Role of External and Internal Calcium on Heterocarrier-Mediated Transmitter Release

Abstract: Release-regulating heterocarriers exist on brain nerve endings. We have investigated in this study the mechanisms involved in the neurotransmitter release evoked by GABA heterocarrier activation. GABA increased the basal release of [³H]acetylcholine and [³H]noradrenaline from rat hippocampal synaptosomes and of [³H]dopamine from striatal synaptosomes. These GABA effects, insensitive to GABA receptor antagonists, were prevented by inhibiting GABA uptake but not by blocking noradrenaline, choline, or dopamine transport. Lack of extracellular Ca²⁺ or addition of tetrodotoxin selectively abolished the GABA-evoked release of [³H]noradrenaline, leaving unaffected that of [³H]acetylcholine or [³H]dopamine. 1,2-Bis(2-aminophenoxy)-ethane-N,N,N′,N′-tetraacetic acid acetoxymethyl ester (BAPTA-AM) or vesamicol attenuated the release of [³H]acetylcholine elicited by GABA. Reserpine, but not BAPTA-AM, prevented the effect of GABA on [³H]dopamine release. Autoreceptor activation inhibited the GABA-evoked release of [³H]noradrenaline but not that of [³H]acetylcholine or [³H]dopamine. It is concluded that (a) the release of [³H]noradrenaline consequent to activation of GABA heterocarriers sited on noradrenergic terminals meets the criteria of a conventional exocytotic process, (b) the extracellular [Ca²⁺]-independent releases of [³H]acetylcholine and [³H]dopamine appear to occur from vesicles possibly through involvement of intraterminal Ca²⁺, and (c) autoreceptor activation only affects heterocarrier-mediated vesicular release linked to entry of extracellular Ca²⁺.     

Effects of hypophysectomy, adrenalectomy and corticosterone treatment on uptake and release of putative central neurotransmitters by rat hypothalamic tissue in vitro.

Uptake and release of radiolabeled serotonin, noradrenaline, dopamine, acetylcholine and GABA by rat hypothalamic tissue $\text{in virto}$ were examined following various treatments, which cause drastic changes in the tissue levels of corticosterone. Hypophysectomy affected both uptake and release of most of the neurotransmitters studied. However, adrenalectomy had a more selective effect, changing these processes for serotonin only. The uptake of radiolabeled serotonin by synaptosomes was decreased by about 30%, while its release from tissue slices upon depolarization with 40 mM K⁺ was increased 25%. Both of these changes could be prevented by injecting adrenalectomized rats with corticosterone.It is suggested that corticosteroid hormones might play a modulatory role in maintaining a certain functional activity level in central serotonergic neurons.     

Presynaptic Distribution of the Cholinergic-Specific Antigen Chol-1 and 5''-Nucleotidase in Rat Brain, as Determined by Complement-Mediated Release of Neurotransmitters

Nerve terminals prepared from rat cortex and hippocampus were loaded with seven radioactive putative neurotransmitters (serotonin, noradrenaline, dopamine, gamma-aminobutyric acid, aspartate, glutamate, and taurine). The release of these transmitters, choline acetyltransferase, 3,4-dihydroxyphenylalanine decarboxylase, enolase, and lactate dehydrogenase was monitored during complement-mediated lysis. Three antisera were used: anti-5'-nucleotidase, anti-Chol-1, and anti-rat cerebrum. Anti-5'-nucleotidase serum did not cause the release of any labelled transmitter or of any of the enzymes studied. Anti-Chol-1 serum released choline acetyltransferase and small amounts of enolase and lactate dehydrogenase. Anti-rat cerebrum caused the release of all seven transmitters, choline acetyltransferase, and small amounts of the other three enzymes. It was concluded that 5'-nucleotidase was not present on any of the terminals studied, and that Chol-1 is only present on cholinergic terminals.     

EFFECTS OF AMINO-OXYACETIC ACID, ETHANOLAMINE-O-SULPHATE AND GABA ON THE CONTENTS OF GABA AND VARIOUS AMINES IN BRAIN SLICES

—The effects of amino-oxyacetic acid, ethanolamine-O-sulphate and γ-aminobutyric acid (GABA) on the contents of GABA, noradrenaline, dopamine and serotonin (5-HT) in slices of rat hypothalamus and midbrain were studied in vitro using a simultaneous fluorimetric assay procedure. Following control incubations the levels of 5-HT were raised, while the levels of the other substances remained steady. Amino-oxyacetic acid caused a reduction in the contents of noradrenaline and 5-HT, but had no effect on either GABA or dopamine. Ethanolamine-O-sulphate both raised the GABA content and lowered the noradrenaline content of slices, while the levels of dopamine and 5-HT were not altered. The presence of GABA in the incubation medium produced complex changes in these levels, depending both on the dose of GABA used and the brain area studied. In the hypothalamus, 0·07 mm -GABA caused an elevation in 5-HT, a drop in noradrenaline, and no change in either GABA or dopamine. With 5 mm -GABA, the noradrenaline level was raised slightly above control values and the endogenous GABA level doubled, while 5-HT and dopamine levels were not different from controls. Similar changes in 5-HT and GABA contents were observed with midbrain slices, but noradrenaline and dopamine were not affected. The possible modes of action of amino-oxyacetic acid and ethanolamine-O-sulphate on the amino acid and amine systems in the brain are discussed.     

In Vivo Electrochemical Measurement of the Long-Lasting Release of Dopamine and Serotonin Induced by Intrastriatal Kainic Acid

Abstract: Intrastriatal injection of the glutamate agonist kainic acid (KA) in rats has been used to produce an animal model to investigate the mechanism of acetylcholine and GABA cell death associated with Huntington's disease. In the present study, the time course of low (10⁻⁵ M ) and high (5 × 10⁻³ M ) concentrations of KA on striatal dopamine and serotonin release was studied in freely moving rats by using in vivo voltammetry. The response to low concentrations of KA varied between animals, either increasing dopamine release during the injection or increasing dopamine and serotonin after the injection for an extended time, suggesting that 10⁻⁵ KA is near the threshold for KA toxicity in the striatum in rats. High concentrations of KA suppressed dopamine release during injection, with both dopamine and serotonin release increasing and remaining elevated for 1–4 and 7–21 days, respectively. KA-induced changes were inhibited by 6-cyano-7-nitroquinoxaline-2,3-dione and bicuculline increased the release of dopamine but not serotonin. These findings suggest that KA-induced changes in dopamine release resulted from a disinhibition of dopamine neurons due to KA-mediated toxicity of striatal GABA neurons. An alternate possibility is that the change in dopamine and serotonin release may have arisen from a functional modification or degeneration of presynaptic terminals.     

Pharmacological heterogeneity of release-regulating presynaptic AMPA/kainate receptors in the rat brain: study with receptor antagonists

Presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptors mediating hippocampal [(3)H]noradrenaline or [(3)H]serotonin release, striatal [(3)H]dopamine release and cortical [(3)H]acetylcholine release were pharmacologically characterized using several AMPA/kainate receptor antagonists. The releases of the four transmitters elicited by exposing synaptosomes to AMPA were antagonized by NBQX, indicating that they reflect AMPA/kainate receptor activation. GYKI52466 did not inhibit the AMPA-induced release of [(3)H]noradrenaline, [(3)H]dopamine or [(3)H]serotonin, while it weakly affected the AMPA-mediated release of [(3)H]acetylcholine. On the contrary, LY300164 and LY303070 were potent antagonists able to discriminate among AMPA/kainate receptor subtypes. Both compounds blocked the AMPA receptors mediating [(3)H]dopamine and [(3)H]acetylcholine release. However, LY303070, but not LY300164, inhibited the AMPA-induced release of [(3)H]noradrenaline, while the AMPA-mediated [(3)H]serotonin release was sensitive to LY300164 but not to LY303070. SYM2206 mimicked LY300164 and prevented the AMPA-induced release of [(3)H]dopamine, [(3)H]acetylcholine and [(3)H]serotonin, but not that of [(3)H]noradrenaline. NS102 failed to antagonize the AMPA-induced release of all four transmitters. LY293558 prevented the AMPA-mediated release of [(3)H]noradrenaline, [(3)H]dopamine, [(3)H]acetylcholine or [(3)H]serotonin. Differently, LY377770 did not inhibit the AMPA-mediated release of [(3)H]noradrenaline and [(3)H]acetylcholine, but it potently blocked the AMPA-induced release of [(3)H]serotonin and, less so, of [(3)H]dopamine. AMOA inhibited the AMPA-induced release of [(3)H]serotonin or [(3)H]acetylcholine, but not that of [(3)H]noradrenaline or [(3)H]dopamine. GAMS prevented the AMPA-mediated release of [(3)H]acetylcholine and, more weakly, that of [(3)H]dopamine, but it failed to inhibit the release of [(3)H]noradrenaline or [(3)H]serotonin elicited by AMPA. gamma-DGG did not affect the AMPA-mediated release of any of the four transmitters studied. In conclusion, based on the antagonist profiles obtained, the four receptors here analyzed all belong to the AMPA-preferring subclass of glutamate receptors; however, they appear to differ from each other, probably due to differences in subunit composition. The compounds LY300164, LY303070, LY377770, AMOA and GAMS may be useful to discriminate among AMPA-preferring receptor subtypes.     

Specific lysis of GABAergic synaptosomes by an antiserum to glutamate decarboxylase

An antiserum to pure glutamate decarboxylase (GAD) when incubated with rat cortical synaptosomes in the presence of complement caused release of 33-53% of lactate dehydrogenase (LDH) and 22-41% of total GAD. In addition most of the gamma-aminobutyrate (GABA) present was released. Anti-GAD antiserum alone, or complement alone, were without action. The antiserum plus complement had no effect on noradrenaline or choline uptake, and did not release choline acetylase (ChAT). Anti-ChAT serum plus complement released 30-37% of ChAT and 10-13% of LDH. It prevented choline uptake. This serum did not produce GAD release or prevent GABA, choline or noradrenaline uptake. When cortical synaptosomes were exposed to both antisera plus complement, their actions were strictly additive. The data indicate specific lysis of GABAergic and cholinergic synaptosomal sub-populations.     

The activity of the monoaminergic systems of the rat hypothalamus in acute stress after chronic stressing]

The influence of chronic stress (footshock combined with randomized light flashes) on acute stress-induced (immobilization) release of noradrenaline, dopamine and serotonin in rat lateral hypothalamus was assessed by microdialysis. The chronic stress resulted in an increase and prolongation of the acute stress-induced release of noradrenaline but not of dopamine and serotonin. The increased rate of accumulation of dioxyphenylacetic acid and unchanged accumulation of homovanillic acid (dopamine metabolites) and dopamine during and after the acute stress in chronically stressed animals reflect a rise of synthetic activity of catecholaminergic systems in response to acute stress and reuptake increase. Marked stress-induced increase in hydroxyindoleacetic acid in chronically stressed rats without any changes in the ST dynamics may be regarded in a similar way. A significant increase in potassium-stimulated release of all the studied monoamines was found while their basal level remained unchanged. The conclusions was made that the hyperergic release of neurotransmitters may be the basis of an inadequate response of animals to acute stress, i.e., one of the neurotic symptoms.     

A fraction enriched in dendrodendritic synaptosomes isolated from rat olfactory bulb: Morphology and transmitter release

A fraction enriched in dendro-dendritic synaptosomes was isolated from rat olfactory bulb by a rapid method. Synaptosomes preserved their ultrastructure and showed configurational changes in relation to incubation in physiological ion medium as described earlier in the case of cortical synaptosomes. Dendro-dendritic synaptosomes were larger and contained more mitochondria than cortical synaptosomes. Doublets of terminals synapsing with each other were frequently seen and each terminal contained synaptic vesicles. Oxygen consumption of dendro-dendritic synaptosomes was decreased by ouabain and increased by 2,4-dinitrophenol. High-potassium medium evoked a considerable release of GABA and dopamine but not of noradrenaline or serotonin in accordance with histochemical published data.     

Effects ofp-chlorophenylalanine on cortical monoamines and on the activity of noradrenergic neurons

The catecholamines noradrenaline, dopamine, adrenaline, the indoleamine 5-hydroxy-tryptamine (5-HT; serotonin), and some of their major metabolites were assayed, using high performance liquid chromatography (HPLC), in the neocortex of normal rats as well as in animals in which 5-HT synthesis had been inhibited withp-chlorophenylalanine. Besides important depletions in serotonin and in 5-hydroxyindole-3-acetic acid, noradrenaline levels were significantly reduced, but the content in 3-methoxy-4-hydroxyphenylglycol was increased, indicating an augmented utilization of this amine. The levels of dopamine and 3-methoxytyramine were also reduced, although homovanillic acid and 3,4-dihydroxyphenylacetic acid levels remained constant. The spontaneous unitary activity of identified noradrenergic neurons in the Locus coeruleus was increased, indicating an hyperactivity of this system. These results can be interpreted in relation to functional interactions between the catecholamines and serotonin; i.e.: a decrease in endogenous serotonin results in the loss of a negative feedback control of noradrenaline release.Special Issue dedicated to Prof. Eduardo De Robertis.     

EFFECT OF γ-AMINOBUTYRIC ACID ON BRAIN SEROTONIN AND CATECHOLAMINES

—Intraperitoneal injections of GABA (5 mg/kg) to rats lowered the level of norepinephrine in brain, heart and spleen but not suprarenals and raised that of serotonin in brain. Changes of these monoamines were most pronounced in the hypothalamic region after 20min. A reduction of hypothalamic norepinephrine was also observed 15min following the intracarotid administration of 0·5 mg/kg of GABA. In these experiments there was a concomitant increase in the level of free GABA in the anterior portion of the ventral hypothalamus. Brain dopamine level and 5-hydroxytryptophan decarboxylase, dihydroxyphenylalanine decarboxylase and monoamine oxidase activities were not affected. The 20 per cent increase of endogenous GABA observed in the midbrain 30 min following the administration of amino-oxyacetic acid was accompanied by a sharp fall in norepinephrine level (39 per cent) and an increase in serotonin (20 per cent). In in vitro studies 10–300 μg/ml of GABA were shown to release norepinephrine from cortical and hypothalamic slices, and to inhibit serotonin release without affecting 5-hydroxytryptophan uptake and to have no effect on the release of dopamine from slices of the region of the corpus striatum nor on the activity of the enzymes mentioned. Subcellular studies showed that the particulate:supernatant ratio for norepinephrine was reduced from a control value of 2·04 to 1·75 and that of serotonin was raised from 2·8 to 3·5. Following pretreatment with iproniazid, GABA reduced the raised level of brain norepinephrine to a greater extent than reserpine but not as intensively as amphetamine. The results obtained suggest that these monoamines may be involved in the mechanisms underlying the action of GABA in brain and that the effect of GABA on brain monoamines may be of certain significance in synaptic events.     

Changes in the synaptosomal membranes in the chronic neurotization of rats

A study was made of the changes in synaptosomal membranes and in some synaptic processes under the development of experimental neurosis in rats. Neurotic rats demonstrated changes in the protein/lipid correlation and in the interaction of the fluorescent ANS probe and synaptosomal membranes. This can be accounted for by an increase in the membrane water repellency. The activity of Na, K-ATPase remains unchanged. The rate of noradrenaline, serotonin, dopamine and GABA synaptosomal reverse uptake in neurotic rats was found to be increased.     

Neurotransmitter systems of some brain regions of rats subjected to chronic morphine intoxication

The content of neurotransmitters and their metabolites was investigated in brain cortex hemispheres, thalamus and brainstem of rats subjected to chronic morphine intoxication (7–21 days). Morphine administration for 7–14 days was accompanied by changes of the catecholamine system functioning, which was the most pronounced in the thalamus and the brainstem. These changes included increased secretion of dopamine and noradrenaline, their decrease in the brain tissue, and an increased content of their metabolites. The changes in serotonin and GABA content were less pronounced and included a decrease of serotonin level and the increase of the GABA content in different periods of opiate administration.     

Neuromediator mechanisms of the effect of the antihistamine agent dimebone on the brain

Dimebone was shown to inhibit monoamine oxidase (MAO) deaminating dopamine and serotonin, decrease dopamine metabolism in the basal ganglia of the rat brain, increase noradrenaline level and depress dopamine deamination in the hypothalamus. Dimebone first increased and then diminished the release of dopamine in the cortex, with the concomitant MAO activation and the increase in dopamine and noradrenaline levels. The in vitro experiments have demonstrated that dimebone (10(-4)) preferentially inhibited MAO activity, type B and dopamine deamination in homogenates of different rat brain structures. The role of MAO inhibition in the mechanism of dimebone action on the catecholamine metabolism in the brain structures and its stimulating effect on CNS are discussed.     

Changes in levels of monoamines and their metabolites in incompletely ischemic brains of spontaneously hypertensive rats

In order to investigate changes in levels of monoamines and their related substances together with those of other neurotransmitters (acetylcholine and GABA), choline and substances related to energy metabolism (ATP, lactate and glucose) accompanying incomplete cerebral ischemia, a bilateral common carotid artery occlusion model of spontaneously hypertensive rats (SHR) was utilized. Animals were subjected to 1 or 2 h ischemia. Then the concentrations of substances were measured in the cerebral cortex, hippocampus and striatum and compared with control values. Due to the incomplete ischemia, ATP showed a moderate decrease, while lactate and choline increased remarkably, and GABA underwent a moderate increase. With regard to monoamines, both noradrenaline and serotonin levels were reduced in the cerebral cortex and hippocampus, whereas dopamine levels increased in the hippocampus. All monoamine metabolites, i.e. metabolites by monoamine oxidase (MAO), metabolites by catechol-O-methyltransferase (COMT), and metabolites by both MAO and COMT, underwent increases. The 3-methoxytyramine level in particular showed marked increases. Furthermore levels of precursor amino acids as well as 5-hydroxytryptophan rose. Acetylcholine decreased moderately only in the cerebral cortex. Among these changes, sustained increases in all the monoamine metabolites were characteristic in the incompletely ischemic brain, suggesting that both COMT and MAO retain their activities in the incompletely ischemic brain.     

Neurotransmitter Receptor Ligand Binding and Enzyme Regional Distribution in the Pigeon Visual System

The relative importance of acetylcholine, dopamine, endogenous opiates, gamma-aminobutyric acid (GABA), glutamate, glycine, noradrenaline, and serotonin as transmitters in the pigeon visual system was estimated by measuring the activity of choline acetyltransferase (ChAT), glutamic acid decarboxylase (GAD), and aromatic amino acid decarboxylase (AAD) as well as the binding of dihydroalprenolol, etorphine, kainic acid, muscimol, serotonin, spiroperidol, strychnine, and quinuclidinyl benzilate (QNB) in the tectum opticum, nucleus rotundus, ectostriatum, dorsolateral thalamus, and hyperstriatum (Wulst). As a nonvisual reference structure, the paleostriatal complex was included in the examination. The regional distribution of most of these parameters was very similar to data reported in the mammalian CNS supporting the hypothesis that the avian tectofugal and thalamofugal visual systems are homologous to the mammalian tecto-thalamo-cortical and retino-geniculo-striate pathways, respectively. On the basis of the low values of their parameters, some transmitters can be excluded as significant contributors in a number of structures. As a hypothesis for further investigations, the presence of cholinergic and serotoninergic systems in the Wulst, possibly originating in the dorsolateral thalamus and nucleus raphe, respectively, and of glycinergic and dopaminergic terminals in the paleostriatal complex is proposed.     

Decreased noradrenergic and serotonergic reactivity of vas deferens of newborn rats from mothers treated with the serotonin reuptake inhibitor fluoxetine during pregnancy and breast-feeding

Female Wistar rats were treated with the serotonin reuptake inhibitor fluoxetine (10 mg/kg/i.p/day), during pregnancy and breast-feeding, for the study of the corresponding newborn rats. At the end of the preweaning period, the 30-day old litters had their vas deferens removed for testing peripheral sympathetic reactivity, through the following experiments in vitro: (a) concentration-contraction curves for serotonin and for the adrenergic agonists noradrenaline, phenylephrine, clonidine and dopamine or for the indirect agonist tyramine (b) contractions induced by electric field stimulation, as an indicator of sympathetic neurotransmission (c) release of endogenous noradrenaline, measured by real-time determinations on HPLC (d) Ca(+2) time-contraction curves, to check for changes on Ca(+2) translocation. Our results showed that the affinity (pD(2)) for serotonin was strikingly decreased by about 1.5 log units. The pD(2) for adrenergic agonists was decreased by about 0.5 log units, except for dopamine and clonidine. The maximum effects and intrinsic activity were decreased only for dopamine. On the other hand, the response to Ca(+2) and the release of noradrenaline from nerve terminals were not modified. In additional experiments, the mother's body weights were measured, showing a decrease during gestation and a recovery during lactation while the offspring's weights were lower than controls. It is concluded that, besides the alterations on body weights, changes on noradrenergic and serotonergic mechanisms were observed and persisted in the newborn, at least one month after parturition.     

The involvement of GABA(A) receptors in the control of GnRH and beta-endorphin release, and catecholaminergic activity in the ventromedial-infundibular region of hypothalamus in anestrous ewes.

To examine the role of the GABA(A) receptor mediating systems in the control of gonadotropin-releasing hormone (GnRH) release from the ventromedial-infundibular region (VEN/IN) of anestrous ewes, the extracellular concentrations of GnRH, beta-endorphin, noradrenaline (NE), dopamine (DA), 4-hydroxy-3-methoxy-phenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC) were quantified during local stimulation or blockade of GABA(A) receptors with muscimol or bicuculline respectively. In most animals stimulation of GABA(A) receptors significantly attenuates GnRH release with concomitant increase of beta-endorphin and DA release, and MHPG and DOPAC levels. Blockade of the GABA(A) receptors generally did not affect GnRH and NE release but inhibited in most animals beta-endorphin release and decreased dopaminergic activity. These results suggest, that GABA may suppress GnRH release directly by GABA(A) receptor mechanism on the axon terminal of GnRH neurons or indirectly by GABA(A) receptor processes activating beta-endorphin-ergic and dopaminergic neurons in the VEN/NI. On the basis of these results in could not be distinguish between these two events. The decrease in extracellular beta-endorphin and dopamine concentration without evident changes in the GnRH level during GABA(A) receptor blockade may suggest that other neuronal systems are involved in this effect.     

Possible involvement of serotonin receptors in the facilitatory effect of a hallucinogenic phenethylamine on single facial motoneurons

2,5-Dimethoxy-4-methylamphetamine (DOM, "STP") is a potent hallucinogen, proposed to be a serotonin receptor agonist. Its effects have not previously been tested upon central neurons where serotonin is excitatory and serotonin antagonists are effective. Extracellular single unit recordings were obtained from facial motoneurons in anaesthetized rats, and drugs were applied from five-barrelled micropipettes by iontophoresis. Facial motoneurons were commonly silent. During subthreshold application of glutamate, firing could be induced by dopamine and DOM. As reported by others, serotonin and noradrenaline also excited facial motoneurons under these conditions. Methysergide antagonized responses to serotonin and DOM but not those to noradrenaline; methysergide could not usually discriminate between responses to serotonin and dopamine. Ketanserin reversibly antagonized (but could not discriminate between) responses to serotonin, dopamine, and noradrenaline. Chlorpromazine antagonized responses to dopamine at doses that did not alter serotonin-induced excitation, and responses to DOM were not reduced by doses of chlorpromazine, that had no local anaesthetic effect on action potentials elicited by DOM and serotonin. These results suggest that DOM is an agonist on at least one type of central serotonin receptor. This receptor may also be a ketanserin (5-HT2) binding site.     

Effects of serotonin and catecholamines on RNA synthesis in planarians; in vitro and in vivo studies

The effects of serotonin, dopamine and noradrenaline on RNA synthesis, estimated by the incorporation of [3H]orotic acid, were studied on regenerating fragments of planarians. Serotonin was observed to inhibit, whereas dopamine and noradrenaline had no apparent action. These three neurohormones and their antagonists were also tested on planarian cell cultures, using [3H]-uridine as tracer. RNA synthesis, inhibited by serotonin, methiothepine (serotonin antagonist) and fluphenazine (dopamine antagonist), was shown to be restored by dopamine. The effects of serotonin, dopamine and their antagonists, are discussed in relation to the adenylate cyclase system.