Changes in the neuronal plasma membrane during synaptogenesis.

During synaptogenesis the plasma membrane of neurons undergoes considerable changes and large portions of it develop to synaptic membranes. This transformation is brought about by biochemical and morphological changes. The aim of the present investigation was to study by morphological methods the in vivo changes of some basic components of the neuronal membranes during the early postnatal period, when active synaptogenesis occurs. The cerebral cortex of Wistar rats was used for preparation of either growth cones (up to postnatal day 10) or synaptosomes (postnatal days 14-30). Our studies were focused on the changes occurring in integral membrane proteins and cholesterol domains, and in certain carbohydrate residues and anionic sites. In the first days after birth relatively few intramembranous particles are found in the plasma membranes of growth cones, few and small cholesterol domains, scarce lectin-binding and anionic sites. During the following days there is a clear tendency for increase of the number of all of the studied structures until the appearance of typical synaptic membranes. Throughout the studied developmental period the protein and the cholesterol molecules were found to occupy distinct membrane domains. The structure of the developing neuronal membrane, poor on proteins and cholesterol, and its maturation to the fully functional synaptic membrane is discussed.

Comparison of the Lowry and the Bradford protein assays as applied for protein estimation of membrane-containing fractions.

Although the Bradford protein estimation assay has found wide distribution, it has a number of drawbacks, which in some cases have been shown to produce erroneous results upon comparison to other, more precise, methods for protein estimation. It was found that the underestimation of the protein content of membrane-containing fractions cannot be overcome by pretreatment with NaOH or the detergents employed (Triton X-100, sodium dodecyl sulfate, 3[(3-cholamidopropyl)-dimethylammonio]propanesulfonic acid) and the protein estimates obtained do not agree with estimates obtained by the Lowry assay. Upon storage of fractions at -20 degrees C there is a considerable loss of dye binding activity, varying in accordance with the membrane content of the fractions, reaching up to 58% in the case of membrane-enriched fractions stored at -20 degrees C for 15 days. Pretreatment with the employed agents brought about an equal increase of dye binding capacity, specific for the individual fractions; however, none of these agents could recover the dye binding activity lost during several days of storage at -20 degrees C. It is suggested that the straightforward Bradford procedure has a rather limited scope of application, particularly concerning membrane-containing samples, and requires preliminary studies to determine its applicability according to the nature of the biological material examined.

Correlation between concentration of cholinesterases and the resistance of animals to organophosphorus compounds.

The molar concentrations of the catalytic sites of serum cholinesterase (ChE--EC 3.1.1.8.) and cholinesterases (ChEs-acetylcholinesterase (AChE)--EC 3.1.1.7. and ChE) from brain and perfused liver of male birds, rats, swine and sheep were determined. A positive correlation between the molar concentrations of the catalytic sites of ChEs and the resistance of the animals to some organophosphorus compounds (OPhCs) was found. In addition, the present study also showed that the difference of the molar concentrations of catalytic sites of ChEs in the brain, blood serum and liver can cause varied resistance to some OPhCs.

Developmental changes of proteins and concanavalin A reactive glycoproteins in growth cones from rat forebrain.

Growth cones were isolated from the forebrains of 1, 5 and 9 days-old rats. The ultrastructural characterization of the obtained subcellular fractions reveals that two of them (GC1 and GC2) contain predominantly growth cones. It was found that the protein content of the membranes contained in these fractions increases 7.5 times, while in whole forebrain the increase is only 3 times, showing that during the studied developmental period there is a predominant protein enrichment of the specialized brain structures (e.g. growth cones). Electrophoretic studies show that there are characteristic changes of the Coomassie Brilliant Blue R250 staining and concanavalin A reactive protein profiles. Comparison of the protein patterns of growth cones to those of synaptosomes from mature forebrain reveal a number of bands, which appear to be characteristic for one of these structures. The possible roles of the developmentally controlled proteins in the processes of synaptogenesis is discussed.

Changes of soluble and membrane proteins of rat brain during pre- and postnatal development.

Membrane and soluble protein fractions were obtained from forebrain, midbrain and hindbrain of embryos or neonatal rats. The amount of the protein of the corresponding brain parts was followed up as a function of DNA content. Age-related changes of the concentration of over 50 protein bands were observed in all three brain parts. There are also bands which tend to disappear (or appear) at distinct stages of development. In each of the brain parts there are bands showing age-dependent changes characteristic for this part. A common feature of the changes of the membrane protein patterns of forebrain and midbrain observed during development is a decrease of the concentration of proteins with lower molecular mass (below 40 kD), while proteins of higher molecular mass become better pronounced. Compared to forebrain and midbrain the hindbrain has a relatively conservative protein composition throughout development.

Mistletoe lectin I binding sites on the synaptosomes of the rat cerebral cortex.

By means of combined lectinological and immunological methods were demonstrated mistletoe lectin I binding sites on rat cerebral cortex synaptosomes. The mistletoe lectin I binds specifically D-galactose. Galactosyl residues were established on the junctional and nonjunctional synaptosomal membrane, on the synaptic vesicles, mitochondria and on myelin contamination. The relative number of mistletoe lectin I receptors per unit area of synaptosomal membrane was calculated.

Distribution of negative charges of rat brain synaptosomes established by means of protamine-ferritin conjugate.

The negative electric charges on the synaptosomes from rat cerebral cortex were studied by means of protamine-ferritin conjugate. The synaptic vesicles in some synaptosomes were heavily labelled with the positively charged conjugate. The synaptosomal membranes including presynaptic and postsynaptic membranes were also stained but in a lesser degree. It was established that the major dense line and the intraperiod line of myelin contaminants were labelled, too.

[Effect of motor regimes on water-soluble and membrane proteins and cholinesterase activity in subcellular fractions of rat brain tissue]. / Vliianie dvigatel'nykh rezhimov na nodorastvorimye i membrannye belki i kholinesteraznuiu aktivnost' subkletochnykh fraktsii tkani golovnogo mozga krys.

Albino rats were kept for a year under conditions of everyday motor loading or of a constant hypokinesia. An increase of the motor activity results in rise in the acetylcholinesterase activity determined in the synaptosomal and purified mitochondrial fractions while hypokinesia induces a pronounced decrease in this enzyme activity. The butyrylcholinesterase activity somewhat decreases in the synaptosomal fraction after hypokinesa but does not change under the motor loading regime. Motor loading causes an increase in the amount of synaptosomal water-soluble proteins possessing an intermediate electrophoretic mobility and seem to correspond to the brain-specific protein 14-3-2. In the synaptosomal fraction the amount of membrane proteins with a low electrophoretic mobility and with the cholinesterase activity rises. Hypokinesia, on the contrary, decreases the amount of these membrane proteins.

[Liberation of adenosine triphosphate after depolarization of the Torpedo electroplaque by potassium chloride]. / Libération d'adénosine triphosphate après dépolarisation de l'électroplaque de torpille par le chlorure de potassium.

The release of ATP after potassium depolarization was measured on fragments of electric tissue incubated in a solution containing the firefly extract. Light emission was proportional to the extracellular KCL concentration. In contrast to the release of ATP after single nerve impulses, the release after direct KCL depolarization was insensitive to curare of eserin.

Subcellular distribution of LDH isoenzymes in neuronal- and glial-enriched fractions.

The LDH isoenzymes of cell organelles (nuclei, mitochondria, microsomes, cytosol) obtained from neuronal- and glial-enriched fractions were determined by disc polyacrylamide electrophoresis and stained for enzyme activity. The quantitative estimation was made either by elution of isoenzymes or by densitometric measurements. The neuronal fraction was found to contain mainly H forms of LDH. The glial fraction also contained H forms but its M/H ratio was higher than that of the neuronal fraction. The cell organelles displayed some specificities with respect to the LDH isoenzymes distribution. The nuclei contained mainly M forms, the mitochondria H forms and the glial microsomal fraction M forms. It is suggested that the different LDH isoenzymes are related to the specific function of the different cell organelles.

[Differences in the enzymatic activity of mitochondria from enriched neuronal and glial fractions]. / Razlichiia v aktivnosti fermentov mitokhondrii iz obogashchennykh neironal'noi i glial'noifraktsii

Studies were performed in the activities of certain enzymes from oxidoreductase group: cytochrome c-oxidoreductase (EC 1.6.99.3), succinate dehydrogenase succinates: cytochrome c-oxidoreductase (EC 1.3.99.1), cytochrome oxidase (EC 1.9.3.1) and malate dehydrogenase (EC 1.1.1.37) in mitochondria from neuronal and glial-enriched fractions. The mitochondrial fraction purity was observed by the electron microscope. The enzyme activity of the glial mitochondrial fraction was much higher than that in the neuronal mitochondria. Malate dehydrogenase from glial enriched fraction consists of three isoenzymes, while neuronal mitochondria had two isoenzymes of malate dehydrogenase. The neuronal mitochondria were found to be more stable to lubrol and digitonin.

Strand breakage in rat brain DNA and its repair induced by ethylnitrosourea in vivo.

The damage and repair of rat brain DNA was studied in vivo after a single carcinogenic dose of ethylnitrosourea. Fragmentation of the brain DNA produced by this carcinogen was demonstrated on alkaline sucrose gradients. By the 24th hrs after treatment with ethylnitrosourea the single-strand damage to DNA was not completely repaired. As the highly differentiated cells of the central nervous system do not proliferate, it is possible that during brain carcinogenesis delayed repair of DNA of primitive cells might be needed for the formation of tumor anlage.