Submicroscopic changes of the synapse after nerve section in the acoustic ganglion of the guinea pig; an electron microscope study

The degenerative changes of the synaptic regions after nerve section have been studied with the electron microscope in the interneuronal synapse of the ventral ganglion of the acoustic nerve of the guinea pig. Fixation with buffered osmic tetroxide was carried out 22, 44, and 48 hours after destruction of the cochlea on one side; the contralateral ganglion being used as control. The submicroscopic organization of normal axosomatic and axodendritic synapses is described. In the synaptic ending four morphological components are recognized: the membrane, the mitochondria, the synaptic vesicles (19, 20), and the cytoplasmic matrix. The intimate contact of glial processes with the endings and with the surface of the nerve cell is described. At the level of the synaptic junction there is a direct contact of the limiting membranes of the ending and of the cell body or dendrite. Both contacting membranes constitute the synaptic one with a total thickness of about 250 A. This membrane has regions of higher electron density where the synaptic vesicles come into intimate contact and fuse with it. Definite degenerative submicroscopic changes in the nerve endings were observed after 22 hours of destruction of the cochlea and were much more conspicuous after 44 and 48 hours. After 22 hours there is swelling of the ending and decreased electron density of the matrix. Most synaptic vesicles have disappeared or seem to undergo a process of clumping and dissolution. Some mitochondria also show signs of degeneration. After 44 hours the synaptic vesicles have practically disappeared; mitochondria are in different stages of lysis; the membrane of the ending becomes irregular in shape, and there is shrinkage and in some cases detachment of the ending. No changes in the postsynaptic cytoplasm were observed. These observations and particularly the rapid lysis of the synaptic vesicles are discussed in correlation with data from the literature indicating the early alteration of synaptic function and the biochemical changes occurring after section of the afferent nerve. The hypothesis that the synaptic vesicles may be carriers of acetylcholine or other active substances (19, 20) and that they may act as biochemical units in synaptic transmission is also discussed.(2)     

SUBMICROSCOPIC CHANGES OF THE SYNAPSE AFTER NERVE SECTION IN THE ACOUSTIC GANGLION OF THE GUINEA PIG. AN ELECTRON MICROSCOPE STUDY

The degenerative changes of the synaptic regions after nerve section have been studied with the electron microscope in the interneuronal synapse of the ventral ganglion of the acoustic nerve of the guinea pig. Fixation with buffered osmic tetroxide was carried out 22, 44, and 48 hours after destruction of the cochlea on one side; the contralateral ganglion being used as control. The submicroscopic organization of normal axosomatic and axodendritic synapses is described. In the synaptic ending four morphological components are recognized: the membrane, the mitochondria, the synaptic vesicles (19, 20), and the cytoplasmic matrix. The intimate contact of glial processes with the endings and with the surface of the nerve cell is described. At the level of the synaptic junction there is a direct contact of the limiting membranes of the ending and of the cell body or dendrite. Both contacting membranes constitute the synaptic one with a total thickness of about 250 A. This membrane has regions of higher electron density where the synaptic vesicles come into intimate contact and fuse with it. Definite degenerative submicroscopic changes in the nerve endings were observed after 22 hours of destruction of the cochlea and were much more conspicuous after 44 and 48 hours. After 22 hours there is swelling of the ending and decreased electron density of the matrix. Most synaptic vesicles have disappeared or seem to undergo a process of clumping and dissolution. Some mitochondria also show signs of degeneration. After 44 hours the synaptic vesicles have practically disappeared; mitochondria are in different stages of lysis; the membrane of the ending becomes irregular in shape, and there is shrinkage and in some cases detachment of the ending. No changes in the postsynaptic cytoplasm were observed. These observations and particularly the rapid lysis of the synaptic vesicles are discussed in correlation with data from the literature indicating the early alteration of synaptic function and the biochemical changes occurring after section of the afferent nerve. The hypothesis that the synaptic vesicles may be carriers of acetylcholine or other active substances (19, 20) and that they may act as biochemical units in synaptic transmission is also discussed.²     

Lipids and proteolipids in isolated subcellular membranes of rat brain cortex

The cerebral cortex of the rat was submitted to an extensive cell fractionation schedule and in the various fractions, protein, proteolipid protein, total phospholipids, cholesterol, galactolipids, plasmalogens, and gangliosides were determined. With increasing purification the different isolated membranous structures: i.e. myelin, nerve ending membranes, synaptic vesicles, mitochondria, and microsomes, show a definite biochemical specialization reflected in their lipid composition. The presence of gangliosides in some nerve ending membranes is confirmed, and the possible functional role of these acid glyco-lipids is discussed. The importance of proteolipids as structural components of membranes is recognized. The richness of these compounds in myelin is confirmed and a special localization in the nerve ending membranes is indicated. Analysis of the molar ratios of the different lipids and proteins in the isolated membranes demonstrates that each one has a specific pattern of molecular organization. This pattern is discussed in relation to the macromolecular structures revealed by electronmicroscopy and some of the molecular models postulated for cell membranes.     

Marker enzymes,phospholipids and acyl group composition of a somal plasma membrane fraction isolated from rat cerebral cortex: a comparison with microsomes and synaptic plasma membranes

Subjecting brain homogenates to differential speed and sucrose density gradient centrifugation resulted in the isolation of a membrane fraction from the post-mitochondrial supernatant with properties and marker enzyme profiles typical of plasma membranes. This membrane fraction is compared with the microsomes and the synaptic plasma membranes isolated from synaptosomes. Like the synaptic plasma membranes, membranes obtained from the post-mitochondrial supernatant were enriched five-fold in 5′-nucleotidase activity. However, the latter membranes were lower in (Na⁺, K⁺)-ATPase activity and higher in NADPH-cytochrome C reductase activity as compared to the synaptic plasma membranes. The post-mitochondrial plasma membranes were also different from the microsomes in their respective marker enzyme activities. Electron microscopic examination indicated largely membranous vesicles for both plasma membrane fractions with little contamination by myelin, mitochondra and intact synaptosomes. The phospholipid and acyl group profiles of the two plasma membrane fractions were surprisingly similar, but they were different from the characteristic profiles of myelin and mitochondria. It is concluded that plasma membranes isolated from the post-mitochondrial supernatant fraction are derived largely from neuronal and glial soma and are thus designated the somal plasma membrane fraction.     

Isolation of synaptic plasma membrane from brain by combined flotation-sedimentation density gradient centrifugation

A method is described for the subcellular fractionation of brain to obtain a preparation highly enriched in synaptic plasma membranes. The enriched fraction is recovered from the interface of a two-step sucrose density gradient on which a hypotonically lysed crude mitochondrial fraction from brain has been separated by simultaneous sedimentation and flotation centrifugation. Enzyme marker activities associated with the neuronal plasma membrane are enriched in the synaptic plasma membrane-containing fraction while less than 10% of enzyme markers associated with the major probable contaminants, myelin and mitochondria, are found in the same fraction. Morphological examination of the enriched fraction suggests that about 80% of the profiles are recognisably synaptic in origin. Compared to previously described methods for obtaining synaptic plasma-enriched fractions of equivalent purity, the procedure reported here is simpler, shorter, and of greater capacity.     

The heterogeneity of rat brain mitochondria isolated on continuous sucrose gradients

Abstract— The distribution of a series of enzymes in the post-nuclear supernatant of rat brain homogenates was investigated following continuous density-gradient centrifugation. The enzymes studied were acetyl coenzyme A synthetase, glutamic dehydrogenase, glutamine synthetase, glutaminase I, succinic dehydrogenase and monoamine oxidase. Each of these enzymes with the exception of glutamine synthetase appears predominantly in the mitochondrial region of the gradient. Although about 20 per cent of this enzyme is present in the crude mitochondrial pellet, on density gradient centrifugation no special association of glutamine synthetase with any of the mitochondrial fractions was observed. Each of the other enzymes studied was found to have a characteristic distribution in the gradient; this suggests that brain mitochondria may be heterogeneous both in buoyant density and in their enzyme content. Three principal fractions are described: (i) dense particles containing high concentrations of acetyl coenzyme A synthetase and glutamic dehydrogenase; (ii) a fraction comprising the bulk of the mitochondria with high levels of monoamine oxidase, succinic dehydrogenase and glutaminase I; and (iii) particles in the synaptic ending region of the gradient characterized by relatively high levels of monoamine oxidase and succinic dehydrogenase and containing only small amounts of the other enzymes studied. If the mitochondrial heterogeneity that is observed on centrifugation reflects the existence within brain cells of mitochondria with specialized function, a partial explanation may be available for multiple pools of tricarboxylic acid cycle intermediates which have been postulated from isotopie labelling experiments.     

Subcellular distribution of the transmissible agent in Creutzfeldt-Jakob disease mouse brain

To determine the intracellular localization of the Creutzfeldt-Jakob disease (CJD) agent in mouse brain, cerebrum tissue of the mouse brain affected with the Fukuoka-1 strain was separated into six subcellular fractions (microsome, nerve ending, myelin, mitochondria, nucleus, and soluble fractions) by differential sucrose density gradient, and then the CJD infectivity of these fractions was examined. Serially diluted samples of each subfraction were inoculated intracerebrally into groups of BALB/c mice, and the infectivity was determined as to end point titration value, incubation period, and number of affected mice. On the basis of the protein content, the highest CJD infectivity was observed in the microsomal fraction. The nerve ending (synaptic plasma membrane) and myelin fractions were also infective. The mitochondria and nucleus fractions showed the lower infectivity. The infectivity of the soluble fraction was the lowest among the six subcellular fractions. From the findings obtained in this study two possibilities as to the intracellular localization of CJD agent were suggested: 1) the transmissible agent of CJD is closely associated with surface membranes of neuronal and/or glial cells, including their processes; 2) the CJD agent is diffusely present intracellularly, including in the surface membranes, but for manifestation of infectivity the agent needs membrane components as prerequisite factors.     

Distribution and metabolism of glycoproteins and glycosaminoglycans in subcellular fractions of brain.

The distribution, carbohydrate composition, and metabolism of glycoproteins have been studied in mitochondria, microsomes, axons, and whole rat brain, as well as in various synaptosomal subfractions, including the soluble protein, mitochondria, and synaptic membranes. Approximately 90% of the brain glycoproteins occur in the particulate fraction, and they are present in particularly high amounts in synaptic and microsomal membranes, where the concentration of glycoprotein carbohydrate is 2-3% of the lipid-free dry weight. Treatment of purified synaptic membranes with 0.2% Triton X-100 extracted 70% of the glycoprotein carbohydrate but only 35% of the lipid-free protein residue, and the resulting synaptic membrane subfractions differed significantly in carbohydrate composition. The glycoproteins which are not extracted by Triton X-100 also have a more rapid turnover, as indicated by the 80-155% higher specific activity of hexosamine and sialic acid 1 day after labeling with [3H]glucosamine in vivo. The specific activity of sialic acid in the synaptosomal soluble glycoproteins 2 hr after labeling was greater than 100 times that of the synaptosomal particulate fraction, whereas the difference in hexosamine specific activity in these two fractions was only twofold, and by 22 hr there was little or no difference in the specific activities of sialic acid and hexosamine in synaptosomal soluble as compared to membrane glycoproteins. These data indicate that sialic acid may be added locally to synaptosomal soluble glycoproteins before there is significant labeling of nerve ending glycoproteins by axoplasmic transport. Fifty to sixty percent of the hyaluronic acid and heparan sulfate of brain is located in the various membranes comprising the microsomal fraction, whereas half of the chondroitin sulfate is soluble and only one-third is in microsomal membranes. When microsomes are subfractionated on a discontinuous density gradient over half of the hyaluronic acid and chondroitin sulfate are found in membranes with a density less than that of 0.5 M sucrose (representing a six- to sevenfold enrichment over their concentrations in the membranes applied to the gradient), whereas half of the heparan sulfate is present in membranes with a density greater than that of 0.8 M.     

Preparative scale isolation of basal-lateral plasma membranes from rat intestinal epithelial cells.

A simple, efficient procedure is described for the preparative scale isolation of basal-lateral membranes from the rat intestinal epithelium. The intestinal mucosa was mildly homogenized and soluble protein and RNA were separated from the homogenate by differential centrifugation. The basal-lateral membranes were then separated from nuclei, mitochondria, and brush border membranes by differential centrifugation in a medium close to the equilibrium density of the basal-lateral membranes. Final purification of the basal-lateral membranes was achieved on a linear density gradient in a high-capacity zonal rotor. The final product (usually at least 40 mg protein) represented a 34% yield of basal-lateral membranes purified 18-fold with respect to protein, 26-fold with respect to brush border membranes, and 53-fold with respect to mitochondria.     

Preparative Scale Isolation of Basal-Lateral Plasma Membranes from Rat Intestinal Epithelial Cells

A simple, efficient procedure is described for the preparative scale isolation of basal-lateral membranes from the rat intestinal epithelium. The intestinal mucosa was mildly homogenized and soluble protein and RNA were separated from the homogenate by differential centrifugation. The basal-lateral membranes were then separated from nuclei, mitochondria, and brush border membranes by differential centrifugation in a medium close to the equilibrium density of the basal-lateral membranes. Final purification of the basal-lateral membranes was achieved on a linear density gradient in a high-capacity zonal rotor. The final product (usually at least 40 mg protein) represented a 34% yield of basal-lateral membranes purified 18-fold with respect to protein, 26-fold with respect to brush border membranes, and 53-fold with respect to mitochondria.     

Distinct platelet-activating factor binding sites in synaptic endings and in intracellular membranes of rat cerebral cortex

The binding of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC or PAF, platelet-activating factor) to synaptic plasma membranes, microsomal membranes, and other rat cerebral cortex subcellular fractions was studied. Using several PAF-binding antagonists, three distinct sites were identified. Two of them were in intracellular membranes (microsomes) and one in synaptic plasma membranes. Microsomal membranes were prepared after obtaining a 43,500 x g pellet from the postmitochondrial supernatant and subsequent centrifugation at 105,000 x g of the resulting supernatant. Most plasma membrane markers were retained in the 43,500 x g pellet (Sun, G.Y., Huang, H.-M., Kelleher, J.A., Stubbs, E.B., Sun, A. Y. (1988) Neurochem. Int. 12, 69-77). Microsomes were purified by density-gradient centrifugation and marker enzymes showed relatively very low contamination by plasma membrane markers. Myelin and mitochondria were devoid of specific PAF binding. A site displaying the highest PAF-binding affinity reported to date in all cells and membranes (KD = 22.5 +/- 1.7 pM and Bmax 8.75 = fmol/mg protein), was found in the microsomal fraction. There was a second binding site in microsomal fractions (KD = 25.0 +/- 0.8 nM and Bmax = 0.96 pmol/mg protein. Ca2+ decreases PAF affinity for the microsomal binding sites. The third binding site displays relatively low specific PAF binding and is present in synaptosomal plasma membranes. Moreover, displacement curves by a wide variety of PAF antagonists indicated different affinities for each of the binding sites described here. These results indicate that PAF-binding sites are heterogeneous in rat cerebral cortex, and they imply that the microsomal membrane sites may be involved, at least in part, in intracellular events such as gene expression.     

真鲷视网膜结构及其视觉特性研究：Ⅱ视网膜光感受细胞超微结构研究

对真鲷光感受细胞的超微结构进行观察,结果表明：视杆外段膜盘为游离膜盘,视锥外段膜盘则为连续的膜结构,视锥和视杆均含有连接纤毛和辅助外段。花萼状突起起源于内段。椭体内充满线粒体,无球状小体。双锥椭圆体并生膜为六层,视锥内段无鳍状突起,视锥突触带,在明适应视网膜中数量增多,在暗适应视网中数量减少,视杆突触带在这两种适应网膜中数量不变,每一杆小球只有一个突触带,而锥小足有４－６个突触带。     

Release of the phophodiesterase activator by cyclic AMP-dependent ATP:protein phosphotransferase from subcellular fractions of rat brain

The subcellular distribution of the endogenous phosphodiesterase activator and its release from membranes by a cyclic AMP-dependent ATP:protein phosphotransferase was studied in fractions and subfractions of rat brain homogenate. These fractions were obtained by differential centrifugation and sucrose density gradient; their identity was ascertained by electron microscopy and specific enzyme markers.In the subcellular particulate fractions, the concentration of activator is highest in the microsomal fraction, followed by the mictochondrial and nuclear fractions. Gradient centrifugation of the main mitochondrial subfraction revealed that activator was concentrated in those fractions containing mainly synaptic membranes.Activator was released from membranes by a cyclic AMP-dependent phosphorylation of membrane protein. The release of activator occurred mainly from the mitochondrial subfractions containing synaptic membranes and synaptic vesicles.The data support the view that a release of activator from membranes may be important in normalizing the elevated concentration of cyclic AMP following persistent transsynaptic activation of adenylate cyclase.     

The effect of pressure on fetal and neonatal liver mitochondrial membranes

The effects of pressure on late fetal and neonatal rat liver mitochondria have been investigated. High hydrostatic pressure, as produced by isopycnic centrifugation in sucrose and glycogen gradients, altered the mitochondrial membranes of 1- and 7-day-old rats. Most of the mitochondrial enzymes, chosen for their known submitochondrial location, had a trimodal distribution in the sucrose gradients. In the glycogen gradients, a shift of the mitochondria to a lower density was noticed. Fetal liver mitochondria were resistant to the hydrostatic pressure exerted during isopycnic centrifugation experiments under different conditions such as sucrose and glycogen density gradients. The submitochondrial compartment tracer enzymes exhibited an unimodal distribution. Experimental temperatures set at 15 degrees C had a protective effect from hydrostatic pressure alterations in the neonatal liver mitochondria, whereas no effects were noticeable in the fetal mitochondria. Experiments in a hydraulic compression chamber showed that outer membranes of fetal mitochondria were more fragile and the inner membranes more resistant to compression than in the early stages after birth.     

Separation of enriched synaptosomes, synaptic vesicles and synaptic plasma membranes]

A rapid and simple method is described for separation of intact synaptosomes, synaptic plasma membranes and vesicles. Two synaptosome fractions were obtained by modified differential centrifugation. The rate zonal zentrifugation in a linear sucrose gradient (very low density) is suitable to obtain fractions highly enriched in synaptic plasma membranes and vesicles. Examination of the prepared fractions was done by enzyme marker activities and electron microscopy     

Release of the phosphodiesterase activator by cyclic AMP-dependent ATP:protein phosphotransferase from subcellular fractions of rat brain.

The subcellular distribution of the endogenous phosphodiesterase activator and its release from membranes by a cyclic AMP-dependent ATP:protein phosphotransferase was studied in fractions and subfractions of rat brain homogenate. These fractions were obtained by differential centrifugation and sucrose density gradient; their identity was ascertained by electron microscopy and specific enzyme markers. In the subcellular particulate fractions, the concentration of activator is highest in the microsomal fraction, followed by the mitochondrial and nuclear fractions. Gradient centrifugation of the main mitochondrial subfraction revealed that activator was concentrated in those fractions containing mainly synaptic membranes. Activator was releasted from membranes by a cyclic AMP-dependent phosphorylation of membrane protein. The release of activator occurred mainly from the mitochondrial subfractions containing synaptic membranes and synaptic vesicles. The data support the view that a release of activator from membranes may be important in normalizing the elevated concentration of cyclic AMP following persistent transsynaptic activation of adenylate cyclase.     

Identification of Synapse Specific Components: Synaptic Glycoproteins, Proteins, and Transmitter Binding Sites

Synaptic junctions (SJ) were prepared from synaptic plasma membranes (SPM) by extraction with Triton X-100 and density gradient centrifugation. These SJs were enriched in certain Concanavalin A (Con A) binding glycoproteins, the 52,000 Mr postsynaptic density (PSD) protein, and receptor sites for L-glutamate, L-aspartate, kainic acid (KA) but not quinuclidinyl benzilate (QNB). Various other membrane fractions were extracted by means of the same procedure. Those fractions prepared from light SPMs and crude myelin contained identifiable synaptic junctions and were also highly enriched in the synaptic components. The SJ-like fraction from mitochondria did not contain any of the characteristic synaptic macromolecules. However, this fraction from microsomes contained levels of the 52,000 Mr PSD protein and binding sites for L-glutamate (L-Glu) and L-aspartate (L-Asp) similar to true synaptic junctions, although the Con A binding glycoproteins and KA binding sites were nearly absent. On the basis of electron microscopy, the SJ-like fraction from microsomes did not contain structures recognizable as SJs. Thus, the Con A binding glycoproteins and KA binding sites appear to be excellent markers for the SJ.     

Identification and Topography of Substrates for Protein Carboxyl Methyltransferase in Synaptic Membrane and Myelin-Enriched Fractions of Bovine and Rat Brain

The major components of crude brain synaptosomes (synaptic membranes, mitochondria, and myelin) have been separated and analyzed by polyacrylamide gel electrophoresis for the presence of proteins that serve as substrates for protein carboxyl methyltransferase. Of the three fractions, synaptic membranes contain the largest number of individual methyl acceptors (at least seven), while mitochondria contain no well-defined methyl acceptors. Undisrupted myelin contains a single major methyl acceptor with a very low apparent molecular weight. The patterns of protein methylation in synaptic membranes prepared from cerebral cortex, hippocampus, striatum, thalamus, and tectum showed marked differences; however, these differences could largely be explained by differential degrees of myelin contamination in synaptic membranes from the different regions. The effect of trypsin pretreatment on the carboxyl methylation of intact and lysed synaptosomes was studied to estimate the sidedness of the major methylation sites on synaptic membranes. One of the methyl acceptors (Mr 48K) appears to be facing the intracellular surface of the synaptosome, but most sites appear to be outward facing.     

ISOLATION OF NERVE ENDINGS FROM THE POSTERIOR PITUITARY GLAND : Electron Microscopy of Fractions Obtained by Centrifugation

Bovine posterior pituitary glands were homogenized in 10 per cent sucrose and fractionated by differential centrifugation. The following centrifugation procedure resulted in the most satisfactory separation: 1000 g for 15 minutes—nuclei, connective tissue, basement membranes with associated endothelium, giant nerve endings, and whole pituicytes; 4200 g for 15 minutes—free nerve endings, including Herring bodies; 17,000 g for 15 minutes—mitochondria; 68,000 g for 15 minutes—neurosecretory granules. Electron microscopic examination was carried out on whole tissue and on the isolated fractions. Isolated nerve endings were examined also by negative staining techniques. Isolated nerve endings retain an apparently normal complement of mitochondria, neurosecretory granules, and microvesicles ("synaptic" vesicles). The free nerve endings closely resemble those observed in sections of intact posterior pituitary tissue. Free microvesicles were not observed in any of the fractions isolated and apparently sediment at centrifugal forces higher than those employed in this study.     

大白鼠脑干听觉中枢的研究下丘中心核内神经突触的特点

1.大白鼠下丘中心核(the Central Nucleus of the Inferior Colliculus,ICCN)内神经末稍以群体的形式有在,神经突触排列的类型主要为系列突触.2.末稍群体(Clustered ending)中轴突终末内含有多种类型的突触小泡.3.ICCN内具有不对称突触与对称突触两种类型的突触结构.4.在ICCN内,突触前终末有大量的突触小泡聚集,并且在突触后常有1—2个大线粒体靠近突触后膜.5.以上结果表明了脑干听觉中枢下丘中心核的结构及其突触连结的模式;突触的结构及其特点,这是频有意义的.