Glial Plasma Membranes Research Articles

Trans-glial channels in ventral nerve roots of crayfish.

The sheath around the roots of the sixth abdominal ganglion in the ventral nerve cord of the crayfish consists of concentric layers of thin glial processes alternating with wide clefts containing filamentous connective tissue. Regions of each glial lamella are perforated by single, short, tubular channels: the trans-glial channels. In thin plastic sections examined in the electron microscope, the channels appear as slits that are 240 A wide and 450-550 A long which traverse glial lamellae less than 1,500 A thick. Branched tubular channels cross glial sheets that are thicker than 1,500 A. The thickest glial wrap is adaxonal; it closely encapsulates individual axons and its cell membrane is separated from the axolemma by a collagen-free space of only 150 A. The adaxonal glial cytoplasm contains unique, three-dimensional networks of interconnected tubules. Separate tubular lattices occur along these thicker processes. In replicas of freeze-fractured sheaths, the outer half of the plasma membrane belonging to the thin glial sheets exhibits many volcano-like protrusions which represent cross fractures through the necks of trans-glial channels. Corresponding depressions on the inner half of these membranes are sites where the plasma membrane invaginates to form the channels. Although some channels are randomly dispersed, others are lineraly positioned in restricted areas across successive glial layers. The number of channels is far more readily appreciated in replicas than in thin sections. The average frequency of channels is 16 per mu2 (range 8 to 33) in normal roots and does not differ significantly from the average of 13 per mu2 in proximal stumps of roots fixed three to four weeks after the roots were cut. The channels are not precisely aligned from one glial layer to the next but do appear to coincide approximately with the adaxonal tubular lattice. The combination of trans-glial channels and adaxonal tubular lattices may provide a complex conduit that could facilitate a rapid, passive flow of electrolytes and nutrients across the nerve sheath to the axonal surface. Horseradish peroxidase solutions bathing the ventral roots enter the trans-glial channels, extracellular clefts and finally the tubular lattices. This distribution supports the proposed role of the channels in a rapid extracellular passage of solutes. The channel profiles have a range of forms consistent with the supposition that they are not static but continually reforming. There are indications that, proximal to the cut, the areas of glial plasma membrane with channel profiles contain more junctional complexes between regenerating cells than between glial cells of normal sheaths. The channel profiles and aggregates of particles belonging to junctions are closely associated when they occupy the same region of the membrane.

The subcellular distribution of polyphosphoinositides in myelinated and unmyelinated rat brain

1. 1. The distribution of polyphosphoinositides from 7- and 34-day-old rat brain was investigated in subcellular fractions prepared by differentia! and density gradient centrifugation. Fractionation in an alkaline medium produced greatly improved recoveries ofdiphosphoinositide and triphosphoinositide, especially in the synaptosomerich and microsomal fractions. 2. 2. On fractionation of 34-day-old brain, over half of the diphosphoinositide and more than three-quarters of the triphosphoinositide were recovered in myelin-containing fractions. The molar ratio of di- to triphosphoinositide in the myelin-rich band from the density gradient was 0.47. The corresponding ratio in the synaptosome-rich band was 1.96. During myelination, the amount of each polyphosphoinositide did not change in the synaptosomal fraction and declined slightly inthemicrosomes. 3. 3. In primary fractions from 7-day-old brain, more than 60 % of each lipid was recovered in the crude mitochondrial pellet. When this pellet was further fractionated, the greatest amounts of the compounds were found in the synaptosome-rich fraction. Both polyphosphoinositides were most enriched, more than 3-fold on a protein basis as compared to whole homogenate, in a band which migrated to a position on the gradient occupied by a myelin-rich fraction when mature rat brains are used. 4. 4. The distribution of polyphosphoinositides on the density gradient resembled those of acetylcholinesterase and 5′-nucleotidase much more than those of lactic acid dehydrogenase and cytochrome c oxidase. 5. 5. Evidence from marker enzyme data and electron microscopic examination of the density gradient fractions leads us to suggest that in unmyelinated rat brain di-and triphosphoinositides are concentrated in neuronal and glial plasma membranes.

Highly Purified Synaptosomal Membranes from Rat Brain: PREPARATION AND CHARACTERIZATION

Synaptosomal plasma membranes were isolated from whole rat brain by a method derived from several existing ones. The procedure involves the sequential centrifugation of a crude mitochondrial fraction through a discontinuous sucrose gradient and a continuous cesium chloride gradient. The material is then subjected to osmotic lysis and centrifuged through an additional discontinuous sucrose gradient. The synaptosomal membranes prepared in this way contain only very small amounts of mitochondria, endoplasmic reticulum, myelin, axonal and glial plasma membranes, and lysosomes; the level of contamination was estimated by assay for appropriate marker enzymes, and by assaying the final membrane preparation for radioactivity following the addition of previously labeled radioactive contaminants at the start of the isolation procedure. On the basis of these criteria, the contaminants examined appear to account for less than 10 to 15% of the protein in the preparation. The isolated membranes were sequentially extracted with Triton X-100 and sodium dodecyl sulfate; as previously described, the Triton-soluble fraction contains those portions of the synaptosomal membrane not associated with the synaptic junction, whereas the sodium dodecyl sulfate dissolves the junctional complex. The fractions were subjected to electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate, and the gels were stained for protein with Coomassie brilliant blue. The results indicate that some proteins are common to both fractions, but that each fraction contains several unique protein species. The membrane preparation also contains RNA and DNA in limited amounts. The bulk of the RNA resembles mitochondrial RNA with respect to its mobility in polyacrylamideagarose composite gels.

The formation of capillary basement membranes during internal vascularization of the rat's cerebral cortex.

The vascularization of the parietal-temporal region of the cerebral hemispheres has been studied in a total of 50 rats from day 11 of gestation up to adults. The first extracerebral vessels constituting the primary perineural vascular network and the first intracerebral vessels on day 12–14 of gestation show sinusoid characteristics, i.e. irregular thickness of the endothelial wall perforated by fenestrations or small holes and showing, if at all, the beginning accumulation of basement membrane (BM) material. No paired vessels have been observed which would be expected if internal vascularization of cortical anlage starts by penetrating loops. Immature capillaries developing by sprouting (and) from the preexistent vessels begin to appear at about day 15 of gestation. The further differentiation of the terminal vascular bed and the establishing of the definitive architecture is accompanied by the maturation of cortical tissue, i.e. diminution of extracellular spaces, differentiation of perivascular, astroglial and neuronal elements including the development of synapses. The continuous process of BM-formation from the first appearance until the postnatal thickening is described by four successive stages: Stage 1. Local accumulations of fine filamentous material between endothelium and opposite perivascular surfaces of sinusoids and sprouts. Stage 2. Delicate networks of filaments attached on endothelial, pericytal and adjacent glial plasma membranes (PM) of immature capillaries, plaques of lamina densa in narrow perivascular clefts. Stage 3. A thin continuous lamina densa adapted to the PM; its filaments are arranged parallel to the cell surface. In this plane they run randomly. In the lamina rara only few filaments run to the PM mainly perpendicular: stage of immature cortical capillaries. Stage 4. Thickened lamina densa, condensed filamentous pattern; narrow zone of lamina rara: stage of mature cortical capillaries. Coincidental with the rapid thickening of BM in the 3rd–4th postnatal week the characteristics of capillary growth change: The intensive sprouting is finished and the capillary length increases nearly proportionally to tissue volume later on. It is suggested that the BM plays a role in regulating differentiation and mitotic division of the adjacent cells.

Changes in the osmiophilia of myelin and lipid content in the kitten optic nerve.

Abstract—Myelin osmiophilia has been shown to develop significantly later than myelin staining by Luxol fast blue and Sudan black, in the developing kitten optic nerve. These histological changes are accompanied by alterations in the lipid composition of the optic nerve. Although myelination commences at about 10 days post partum in the nerve the appearance of cerebrosides is unexpectedly delayed. Changes in fatty acid chain length and lipid composition of optic nerve are consistent with the suggestion that ‘early’ myelin may be unchanged glial plasma membrane.

The thoracic glanglia of the grasshopper, Melanoplus differentialis: fine struc- ture of the perineurium and neuroglia with special reference to the intracellular distribution of phosphatases.

The thoracic ganglia of Melanoplus are encapsulated by a number of consecutive protective sheaths. These include an outer layer of pigment cells, an acellular neural lamella composed partially of collagenous fibres, and an inner perineurium. The perineurial cells contain numerous osmiophilic granules; these are extracted by glutaraldehyde fixation after which the cells have a vacuolated appearance. Beneath the perineurial sheath lies the ganglion proper, in which neuroglial cells ensheath neurones that are arranged peripherally around a central neuropile. The neuroglia consist of attenuated cells with prominent cytoplasmic processes extending around, as well as into, the cytoplasm of the nerve cell bodies. Membrane-delimited dense bodies or “gliosomes” are present in the glial cytoplasm; these appear to be lysosomes for they contain cytochemically demonstrable acid phosphatase activity in incubated sections. These lysosomes differ from those of most vertebrates in that, in addition to hydrolyzing nucleoside monophosphates at acid pH, they also hydrolyze nucleoside diphosphates and triphosphates at neutral pH. Reaction product from adenosine triphosphatase activity is seen in both the glial plasma membrane and in the cisternae of the rough endoplasmic reticulum, as is reaction product due to thiamine diphosphatase. This enzymatic activity in the membranes of the glial cells provides a useful way of “marking” the cells for both light and electron microscopy. The possible signifcance of membrane-associated phosphatase activity in a cell whose function appears to be at least in part trophic, is discussed.

The lipid composition of rat brain myelin and subcellular fractions during development.

1. The lipids of whole brain and subcellular fractions of the rat were analysed during development. 2. The deposition of cholesterol occurred in two phases, one related to increasing wet weight of the brain and the second to myelination. Cerebroside accumulation was related only to myelination. 3. The composition of myelin isolated from 12-day-old rat brain was different in some respects from that of the adult. In the former there was an increase of phospholipid in relation to cholesterol and a marked deficiency in cerebroside. 4. It is suggested that early myelin is extruded glial plasma membrane, which only later becomes mature myelin.

Myelinogenesis in the rat brain.

MORPHOLOGICAL evidence indicates that myelin is formed in the central nervous system by the wrapping of macroglial plasma membrane about the axon1,2. Proliferation of glial cells occurs shortly before myelination3, and connecting processes between oligodendrocytes and the developing myelin sheath have been noted4. This concept is supported by experiments with isotopes, which suggest that lipid from the microsomal fraction, which would include glial plasma membrane, is utilized in myelin formation5. Myelin would therefore be expected to have the same or similar chemical composition as that of the oligodendroglial plasma membrane and myelin lipids would be present in the brain during the stage of glial proliferation immediately before the first appearance of myelin rings. Analysis of glial-rich fractions from adult rat brain (Table 1), however, shows that they contain much less cerebroside and more phospholipid than is present in myelin, and hence that the composition is similar to that of most other tissue membrane systems. This same type of lipid composition is found in glial tumour tissue (Table 2) and in the glial-rich brain just before the onset of myelination9. In order to examine the sequence of biochemical events further, myelin has been isolated from the brains of rats from 10 days old onwards. The early myelin fraction was found to have a low cerebroside content and to be rich in phospholipid and thus similar to the lipid composition of glia (Table 1). Only when rats were 30 days old was the adult myelin lipid ratio attained. Morphological, enzyme and chemical examination of myelin from 12 day old rats does not suggest that this fraction is heavily contaminated by other subcellular fractions. Thus little succinic dehydrogenase and ATPase activity and only small amounts of nucleic acids are found in the preparation. It therefore seems possible that early myelin consists essentially of oligodendroglial plasma membrane and that later insertion of cerebroside into the myelin lamella produces the stable mature membrane structure. This hypothesis, which attributes a key role to cerebroside, may be pertinent to neurological diseases in which defects in cerebroside content have been noted.