G. Vincendon

High affinity binding site for γ-hydroxybutyric acid in rat brain

Abstract The existence of a specific synthesizing enzyme for γ-hydroxybutyric acid in rat brain has recently been reported. Here, for the first time, we demonstrate the presence of a high affinity, apparently specific binding site for this compound in the same tissue. This binding does not require Na + and takes place optimally at pH 5.5. The bound γ-hydroxybutyric acid is not displacable by GABA or baclofen. We report here on some structurally related compounds of GHB with a similar or better binding capacity than GHB itself. The number of binding sites increases with age up to adulthood and differs depending on the brain region. In primary tissue cultures of pure chicken neurones and glia, γ-hydroxybutyric acid binding occurs exclusively-- in the neuronal preparations.

Adult rat brain synaptic vesicles II. Lipid composition

Abstract The lipid composition of synaptic vesicles isolated from adult rat brain was determined. Vesicles contained cholesterol and phospholipid but very little ganglioside, galactolipid, free fatty acid and triglyceride was detected. Ethanolamine and choline phosphoglycerides were the dominant phospholipids. Lysophosphatidyl choline was present in very low amounts. The fatty acid composition of the phosphoglycerides was characterized by high levels of docosahexaenoic acid in the ethanolamine and serine phosphoglycerides, and the absence of long chain fatty acids from the sphingomyelins. All the characteristic features of the lipid composition of the synaptosomal plasma membrane (with the exception of the ganglioside content) were seen in the synaptic vesicle lipids. The results are discussed in terms of the exocytosis mechanism of transmitter release.

Immunocytochemical evidence for the existence of GABAergic neurons in the nucleus raphe dorsalis. possible existence of neurons containing serotonin and GABA

It has been established that nerve cell bodies of the nucleus raphe dorsalis (NRD) belong to ascending 5-hydroxytryptamine systems. These neurons could be modulated by GABAergic interneurons or interposed GABA neurons. A high glutamate decarboxylase (GAD) activity in the NRD and a specific high-affinity uptake mechanism for GABA suggest the presence of GABA synthesizing elements in the NRD. Anti-GAD antibodies were used by an immunocytochemical procedure to demonstrate the presence of GABAergic elements. Anti-GAD antibodies were previously tested in the cerebellum and substantia nigra. Large amounts of GAD-positive reaction product were observed in the cytoplasm of some neurons (fusiform, ovoid or multipolar) or appeared as punctate deposits apposed to dendrites, soma and dispersed in the neuropil of the NRD. At the electron microscopic level, GAD-positive reaction product was observed within the cytoplasm of numerous somata in sections from colchicine-treated rats. GAD-positive staining was observed in numerous fibers or axonal terminals and two types of morphologically different fibers could be distinguished. The first displays small clear vesicles and few large granular vesicles (LGV) (80-100 nm), the second displays only clear round vesicles (40-60 nm). After 5,7-dihydroxytryptamine treatment (a neurotoxic for 5-HT terminals), the immunocytochemical labeling is much decreased. Some reactive neurons are still dispersed in the nucleus but the fibers containing LGV are no longer observed. These results strongly suggest that some neuronal elements in the NRD are morphologically, pharmacologically and anatomically similar to 5-HT neurons described at this level. Such cell elements could possess a double GABA and 5-HT potentiality. If this is not the case, a population of GABA neurons could be sensitive to 5,7-DHT and so have the capacity to take up 5-HT. The other reactive elements, insensitive to 5,7-DHT, could represent the GABAergic interneurons postulated at this level. Numerous GAD positive fibers or axon terminals were observed in synaptic contact with dendrites, axons or soma of other neurons. The chemical nature of the neuronal postsynaptic elements remains unknown. These findings strongly support the hypothesis for GABA-mediated inhibition in the NRD.

Postnatal development of rat cerebellum: Massive and transient accumulation of concanavalin a binding glycoproteins in parallel fiber axolemma

Modifications of protein-bound sugars during postnatal development of rat cerebellum were studied. Glycoprotein-bound mannose accumulates, in the particulate fractions, at an earlier age than the bulk of glycoprotein sugar. This corresponds to a transient and massive accumulation of glycoproteins which bind to Concanavalin A (Con A). These glycoproteins were localized by using fluorescent Con A and the horseradish peroxidase-Con A method. Cerebellar white matter and the molecular layer bind massive amounts of Con A. The binding in the molecular layer is transient. It follows the same time course as the Con A-binding glycoproteins of particulate fractions, and it is largely confined to the axolemma of parallel fibers. Only growing or newly formed parallel fibers bind Con A. The disappearance of the binding is simultaneous with the maturation of parallel fibers and their synapse formation. These phenomena can be related to fiber growth and maturation and, also, to synapse formation. The possibility of a specific role of Con A-binding glycoproteins is discussed.

Specific and Artefactual Cellular Localizations of S100 Protein: An Astrocyte Marker in Rat Cerebellum

Immunohistological techniques with specific antisera have been used to reinvestigate the problem of cellular localization of the neurospecific protein S100. In adult rat cerebellum, this protein is exclusively localized in astrocytes as shown by revealing in the same tissue secretion S100 protein and an oligodendrocyte marker, the isoenzyme II of carbonic anhydrase. The exclusive astroglial localization of S100 protein was confirmed by immuno-electron microscopy. A number of technical artefacts which may be at the origin of the claimed localization of S100 protein in neuronal cytoplasm and nuclei were also determined and described.

Evidence for cysteine sulfinate as a neurotransmitter

The Na+-independent binding of L-[3H]cysteine sulfinate and L-[3H]cysteine sulfinate uptake were investigated in rat brain membranes and vesicles. Specific binding of L-[3H]cysteine sulfinate was saturable and occurred by a single high affinity process with a Kb of 100 nM +/- 9 and a capacity (Bmax) of 2.4 +/- 0.22 pmol/mg protein. Sodium ions were found to have a biphasic effect; low concentrations (in the range of 0.1-3 mM) induced a marked inhibition of the binding whereas higher concentrations (10-300 mM) resulted in a dose-dependent stimulation of binding. The inhibition potency, expressed as the Ki values of a wide range of compounds with known pharmacological activities was tested. L-Cysteine sulfinate was the most potent inhibitor being 3-fold more potent than L-glutamate and 80 times more potent than L-aspartate. The regional distribution of the binding of L-[3H]cysteine sulfinate in the brain was found to be heterogeneous. These results provide the first evidence for an interaction of cysteine sulfinate with specific receptor sites on the synaptic membrane. The rate of L-[3H]cysteine sulfinate uptake shows a biphasic dependence on the concentration of L-cysteine sulfinate, corresponding to a high affinity (27.2 microM) and a low affinity (398 microM) transport system. The maximum L-[3H]cysteine sulfinate uptake is reached at 2 min. The reversibility of this transport was demonstrated. The L-[3H]cysteine sulfinate uptake increases as a function of the sodium concentration. Chloride and potassium ions stimulate the uptake. The decrease or increase in the electrical membrane potential (delta psi) caused by replacing the chloride ions by the sulfate or sulfocyanate ions respectively leads to a decrease or increase in the rate of uptake. Increase in the extravesicular osmolarity leads to a decrease in the extent of L-[3H]cysteine sulfinate accumulation. Amino acids with an acidic group in position omega were found to be potent inhibitors (the most potent being L-aspartate). The length of the carbon chain also has a bearing on the inhibitory effect. The regional distribution of L-[3H]cysteine sulfinate uptake in the brain was heterogeneous. These results demonstrate the existence of a high affinity system which may correspond to the transmitter inactivation. Binding and uptake sites are distinguishable as evidenced by the affinity constants, the ionic and pharmacological effects and the different regional distributions in the brain. Finally, these results give further evidence for a neurotransmitter role of L-cysteine sulfinate.

PURIFICATION AND SOME PROPERTIES OF S-100 PROTEIN FRACTIONS FROM SHEEP AND PIG BRAINS

Abstract— The S‐100 protein fraction of pig and sheep brain was purified in 40 per cent yield by a modification of the procedure of Moore (1965), which avoided selective loss of S‐100 components. The S‐100 fraction of both pig and sheep is a mixture of proteins as indicated by acrylamide gel electrophoresis and N‐terminal amino acid analysis. Differences in amino acid composition, electrophoretic heterogenity and N‐terminal analysis were found.

A Biochemical and Immunohistological Study of S100 Protein in Developing Rat Cerebellum

Specific immune sera, one directed against S100 protein and one against glial fibrillary acidic protein (both proteins are specific astrocytic markers in rat cerebellum), were used in immunohistological methods to follow the formation and maturation of astrocytes during the postnatal development of rat cerebellum. Throughout development, S100 protein is detected only in astrocytes. Immunofluorescent Golgi epithelial cells and other astrocytes are detected in the cerebellum of newborn rat, their number increases rapidly between birth and the 10th postnatal day and then more slowly until the end of the 3rd week. In contrast, the increase in S100 protein level, measured in cerebellar homogenates by radioimmunoassay, was small until the 10th postnatal day. The greater accumulation subsequently found corresponds to an increase in the content of each astrocyte during the process of maturation.

Purification and properties of the membrane-bound acetylcholinesterase from adult rat brain

The membrane-bound acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) from adult rat brain has been purified to homogeneity using sequential affinity chromatography on Con A-Sepharose and on dimethyl-aminoethylbenzoic acid-Sepharose 4B followed by DEAE-cellulose chromatography. The yield of the purified enzyme (specific activity: 3068 U/mg protein) is higher than 50%. Polyacrylamide gel electrophoresis in the presence of Triton X-100 gives only one band with acetylcholinesterase activity. With the exception of electrofocusing and pore gradient electrophoresis, where a multiple band pattern was detected (which seems to be artefactual), the enzyme appears to be homogeneous. Gel filtration and sucrose density gradient centrifugation in the presence of Triton X-100 give only one symmetrical peak, with a calculated molecular weight of 328 000. Since polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) and mercaptoethanol gives only one band with a molecular weight of 74 500, a tetrameric structure can be postulated for the membrane-bound acetylcholinesterase from rat brain.

UP-REGULATION OF DOPAMINE D2 RECEPTOR MRNA IN RAT STRIATUM BY CHRONIC NEUROLEPTIC TREATMENT

The effect of prolonged administration of antagonists on rat striatal dopamine D2 receptor binding and mRNA content was examined. Both haloperidol (2 and 4 mg/kg) and sulpiride (10 mg/kg) induced a significant rise in total D2 and D2(444) mRNA level and in Bmax. Regulation of messenger RNA accumulation is thus an important determinant of dopamine D2 receptor density.