Georgina Rodriguez de Lores Arnaiz

ISOLATION OF SYNAPTIC VESICLES AND STRUCTURAL ORGANIZATION OF THE ACETYLCHOLINE SYSTEM WITHIN BRAIN NERVE ENDINGS

THE DISCOVERY of synaptic vesicles as the most significative component of nerve endings (DE ROBERTIS and BENNETT, 1954, 1955) led to the suggestion that they could be the site of storage and synthesis of transmitter substances. Since then this concept has become associated with the notion of a “quantized” release of acetylcholine at the neuromuscular junction (DEL CASTILLO and KATZ, 1956). At the same time several indirect indications of the role of these vesicles in synaptic transmission were demonstrated (DE ROBERTIS, 1959). The stimulation of some cholinergic terminals by DE ROBERTIS and VAZ FERREIRA (1957) and subsequent electronmicroscope observations suggested that within the endings a balance exists between the formation and release of synaptic vesicles, related to the frequency of stimulation. The isolation of such a subcellular component remained as the most direct approach to the elucidation of the chemical nature and physiological significance of synaptic vesicles. An approach to such a goal was made independently by DE ROBERTIS et al. (1960, 1961a) and GRAY and WHITTAKER (1960) with the demonstration that the so-called mitochondrial fraction of the brain contained a considerable number of isolated nerve endings in addition to free mitochondria and myelin. In our first paper we reported an attempt to isolate synaptic vesicles by disrupting the endings mechanically and subfractionating the microsomes in a continuous gradient. However the vesicles isolated in one of the four microsomal fractions were not as regular in size as the true synaptic ones. At that time we postulated that: “the further purification of intact synaptic terminals may provide a good starting point for the isolation of pure synaptic vesicles”. We also stated that : “when this is done, the synaptic vesicles will be probably found in one of the submicrosomal fractions”. More recently a technique was developed in our laboratory which allows the subfractionation of the mitochondrial fraction (Mit)? into five separated layers which in order of increased density are essentially composed of: A, myelin; B, fragments of endings and membranes; C , cholinergic nerve endings; D, non-cholinergic nerve endings; and E, free mitochondria (see methods and electronmicrographs in DE ROBERTIS et al., 1962a). The two subfractions C and D of nerve endings provided an excellent material for the isolation of synaptic vesicles. However it was observed

Isolation of different types of synaptic membranes from the brain cortex

Abstract A technique based in the osmotic shock of the crude mitochondrial fraction of the rat brain cortex, followed by differential and gradient centrifugations permits the isolation of synaptic membranes. These are characterized by electron microscopy, the uptake of C14-d-tubocurarine and the presence of membrane bound enzymes i.e. acetylcholinesterase, Na+K+ activated adenosintriphosphatase, K+ activated p-nitrophenylphosphatase, glutamine synthetase and monoaminoxidase. Two main types of synaptic membranes: one cholinergic and the other non-cholinergic may be differentiated on the bases of their different specific gravity, the C14-d-tubocurarine uptake and the distribution of the various enzymes.

Isolation of synaptic vesicles from nerve endings of the rat brain.

SINCE the discovery of ‘synaptic vesicles’ as a specific component of synapses1,2 it was suggested that they could be associated with the storage and possibly with the synthesis of transmitter substances. This concept was supported by degeneration experiments3 and specially by the stimulation of some cholinergic terminals4, which suggested that within the ending a balance exists between the formation and release of synaptic vesicles. However, the isolation of such a sub-cellular component remained as the most direct approach to the elucidation of the physiological significance and chemical nature of these vesicles.

5-HYDROXYTRYPTOPHAN DECARBOXYLASE ACTIVITY IN NERVE ENDINGS OF THE RAT BRAIN*

IN RECENT years the localization in nerve endings and synaptic vesicles, in addition to the physiological action, has become one of the main criteria for identifying a substance as a ‘transmitter’ in brain. In a study of the ACh-system in rat brain the three main components ACh, AChE (DE ROBERTIS, PELLEGRINO DE IRALDI, RODRIGUEZ DE LORES ARNAIZ and SALGANICOFF, 1962u), and ChAc (DE ROBERTIS, RODRIGUEZ DE LORES ARNAIZ, SALGANICOFF, PELLEGRINO DE IRALDI and ZIEHER, 1963), were found to be concentrated in a special population of nerve endings contained in a submitochondrial fraction. Under the electron microscope a synaptic complex was recognized which comprises the nerve ending with the two synaptic membranes attached by the intersynaptic filaments and the subsynaptic web (DE ROBERTIS et ul., 1961). The finer localization of the ACh-system within the synaptic complex showed that while both ACh and ChAc were mainly related to the synaptic vesicles, AChE appeared to be mostly concentrated in the synaptic membranes. Similar studies on two components of the 5-HT-system, M A 0 and 5-HT, showed that the inactivating enzyme is exclusively localized in mitochondria, following exactly the distribution of SDH (RODRIGUEZ DE LORES ARNAIZ and DE ROBERTIS, 1962), and 5-HT is concentrated in microsomes and in the same population of nerve endings containing the ACh-system (ZIEHER and DE ROBERTIS, 1963). Upon disintegration of the synaptic complex by osmotic shock (DE ROBERTIS et al., 19623) MA0 remained within the mitochondria and 5-HT was evenly distributed among the resulting fractions. A study of the localization of 5-HTP-D, the enzyme that forms 5-HT by decarboxylation of 5-HTP (UDENFRIEND, CLARK and TITUS, 1953), first seemed hopeless in view of the reports in the literature indicating that this is a completely soluble enzyme (CLARK, WEISSBACH and UDENFRIEND, 1954; BOGDANSKI, WEISSBACH and UDENFRIEND, 1957; UDENFRIEND, BOGDANSKI and WEISSBACH, 1957; PAGE, 1958; ROSENGREN, 1960). But in preliminary experiments on rat brain, in which we used the homogenization technique currently employed in our laboratory, we

5-Hydroxytryptophan-decarboxylase activity in normal and denervated pineal gland of rats☆

Abstract The 5-hydroxytryptophan decarboxylase was determined in normal and denervated pineal glands. In the experimental glands the determination was made at 2.5, 5, 10, 12 and 30 days after bilateral gangliectomy of the superior sympathetic ganglia. The results show an increase in enzyme activity in the denervated gland which may be as large as 150 per cent after 2.5 days and persists until 30 days. These results are discussed in relation with previous work (12) in vivo which showed that denervation produces a definite decrease in the synthesis of serotonin after injection of 5-hydroxytryptophan. The tentative hypothesis is postulated that the pineal nerves contain an inhibitory substance of the enzyme which disappears after denervation.

Levels of norepinephrine in rat cerebellum after administration of the convulsant 3-mercaptopropionic acid.

The level of norepinephrine in rat cerebellum was assayed after the administration of the convulsant 3-mercaptopropionic acid. Decrease in norepinephrine was observed after the onset of seizure. The results were compared with those of GABA which were diminished both during and after convulsions. The decrease of GABA was prior to that of norepinephrine.