Amperometric characterization of exocytotic events from single mast cells: dependence on external and internal Ca++ sources.

Mast cells exocytotically release histamine/serotonin in response to different secretagogues. We have used substance P and compound 48/80 to study the Ca++ dependency of serotonin exocytosis from peritoneal mast cells using carbon fiber amperometric techniques. The exocytotic release pattern consists of a burst of events superimposed on a slow, transient, amperometric current baseline increase. Cellular re lease parameters (number, frequency and total charge of amperometric events) and individual event characteristics (charge integral, half width and peak amplitude) were similar for the two secretagogues used. Zero Ca++ conditions greatly reduced, without completely abolishing,cellular release parameters. Cyclopiazonic acid, an inhibitor of the endoplasmatic Ca++ ATPase, reduced the cellular exocytotic capacity and diminished the amplitude of individual exocytotic events more effectively than the 0 Ca++ condition. The cyclopiazonic acid effects occurred in the presence of external Ca++, indicating that this condition is not sufficient for maintaining full exocytotic capacity. The results confirm the importance of intracellular Ca++ for exocytotic activation. For the first time evidence is presented that the integrity of intracellular Ca++ pools determines the amplitude and frequency of individual exocytotic events. Saponin, a non-specific detergent, also induced quantal release similar to that obtained with substance P and compound 48/80. This release was not dependent on extracellular Ca++, but cyclopiazonic acid significantly reduced individual exocytotic release.

Pharmacological modulation of endogenous dopamine and DOPAC outflow from nucleus accumbens.

The release of endogenous DA and DOPAC from nucleus accumbens slices were studied measuring net outflow of DA and DOPAC in the superfusate of static chambers, to analyze the correlation between DA and DOPAC outflows and identify which DA stores may serve as possible sources for DOPAC formation. Under resting conditions, or following stimulation with low (< 15 mM) KCl concentration, DOPAC outflow was greater than DA. When DA release was stimulated by higher (> 25 mM) KCl concentrations, DA outflow increased, proportionally more than DOPAC. In the virtual absence of Ca2+ in the Krebs solution DA outflow, induced by 25 mM KCl, was reduced to about 10%, while DOPAC outflow was only reduced to 45%. When the synthesis of DA was inhibited with alpha-MPT, DA and DOPAC outflow were unchanged during the first stimulation period. During a second stimulation period, however, their outflow were significantly reduced. Nomifensine, a DA uptake inhibitor, increased the basal DA outflow by about 100%, but only blocked DOPAC basal outflow by about 25%. The 25 mM KCl stimulated DA outflow was not affected by Nomifensine, while the stimulated DOPAC outflow was reduced by about 50%. These results demonstrate that there is a weak correlation between the outflows of DA and DOPAC, suggesting a complex relationship between the mobilization of the different DA pools and DOPAC outflow. The formation of DOPAC from some of these pools, appear to be dependent on the stimulation levels and on the pharmacological manipulation of the tissue.

Ca2+ dependency of serotonin and dopamine release from CNS slices of chronically isolated rats.

Much evidence points to a significant involvement of the classical neurotransmitters 5-HT and DA in affective disorders with possible changes in different structures of the CNS and also at different levels of the signal transduction chain, i.e., receptor, synthesis, uptake or release. We have used chronic isolated housing as an animal model of depression. These isolated rats enabled the study of KCl-induced release of 5-HT and DA from nucleus accumbens, prefrontal cortex and hippocampal slices. The following questions were addressed: first, if there is a change in the depolarization dependent release of DA and 5-HT from these CNS structures, and second, if the release is through the classical exocytotic mechanism. A significant increase in KCl stimulated release of 5-HT was observed in chronically isolated animals when compared to controls. 5-HT release was completely abolished from controls or isolated animals, when slices were incubated with Krebs containing zero Ca2+/10 mM Mg2+, the inorganic Ca2+ channel blockers, Cd2+ or Ni2+ and the calmodulin inhibitor, trifluoperazine. The organic Ca2+ channel blockers nifedipine and D-600 were less effective in inhibiting the stimulated 5-HT release. KCl stimulated DA release was only significantly increased from hippocampus slices, of isolated, but not control animals. This release was also highly Ca2+-dependent. The basal release of DA and 5-HT was similar in control and isolated animals and was not affected by the Ca2+ channel antagonists. The results suggest that extracellular Ca2+-dependent release of 5-HT and, to a lesser degree, of DA, is increased in this chronic animal model of depression in several CNS structures.

Release of [3H]dopamine from rat prefrontal cortex: modulation through presynaptic cholinergic heteroreceptors.

The release of tritiated dopamine ([3H]DA) from slices of the rat prefrontal cortex was studied using a superfusion technique. Release appeared to be voltage-dependent and also dependent on external Ca2+, suggesting the presence of a specific neurotransmitter release mechanism. gamma-Aminobutyric acid (GABA), aspartate, glutamate and serotonin had no effect on either basal DA release or K(+)-stimulated release. Carbachol, a cholinergic agonist, inhibited K(+)-stimulated DA release. The results demonstrate that cholinergic heteroreceptors on dopaminergic terminals of the prefrontal cortex modulate DA release.

Changes in basal and stimulated release of endogenous serotonin from different nuclei of rats subjected to two models of depression.

Basal serotonin (5-HT) release, measured in the presence of imipramine, was significantly reduced in slices of hippocampus, n. accumbens and frontal cortex from rats subjected to forced swim test and chronic isolation. Only in the hippocampus, in the two animal models of depression, K-stimulated release was increased. In the hippocampus and frontal cortex, but not in n. accumbens, tissue content of 5-HT was reduced. This constitutes the first direct demonstration of a decreased tonic release of serotonin from different nuclei during depression. The results also suggest a nuclei specific, facilitator, presynaptic regulation of 5-HT release.

Serotonin containing neurons in the retina of the teleost Eugerres plumieri.

The concentration and localization of serotonin was determined in the retina of the teleost Eugerres plumieri by using high performance liquid chromatography (HPLC) and immunohistochemical techniques. Serotonin and dopamine were measured simultaneously, their concentrations in the retina being 77 +/- 8 and 516 +/- 23 ng/mg tissue respectively. Treatment of the animals with pargyline significantly increased the levels of dopamine and serotonin. When retinas were treated with the neurotoxin 5,6-dihydroxytryptamine, the level of serotonin was reduced by more than 90% while the dopamine content only diminished by 20% when compared to controls. By using immunohistochemistry with a monoclonal anti-serotonin antibody it was possible to localize this amine in cell bodies of a population of amacrine cells with processes extending mainly into a thin layer of the most external lamina of the inner plexiform layer. Very few ramifications were seen projecting to the internal lamina of this layer. When visualized in flat mount preparations, dense arborization of fluorescent processes was observed. This is the first direct evidence that serotonin is apparently present in amacrine cells of the retina of E. plumieri with a distribution of the serotonergic terminals similar to goldfish but somewhat different when compared to other species.

Depolymerisation and rearrangement of actin filaments during exocytosis in rat peritoneal mast cells: involvement of ryanodine-sensitive calcium stores.

Cytoskeletal F-actin associated with synaptic vesicles and granules plays an important role during Ca(2+)-mediated exocytosis. In the present work, we have used amperometry and confocal fluorescence to study the role of internal Ca(2+) in the rearrangement of F-actin (visualised with phalloidin-Alexa 546) during exocytosis in rat mast cells. The F-actin-depolymerising drug, latrunculin A, and the ryanodine receptor agonists ryanodine and caffeine that, per se did not induce exocytosis, enhanced the exocytotic responses elicited by compound 48/80 (C48/80). They also induced cortical actin depolymerisation in the presence or absence of external Ca(2+). Degranulation induced by C48/80 was accompanied by the formation of a cytoplasmic F-actin network. Depletion of internal Ca(2+) with cyclopiazonic acid inhibited latrunculin potentiation of C48/80-stimulated exocytosis and completely blocked the formation of the cytoplasmic F-actin network. This indicates that the mobilisation of Ca(2+) from ryanodine-sensitive intracellular stores plays an important role in the depolymerisation of the cortical F-actin barrier and possibly in the formation of the internal F-actin network during exocytotic activation of peritoneal mast cells.

Release of gamma-[3H]aminobutyric acid from rat olfactory bulb and substantia nigra: differential modulation by glutamic acid.

We have studied the glutamate modulation of gamma-[3H]aminobutyric acid ([3H]GABA) release from GABAergic dendrites of the external plexiform layer of the olfactory bulb and from GABAergic axons of the substantia nigra. In the olfactory bulb, [3H]GABA release was induced by high K+ and kainate, and not by aspartate and glutamate alone. However, when the tissue was conditioned by a previous K+ depolarization, glutamate and aspartate caused [3H]GABA release. The effect of glutamate was significantly enhanced when the GABA uptake mechanism was blocked by nipecotic acid. N-Methyl-D-aspartate and quisqualate did not cause [3H]GABA release under the same conditions. The acidic amino acid receptor antagonist 2-amino-4-phosphonobutyric acid and the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid significantly inhibited the K+-glutamate- and the kainate-induced [3H]GABA release. Mg2+ (5 mM), which blocks the N-methyl-D-aspartate receptors, significantly inhibited the K+-glutamate-induced but not the kainic acid-induced [3H]GABA release. The K+-glutamate-stimulated release, but not the K+-stimulated [3H]GABA release, was strongly inhibited by Na+-free solutions or by 300 nM tetrodotoxin. Apparently the glutamate-induced release of [3H]GABA occurs through an interneuron because it is dependent on the presence of nerve conduction. In the substantia nigra no [3H]GABA release was elicited by any of the glutamate agonists tested. The present results clearly differentiate between the effects of glutamate on the release of [3H]GABA from the substantia nigra and from the olfactory bulb.(ABSTRACT TRUNCATED AT 250 WORDS)

Excitatory sulfur-containing amino acid-induced release of [3H]GABA from rat olfactory bulb.

The effect of L-cysteine sulfinic acid (CSA) and L-homocysteic acid (HCA) on the release of tritiated gamma-amino butyric acid ([3H]GABA), from the external plexiform layer (EPL) of the rat olfactory bulb, was compared with that of glutamate. These amino acids induced release of GABA was strongly inhibited by the glutamate uptake blocker, pyrrolidine-2,4-dicarboxylate (2,4,PDC) (50 microM), while it was not inhibited by the specific GABA uptake blockers nipecotic acid (0.5 mM) or NO-711 (5 microM). Only the HCA induced GABA release was 60% inhibited by beta-alanine (0.5 mM), a glial GABA uptake blocker and 78% by the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (AP-5) (100 microM). The non-NMDA receptor antagonists 6-cyano-2,3-dihydroxy-7-nitro-quinoxaline (CNQX) up to 500 microM had no effect on HCA or CSA stimulated GABA release. These results bring evidence for an excitatory role of HCA and CSA together with glutamate on GABAergic neuronal or glial elements, in the olfactory bulb. This role could be mediated through the reversal of the glutamate or/and the glial GABA transporter and through the activation of a NMDA type receptor.

Neuronal and glial release of [3H]GABA from the rat olfactory bulb.

GABA uptake and release mechanisms have been shown for neuronal as well as glial cells. To explore further neuronal versus glial components of the [3H]gamma-aminobutyric acid ([3H]GABA) release studies were performed with two different microdissected layers of the olfactory bulb of the rat: the olfactory nerve layer (ONL), consisting mainly of glial cells, and the external plexiform layer (EPL) with a high density of GABAergic dendritic terminals. In some experiments substantia nigra was used as a GABAergic axonal system and the trigeminal ganglia as a peripheral glial model. Spontaneous release of [3H]GABA was always lower in neuronal elements as compared with glial cells. A veratridine-evoked release was observed from the ONL but not from the trigeminal ganglia. Tetrodotoxin (TTX) abolished the veratridine-evoked release from the ONL, which also showed a partial inhibition when high magnesium concentrations were used in a Ca2+-free solution. beta-Alanine was strongly exchanged with [3H]GABA from the ONL of animals with the olfactory nerve lesioned and from animals with no lesion; but only a small heteroexchange was found from the external plexiform layer. The beta-alanine heteroexchange was able to deplete the releasable GABA store from the ONL of lesioned animals. In nonlesioned animals and the external plexiform layer, the veratridine-stimulated release of [3H]GABA was not significantly reduced after the beta-alanine heteroexchange. Stimulation of the [3H]GABA release by high concentrations of potassium elicited a higher release rate from axonal terminals than from dendrites or glia. Neurones and glia showed a similar inhibition of [3H]GABA release when a high magnesium concentration was added to a calcium-free solution. When D-600 was used as a calcium-flux blocker no inhibition of the release was observed in glial cells, whereas an almost complete blockage was found in both neuronal preparations (substantia nigra and EPL). These results provide further evidence for differential release mechanisms of GABA from CNS neurones and glial cells.

Glutamate receptor desensitization block potentiates the stimulated GABA release through external Ca2+-independent mechanisms from granule cells of olfactory bulb.

Glutamate stimulated release of [3H]GABA was studied, during receptor desensitization block and its modulation by voltage gated Ca2+ channels, internal Ca2+ mobilization and GABA transport inhibitors from olfactory bulb slices. Under control conditions, glutamate and agonists induced release was strongly inhibited by Mg/0 Ca2+ Krebs and Cd2+ and partially inhibited by Ni2+ and nifedipine. Cyclothiazide, which blocks desensitization of glutamate receptors, potentiated glutamate, kainate, AMPA and quisqualate induced release. This effect was less dependent of entry of external Ca2+, but was inhibited by trifluoperazine and thapsigargin, inhibitors of Ca2+-calmodulin and endoplasmatic Ca2+ ATPase respectively. Nipecotic acid and NO-711, inhibitors of the GABA transporter, were also able to reduce cyclothiazide potentiated release induced by the 4 secretagogues. Under control conditions, glutamate stimulates the release of GABA in cooperation with VDCC. However, during receptor desensitization block, glutamate stimulated GABA release is mainly modulated through mechanisms dependent on internal Ca2+ mobilization and reversal of the GABA transporter.

Study of the mechanism of release of [3H]GABA from a teleost retina in vitro.

gamma-Aminobutyric acid (GABA) is thought to be a neurotransmitter in the vetebrate retina. We studied the voltage and Ca2+ dependency of the process of release of [3H]GABA from the retina of the teleost Eugerres plumieri, using a microsuperfusion technique. Two depolarizing agents, veratridine and high potassium, produced a concentration-dependent release of [3H]GABA. The veratridine effect was inhibited in Na+-free solution, but was not affected by 1 microM tetrodotoxin. A substantial inhibition (about 75%) of the veratridine- and potassium-stimulated release of [3H]GABA occurred in Ca2+-free medium. Inhibitors of the Ca2+ channel, such as Mg2+ (20 mM), La3+ (0.1 mM), and methoxy-verapamil (4 microM-0.4 mM), inhibited the veratridine- and K+-stimulated release. However, Co2+ and Cd2+ caused a potentiation and no change of the K+- and veratridine-stimulated release, respectively. This release process is apparently specific, since both depolarizing agents were unable to release [3H]methionine, a nontransmitter amino acid, under the same experimental conditions. Autoradiographic studies with [3H]GABA, using the same incubation conditions as for the release experiments, showed a high density of silver grains over the horizontal cells with almost no accumulation by amacrine cells and Müller cells. beta-Alanine and nipecotic acid were used as two relative specific inhibitors of the glial and neuronal GABA uptake mechanisms, respectively. Only a small heteroexchange with [3H]GABA was found with beta-alanine, and no inhibition of the subsequent veratridine-stimulated release. On the other hand, nipecotic acid produced a strong heteroexchange with [3H]GABA and lacked the capacity to induce the veratridine-stimulated release of [3H]GABA. These results suggest a voltage- and Ca2+-dependent neuronal release of [3H]GABA from retina.

NMDA and non-NMDA receptor-mediated release of [3H]GABA from granule cell dendrites of rat olfactory bulb.

We have studied the effect of glutamate and the glutamatergic agonists N-methyl-D-aspartate (NMDA), kainate, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on [3H]GABA release from the external plexiform layer of the olfactory bulb. The GABA uptake blocker nipecotic acid significantly increased the basal [3H]GABA release and the release evoked by a high K+ concentration, glutamate, and kainate. The glutamate uptake blocker pyrrolidine-2,4-dicarboxylate (2,4-PDC) inhibited by 50% the glutamate-induced [3H]GABA release with no change in the basal GABA release. The glutamatergic agonists NMDA, kainate, and AMPA also induced a significant [3H]GABA release. The presence of glycine and the absence of Mg2+ have no potentiating effect on NMDA-stimulated release; however, when the tissue was previously depolarized with a high K+ concentration, a significant increase in the NMDA response was observed that was potentiated by glycine and inhibited by the NMDA receptor antagonist 2-amino-5-phosphonoheptanoic acid (AP-7). The kainate and AMPA effects were antagonized by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) but not by AP-7. The glutamate effect was also inhibited by CNQX but not by the NMDA antagonist 2-amino-5-phosphonopentanoic acid (AP-5); nevertheless, in the presence of glycine, [3H]GABA release evoked by glutamate was potentiated, and this response was significantly antagonized by AP-5. Tetrodotoxin inhibited glutamate- and kainate-stimulated [3H]GABA release but not the NMDA-stimulated release.(ABSTRACT TRUNCATED AT 250 WORDS)

Possible neurotransmitter role of noradrenaline in the teleost retina.

The role of dopamine as a neurotransmitter in the retina of different species has been clearly established; however, there is still some controversy as to whether noradrenaline (NA) is present as a neurotransmitter in this tissue. In this study, we show that, under controlled conditions, NA is present in the retina of goldfish at a concentration of 0.15 +/- 0.03 ng/mg protein and its biosynthetic enzyme, dopamine beta-hydroxylase shows an activity of 2.5 +/- 0.2 pmol NA/hr/mg protein. The amount of NA increases to 1.88 +/- 0.24 ng/mg protein in light adapted animals and decreases to undetectable levels in dark adapted ones. By contrast, dopamine-beta-hydroxylase levels are not affected by changes in light conditions. This finding provides further evidence in favor of a neurotransmitter role for NA in vertebrate retina.

Localisation of 3H-GABA in the rat olfactory bulb: an in vivo and in vitro autoradiographic study.

In an attempt to further clarify the localisation of GABAergic elements in the olfactory bulb we have performed, in vivo and in vitro, autoradiographic studies with 3H-GABA (gamma-amino butyric acid) and 3H-DABA (L-2,4 diamino butyric acid). The results have shown a strong labelling with 3H-GABA of the glial cells in all the layers of the olfactory bulb. A high concentration of grains was observed in the periglomerular region. The labelling in the external plexiform layer was uniformly distributed in the neuropile with the strongest activity at the level of the dendritic processes of the granule cells, leaving the mitral cell dendrites and cell bodies almost free of grains. 3H-DABA showed a very similar pattern to 3H-GABA. When olfactory bulb slices were preincubated with beta-alanine the labelling of the glial elements almost disappeared especially at the level of the olfactory nerve layer. The labelling pattern of the other layers of the bulb remained mostly unchanged. This supports the view that a population of periglomerular and granule cells are GABAergic and that beta-alanine competes with GABA uptake sites only in glial cells.

Dopamine and noradrenaline content in fish retina: modulation by serotonin.

The presence of noradrenaline (NA) and the possible interaction of serotonin (5-HT) with dopaminergic and noradrenergic neuronal elements was studied in the retina of the teleost Eugerres plumieri. By means of a histofluorescent technique, paired amacrine cells can be visualized after intravitreal injection of NA or 5,6-dihydroxytriptamine, suggesting their probable catecholaminergic and indoleaminergic nature. With a high-performance liquid chromatographic (HPLC) method and after pargyline treatment of the animal, 6.86 ng/mg protein of dopamine (DA) was detected, while NA content was 0.50 ng/mg protein. The NA levels of the retina increased, whereas the DA levels decreased in a significant manner after in vivo treatment with 5-HT. 5-methoxy-N,N-dimethyltryptamine, a 5-HT agonist, was able to mimic this effect partially, while the agonists tryptamine and quipazine did not affect the levels of DA and NA. The antagonists methysergide, metergoline, and cyproheptadine significantly blocked the 5-HT-induced NA increase, whereas only the first two antagonists were effective in inhibiting the 5-HT-induced DA decrease. The 5-HT modulation of NA and DA is apparently receptor mediated and is not due to a hetero-exchange, since imipramine was not able to block the 5-HT effect. These findings support the suggested interaction between serotoninergic and dopaminergic cells [Negishi et al: J Neurosci Res 5:621-635, 1980; Neurosci Lett 25:1-5, 1981]. Furthermore, they demonstrate the possible modulation which 5-HT exerts on the endogenous levels of NA.

Extrasynaptic vesicular transmitter release from the somata of substantia nigra neurons in rat midbrain slices.

Substantia nigra neurons release dopamine from their somatodendritic regions. A long-unresolved question is whether this release occurs by exocytosis or by a nonvesicular mechanism. We used carbon fiber microelectrodes in a brainstem slice to assay secretion from single cell bodies that had been cleared of connective tissue. Amperometry at the carbon fiber microelectrodes revealed unitary events in approximately 90% of cells in resting conditions. These events had charge integrals ranging from a few femtocoulombs to several hundred femtocoulombs (fC). Local glutamate application enhanced the event frequency by 3.5-fold on average and up to 10-fold in highly responsive cells, although the mean charge integral was not modified. Local application of a high K+-containing saline had effects similar to those of glutamate. The frequency of resting and stimulated amperometric events was much lower at 21-22 degreesC than at 32-35 degreesC. The addition of Cd2+ (50 microM), a blocker of voltage-dependent Ca2+ channels, to the bath solution blocked the stimulatory effects of glutamate. These results suggest that dopamine is released from the somata of substantia nigra neurons by exocytosis and that this mechanism is regulated by neuronal electrical activity. More generally, this study demonstrates the applicability of carbon fiber microelectrodes to the measurement of quantal monoamine secretion in brain slices.