glial processes, as is typical of invertebrates. The fine structural details of the glial cells have been studied in thin sections and in replicas produced by freeze-fracturing where the intramembranous particle (IMP) populations within the lipid bilayer are visible. These consist of both low-profile IMPs as well as prominent ones 6-8 nm in diameter, scattered at random over the lipid interface in the myelinating glia. The larger IMPs on both P and E faces number about 80/mum2 at 16 degrees C in contrast to the IMP density of 400/mum2 in the other glial membranes. After acclimation to 5, 16 and 26 degrees C, the loose myelin glial membranes show variations in the density of their larger IMP population; in animals acclimated over 3 or more weeks to 5 degrees C, the number of these IMPs is significantly (P less than 0.001) less per unit area than in animals acclimated to 16 or 26 degrees C. The size of the particles at 5 degrees C is significantly (P less than 0.001) smaller than those at 16 or 26 degrees C. When animals are subjected to a sudden differential in ambient temperature, from 26 or 16 to 5 degrees C, or from 5 to 26 degrees C, and their giant axons with encompassing glia are fixed and frozen 30 min after this temperature change, the IMP population of the glial membranes remaining does not appear to alter. The differences in the IMP population of the myelinating glial membranes at different temperatures may reflect the extent to which they insulate and/or influence the velocity of impulse propagation.

Locating lipids in the CNS: an historical perspective.

Localization of lipids in the CNS is considered from an historical perspective. General consideration is given to the identification and separation of different parts of the CNS and to the recognition and detection of lipids. Problems associated with each of these aspects are noted. More treatment is given to the localization of gangliosides and the contributions of Leon Wolfe are highlighted.

Comparative studies on glial markers.

Macromolecular markers for glial cells have been sought for a variety of reasons. One of the earliest was the need for a means of assessing the purity of cell and subcellular fractions prepared from nervous tissue. While there is still a requirement for this kind of tool, emphasis has shifted towards seeking information on biochemical differentiation among cells and their functional interactions. A brief general review will be made of glial markers and two of these, 2',3'-cyclic nucleotide 3'-phosphohydrolase (CNP) and glutamine synthetase (GS), will be considered in detail. Until recently studies of markers have been concentrated on the higher vertebrates and those on lower vertebrates and invertebrates have hardly begun. However, such comparative studies may lead to fresh insight into old problems. For example, CNP has long been regarded as a marker for myelin and oligodendrocytes but it has not been possible to attribute a functional role to it and its relation to myelination has remained obscure. The finding that it is present in the glia of a moth Manduca sexta which lacks myelin provides a stimulus for a fresh approach to the problem. Another example is provided by studies on GS. This enzyme is found in astrocyte feet and preliminary results indicate that it is localized also in the perineurial glia of Aplysia ganglia. These results lead to a reconsideration of the perennial question of the possible role of astrocyte feet in barrier mechanisms. Extension of comparative studies may not only raise new questions but also provide some answers.

Isolation and initial characterization of myelin-like membrane fractions from the nerve cord of earthworms (Lumbricus terrestris L).

We report here the isolation of fractions enriched in components of the myelin-like membranes surrounding the giant axons of the earthworm. Lumbricus terrestris L. The composition and purity of the fractions have been assessed using SDS-protein electrophoresis, Western immunoblots, and electron microscopy. Preliminary enzyme assays indicated that the mitochondrial marker, succinate dehydrogenase, has a similar specific activity distribution in earthworm nerve cord and in mouse liver sedimentation velocity fractions, however, the distribution of the total units of activity among the fractions seems to indicate the existence of smaller mitochondria in earthworm nerve cord compared with mouse liver mitochondria. In earthworm nerve cord fractions, Na+/K+ ATPase and Ca2+/Mg2+ ATPase were found to be enriched exclusively in the fraction containing large plasma and myelin-like membranes, while in the mouse liver fractions, the total units of these two enzymes were found to be distributed broadly among fractions. 5'-Nucleotidase activity in the earthworm nerve cord seemed to be restricted to the microsomal fractions (endomembrane network), with a very low activity associated with the large plasma and myelin-like membrane fraction. We have established the presence of keratins or prekeratins in the myelin-like membranes, probably in the form of tonofilaments. However, we could not show that the desmosome-like structures, characteristic of these membranes, are composed of those proteins described for vertebrate epithelial desmosomes.

Effect of acclimation temperature on the axon and fiber diameter spectra and thickness of myelin of fibers of the optic nerve of goldfish.

The optic nerves of common goldfish acclimated to 5 and 25 degrees C were fixed with glutaraldehyde in either phosphate buffer or PIPES with EGTA, post-fixed with osmium tetroxide, and examined by electron microscopy. The axon diameter spectra, from axons measured in electron micrographs and those measured on the electron microscope screen, differ noticeably with acclimation temperature. At the lower temperature, there is a definite shift toward the occurrence of larger fibers compared with the spectrum of the 25 degrees C fish. Although the number of fibers assessed is small compared with the total number in the goldfish nerve, these results confirm our previous study. These findings could be attributed to an increase in the number of new fibers during the acclimation to the higher temperature. We discuss this possibility and on the available evidence find it unlikely. Other changes in the axon and fiber are also seen with acclimation temperature. The axon to fiber diameter ratio, made directly from the electron micrographs, shows that axons from the nerves of the higher acclimation temperature fish possess consistently thicker myelin sheaths than are found for axons in nerves of the lower temperature fish. This finding is also in agreement with results obtained by us from measurements independent of each other.

The effect of temperature- and oxygen-acclimation on phospholipids of goldfish (Carassius auratus L.) brain mitochondria.

Goldfish (Carassius auratus L.) were temperature- and oxygen-acclimated and the composition of the phospholipids and their acyl groups in brain mitochondria was determined. The proportion of ethanolamine to choline phospholipid was greater while the plasmenyl ethanolamine value (P-GPE/D- + P-GPE) was lower at the low acclimation temperature. For the ethanolamine glycerophospholipids, a rise in the ratio n-6/n-3 fatty acyl groups occurred with cold acclimation. No significant change in the ratio was exhibited by phosphatidyl choline. When the oxygen level was increased, at either acclimation temperature, a rise in the GPE/GPC ratio and the plasmenyl ethanolamine value resulted. The n-6/n-3 ratio was generally increased for the ethanolamine classes when the oxygen concentration was raised. The possible significance of these changes is discussed.

The effects of temperature- and oxygen-acclimation on phospholipids of goldfish (Carassius auratus L.) brain microsomes.

Brain microsome phospholipids and their acyl groups, from temperature and oxygen acclimated goldfish, were investigated. At the lower acclimation temperature (5C) the proportion of ethanolamine- to choline-glycerophosphatides (GPE/GPC) was increased, and the proportion of phosphatidal ethanolamine value decreased. A rise in the n-6/n-3 fatty acyl group also occurred in cold acclimation. Irrespective of acclimation temperature, 25 degrees C or 5 degrees C, a partial replacement of GPC by GPE occurred when the concentration of oxygen was increased; conversely the GPE/GPC ratio decreased at the hypoxic level. The plasmalogen GPE content increased as the oxygen concentration was raised. A rise in the n-6/n-3 ratio, for ethanolamine glycerophosphatides and phosphatidyl choline, occurred when the oxygen concentration was increased (hypoxia to hyperoxia). It is concluded that the lipid alterations associated with thermal acclimation are, in part, attributable to the concomitant change in oxygen concentration.

Comparative immunohistochemical study of glial filament proteins (glial fibrillary acidic protein and vimentin) in goldfish, octopus, and snail.

Glial fibrillary acidic protein (GFAP) and vimentin proteins are known to be component proteins of glial filaments in the CNS of many vertebrates. The nature of the filaments present in the glial cells of the goldfish optic tectum and in the CNS of two members of the Mollusca (Helix pomatia and Octopus vulgaris) were investigated using immunocytochemical localization of monoclonal antibodies to GFAP and vimentin. Immunoblots visualized by the alkaline phosphatase method showed cross-reactive protein bands to GFAP and vimentin antibodies in total brain homogenates of the goldfish, octopus, and snail. Immunofluorescence staining of the goldfish optic tectum showed GFAP immunoreactivity, primarily in the ependymal cell processes. Immunogold labelling at the ultrastructural level verified that GFAP antibodies were bound to glial filaments. Immunolabelling of the optic lobe of Octopus vulgaris and the cerebral ganglia of Helix pomatia suggests that a protein exhibiting antigenic properties similar to GFAP is a component protein in the filaments of the protoplasmic and filamentous glia randomly distributed throughout the CNS. Unlike anti-GFAP antibodies, which stained relatively specific to filaments, vimentin staining in the CNS tissues of the three organisms studied did not appear to be exclusive to filamentous structures. As vimentin protein has been shown, in previous studies as well as our own, to exist in many tissue types, this suggests that it does not appear to be confined to glial filaments but is shared with other subcellular components. The proteins GFAP and vimentin which are thought to be well conserved in vertebrate evolution also appear to be expressed in the nervous system of some lower organisms.

Effect of acclimation and fixation temperatures on the number of lamellae and periodicity of myelin in fibres of the optic nerve of goldfish.

Previous findings from our laboratory have shown that the optic nerves of goldfish acclimated to different temperatures differ considerably in their glycerophospholipid composition. This paper describes changes in the morphology of the nerve with different acclimation and fixation temperatures. Optic nerves of 5 and 25 degrees C acclimated fish were excised and fixed at the temperature of acclimation, or at the reverse temperature, and the morphology observed by electron microscopy. Under all temperature conditions considered there is a statistically significant linear relationship between the radius of the axon and the number of myelin lamellae. However, the temperature of acclimation and fixation both influence the regression coefficients for this relationship, the higher the acclimation temperature the lower the coefficient and the higher the fixation temperature the higher the coefficient. The periodicity of the myelin also alters with these temperatures, being greater in the 25 degrees C fish than in the 5 degrees C ones. Myelin sheath thickness is also significantly greater in the 25 degrees C fish. These results are discussed in relation to observed changes in glycerophospholipid composition and conduction velocities.