Maurizio Raiteri

A simple apparatus for studying the release of neurotransmitters from synaptosomes

Abstract A simple superfusion technique for studying the release of neurotransmitters from preloaded synaptosomes is described. As compared with previously described procedures the method has the following advantages: (1) prevention of re-uptake; (2) immediate utilization of preloaded synaptosomes; (3) great flexibility and reliability; (4) continuous determination of release from parallel samples.

Serotonin release is modulated by presynaptic autoreceptors.

The existence of presynaptic autoreceptors controlling the release of 5-hydroxytryptamine (5HT) from serotonergic nerve endings was investigated utilizing superfused hypothalamic synaptosomes. Extracellular 5HT reduced the high K+-induced release of previously accumulated 3H-5HT. The central 5HT receptor blocker methiothepin counteracted the inhibitory effect of 5HT. Other 5HT antagonists (cyproheptadine, methysergide and mianserin) were inactive and may therefore act preferentially at the postsynaptic receptors.

Effect of sympathomimetic amines on the synaptosomal transport of noradrenaline, dopamine and 5-hydroxytryptamine.

The interaction of sympathomimetic amines with the transport of 3H-noradrenaline (3H-NE), 3H-dopamine (3H-DA) and 3H-5-hydroxytryptamine (3H-5-HT) were investigated in rat hypothalamic (3H-NE) and striatal (3H-DA) and 3 H-5-HT) synaptosomes. Modifications in the phenylethylamine structure led to changes in activity towards biogenic amine uptake and release: (a) the introduction of a beta-OH group led to compounds less active in inhibiting uptake and stimulating release of 3H-NE, 3H-DA and 3H-5-HT, with the exception of 3H-NE release which was stimulated more by unlabeled 1-NE than by DA; (b) the introduction of phenolic-OH groups always led to compounds which were stronger uptake inhibitors and releasers of the three biogenic amines; (c) the alpha-methylation increased the potency towards uptake inhibition and release stimulation, with the exception of 3H-NE release: in fact, the releasing activity of phenylethylamine was suppressed by alpha-methylation; (d) the introduction of a -Cl group in the para position selectively potentiated the effects on 3H-5-HT uptake and release and generally depressed those on catecholamine transport.

d-Amphetamine as a releaser or reuptake inhibitor of biogenic amines in synaptosomes.

The effect of d-amphetamine on the release of tritiated norepinephrine (NE), dopamine (DA) and 5-hydroxytryptamine (5-HT) was analyzed in synaptosomes from different brain area. 3H-NE release was unaffected in the hypothalamus, a region which is rich in noradrenergic terminals, and in cerebellum and pons-medulla, but was substantially increased in corpus striatum and moderately in cerebral cortex. 3H-DA release was strongly enhanced in corpus striatum, a region rich in dopaminergic terminals, substantially increased in cerebral cortex, and slightly increased in the hypothalamus. Since the regional pattern of d-amphetamine-stimulated release was similar with the two catecholamines, but the stimulation was greater with 3H-DA than with 3H-NE, and was more evident in areas richer in dopaminergic terminals, it is suggested that the drug can release 3H-DA or artificially stored 3H-NE from dopaminergic terminals, but not 3H-NE, from noradrenergic terminals. d-Amphetamine also seems capable of releasing 3H-5-HT from serotoninergic terminals. In contrast with the two catecholamines, 3H-5-HT release was more enhanced in cerebral cortex than in corpus striatum.

Detectability of high and low affinity uptake systems for GABA and glutamate in rat brain slices and synaptosomes

Abstract High and low affinity uptake systems for GABA and glutamate are detectable in crude mitochondrial and synaptosomal fractions from adult rat brains, in 0.4 mm thick slices from newborn brains and in 0.1 × 0.1 × 2 mm prisms from adult brains. The high affinity system is not detectable in 0.4 mm thick slices from adult brains, being probably masked by the predominant uptake due to the low affinity transport system. It is suggested that the bulk of the low affinity uptake is due to non-synaptosomal structures and is destroyed during the preparation of 0.1 × 0.1 × 2 mm prisms and of subcellular fractions. The effects of the geometry of the slices in the determination of the results obtained is discussed.

RELEASE AND EXCHANGE STUDIES RELATING TO THE SYNAPTOSOMAL UPTAKE OF GABA

Abstract— Synaptosomal release and exchange of [3H]GABA were studied by a superfusion technique which minimizes reuptake. The release of [3H]GABA was increased by depolarizing concentrations of KCl and showed calcium‐dependence. Superfusion with 1‐1000 μm unlabelled GABA caused a dose dependent, saturable increase in the release of radioactivity by homoexchange. The exchange process showed high substrate specificity: among the various amino acids and putative neurotransmitters tested, only γ‐amino‐β‐hydroxybutyric acid was a good stimulator of [3H]GABA release. Superfusion with sodium‐free medium (NaCl replaced by sucrose) virtually abolished homoexchange. Ouabain also increased the release of [3H]GABA, and its action was additive to that of unlabelled GABA.

A reevaluation of veratridine as a tool for studying the depolarization-induced release of neurotransmitters from nerve endings.

The effect of veratridine on neurotransmitter release was studied using rat brain synaptosomes superfused at 37°C. Veratridine (5–75 μ M) caused a concentration-dependent release of [3H]GABA from prelabeled synaptosomes in the presence of 2.7 mM Ca2+. In the whole range of veratridine concentrations, the release of [3H]GABA elicited by the drug was substantially increased rather than decreased in the absence of Ca2+ or with Ca2+ concentrations of 0.45 and 0.9 mM. The release of the amino acid was inhibited more by 5.4 mM than by 2.7 mM Ca2+. The effect on endogenous (chemically measured) GABA was similar to that on [3H]GABA. The inhibitory effect of Ca2+ on the veratridine-induced release of [3H]GABA was consistently seen in a variety of experimental conditions except one, namely when the experiment was run at room temperature (22–23°C) rather than at physiological temperature (37°C). In fact, at 22–23°C the release of GABA evoked by the alkaloid was somewhat potentiated by Ca2+. At 37°C, glutamate appeared to behave similarly to GABA, whereas the veratridine-induced release of [3H]noradrenaline and [3H]dopamaine was largely Ca2+-dependent. The mechanism of the release of transmitters elicited by veratridine is discussed. It is concluded that the evoked release of GABA and glutamate is due more to the veratridine-induced depolarization (Na+ influx) than to the accompanying influx of Ca2+, and it is suggested that the inhibitory effect of Ca2+ on the overall release of amino acids is due to the antagonism exerted by the divalent cation on the veratridine action at the Na+ channel. In contrast, in the case of catecholamines, the influx of Ca2+ would have a prominent role in triggering exocytotic release, whereas the depolarization itself would have slight or no importance.

Comparative study of the effects of mianserin, a tetracyclic antidepressant, and of imipramine on uptake and release of neurotransmitters in synaptosomes

The effects of mianserin, a tetracyclic antidepressant, on uptake and release of [3H]noradrena‐line (3H‐NA), [3H]dopamine (3H‐DA), [3H]‐5‐hydroxytryptamine(3H‐5‐HT) and [3H]γ‐aminobutyric acid (3H‐GABA) in synaptosomes from different areas of the rat brain were investigated in a comparative study with the tricyclic antidepressant imipramine. Mianserin and imipramine were inhibitors of 3H‐NA uptake in the hypothalamus, but could not increase 3H‐NA release from noradrenergic nerve endings. This behaviour was similar to that of (+)‐amphetamine. Both mianserin and imipramine had essentially no effect on 3H‐DA transport mechanisms in striatal synaptosomes. (+)‐Amphetamine, in contrast, strongly affected both 3H‐DA uptake and release. Imipramine was stronger than mianserin in inhibiting 3H‐5‐HT accumulation by striatal synaptosomes. In contrast, mianserin stimulated 3H‐5‐HT release whereas imipramine was ineffective. Mianserin had virtually no inhibitory activity on 3H‐5‐HT uptake by rat blood platelets. Imipramine was a modest inhibitor of 3H‐GABA accumulation by whole brain synaptosomes; mianserin had no effect. Both drugs did not alter 3H‐GABA release. These results indicate that mianserin interferes in a way different from that of tricyclic antidepressants with the neurotransmitter transport mechanisms at the presynaptic level.

Synaptosomal transport processes.

Publisher Summary This chapter provides an overview of synaptosomal transport processes. The synaptosome is a fundamental in vitro preparation for studying the dynamic biochemical aspect of neurotransmission and neurosecretion. It qualifies, in many respects, as a functioning presynaptic entity with an impressive range of metabolic and transmission based properties. Synaptosomes prepared from whole brain or from grossly dissected brain areas are heterogeneous; the purest synaptosomal preparation contains populations of nerve endings synthesizing and utilizing different neurotransmitters. In spite of this heterogeneity, synaptosomes are often preferred to other nervous tissues preparations in studies concerned with properties believed to be specific to presynaptic nerve terminals. In addition, studies on synaptosomal transport processes are performed under a variety of experimental conditions whose importance in the determination of the results obtained has rarely been evaluated. This chapter illustrates some examples in this context and reviews the aspects of neurotransmitter transport at nerve endings.

d-Amphetamine and the release of 3H-norepinephrine from synaptosomes

Abstract The release of 3H-norepinephrine from rat brain synaptosomes was studied by a superfusion technique which prevents re-uptake of the released amine. d-Amphetamine had a minimal stimulatory effect on 3H-norepinephrine release and was a potent uptake inhibitor. It is suggested that d-amphetamine may act primarily by inhibiting norepinephrine re-uptake at adrenergic synapses.