The release of gamma-aminobutyric acid, glutamate, and acetylcholine from striatal slices: a mass fragmentographic study

The release processes of endogenous Acetylcholine (ACh), gamma-aminobutyric acid (GABA), glutamate (Glu) and glutamine (GLN) were studied in superfused guinea-pig caudatal slices. Basal ACh release remained constant for up to 2 h, while the basal release of GABA, Glu and GLN declined to half or less of its initial values after 1 h of superfusion. Electrical stimulation increased the ACh release by 700-800% and that of GABA by 80% whereas it decreased the output of Glu by 50% and failed to modify the GLN efflux. KCl (25 nM) increased the output of ACh by 400%, that of GABA by approximately 500% and decreased that of Glu by 40%. Substituting of CaCl(2) by MgCl(2) in the superfusion medium reduced the basal efflux of GABA, Glu and GLN. Under these conditions, no evoked release of ACh or of GABA was detected, following electrical or KCl stimulation. Tetrodotoxin 5 x 10(-7) decreased the basal ACh release by 60% and increased the GABA efflux by 40%. The toxin abolished the stimulus-evoked ACh efflux but scarcely affected that of GABA. These results are consistent with a possible neurotransmitter role of ACh and GABA in the striatum and show some differences in the ionic mechanisms underlying GABA and ACh release.     

The effects of dopamine on temperature regulation in goldfish

Summary Microinjections of dopamine (DA) were made into specific forebrain loci in goldfish (Carassius auratus: 40–85 g) to study the involvement of DA in behavioral thermoregulation. Injections of 25, 50, 100 and 250 ng DA into the anterior aspect of the nucleus preopticus periventricularis (NPP) led to consistent, dose-dependent decreases in selected temperature was observed following injections of 5 or 10 ng DA. Injections of the control solution were without effect.Injections of DA into other forebrain loci, including the posterior half of the NPP, either had no thermoregulatory effect or had minor thermoregulatory effects which, in comparison to injections into the most effective sites, were inconsistent and required larger doses to obtain. The decrease in selected temperature following injections of 100 ng DA into the anterior NPP was blocked by haloperidol, a dopaminergic antagonist, but not by phentolamine, a noradrenergic antagonist. Injections of haloperidol alone resulted in a minor, but statistically significant, increase in selected temperature.The most sensitive DA sites lie caudal to the sites most sensitive to norepinephrine within the anterior NPP. DA acts on the dopaminergic receptors of central thermoregulatory neurons in the anterior NPP of goldfish. These receptors appear to mediate behavioral responses to excessively warm environments.Abbreviations DA dopamine - NE norepinephrine - NPP nucleus preopticus periventricularis - PBS phosphate buffer solution     

Peroxidation induced changes in synaptosomal transport of dopamine and gamma-aminobutyric acid

The effects of iron-dependent peroxidation on respiration and neurotransmitter transport of brain nerve endings has been studied. Rat brain synaptosomes were peroxidized by exposure to an ADP-Fe/ascorbate system and the protective effect of added Se, Cd, or Zn was investigated with regard to dopamine and gamma-aminobutyric acid (GABA) transport. Peroxidation impaired the respiration of synaptosomes by about 20% and caused a marked increase in dopamine uptake; but in contrast, peroxidation induced a large decrease in synaptosomal uptake of GABA. The increased dopamine transport into synaptosomes was partially prevented by the presence of Zn, Se, or Cd. The presence of Zn, Cd, or Se, in order of decreasing effectiveness, also slowed down ADP-Fe/ascorbate mediated peroxidation of synaptosomes. Peroxidation caused a significant inhibition of veratridine-dependent release of both dopamine and GABA from synaptosomes, but the KCl-dependent release of these neurotransmitters was not effected by peroxidation. These results implicate that peroxidation damage of nerve endings may lead to large changes in neurotransmitter transport thus resulting in an alteration in the function of the central nervous system.     

Multiple benzodiazepine receptors and their regulation by gamma-aminobutyric acid

The interaction of propyl β-carboline-3-carboxylate (PCC) with benzodiazepine receptors in the cerebral cortex of the rat was investigated by direct measurements of [³H]PCC binding and by competitive inhibition of [³H]flunitrazepam (FLU) binding. Initial experiments showed that [³H]PCC binding exhibited characteristics of saturability, stereospecificity and a pharmacological specificity remarkably similar to that of [³H]FLU binding. Analysis of [³H]PCC binding isotherms and PCC/[³H]PCC competition curves revealed the presence of a small population of super high affinity PCC binding sites (K_SH = 30–100 pM) which represents approximately 3–6% of the total sites. When measured by competitive inhibition of [³H]FLU binding, receptor occupancy by PCC was generally consistent with that determined by direct measurements of [³H]PCC binding. Analysis of the PCC/[³H]FLU competition curve revealed the presence of two major populations of high and low affinity PCC binding sites with dissociation constants of 0.54 and 10 nM and relative abundances of 52 and 45%, respectively. Collectively, the results of the [³H]PCC binding isotherm, PCC/[³H]PCC competition curve and PCC/[³H]FLU competition curve are internally consistent when rationalized in terms of three populations of benzodiazepine receptors - super high, high, and low affinity - each having different affinities for PCC and equal affinity for FLU. The effects of γ-aminobutyric acid (GABA) on PCC and FLU binding were investigated, and it was observed that GABA enhanced the binding of FLU to the various receptor subtypes whereas no significant effect of GABA on the binding of PCC was detected.     

Distribution of gamma-aminobutyric acid, glycine, glutamate and aspartate in the cochlear nucleus of the rat

The distributions of gamma-aminobutyric acid (GABA), glycine, glutamate and aspartate were measured in cochlear nuclei of two rats by quantitative histochemical mapping procedures. The levels and distributions in the two rats were comparable, and resembled those previously reported for cat cochlear nucleus. The results are consistent with a concept that these putative transmitter amino acids have similar levels and distributions in the cochlear nucleus among mammals.     

Formation of gamma-aminobutyric acid from glutamate and putrescine in rat hypothalamic and hippocampal synaptosomes and regulation of these processes by glucocorticoids]

It has been shown that single or multiple hydrocortisone and ACTH administrations to intact rats increased GABA content and its synthesis from glutamate and putrescine in synaptosomes of hypothalamus. The letter content was increased by single hormonal administration while multiple hormonal administration and adrenalectomy decreased it. Ornithine decarboxylase activity was increased by single hydrocortisone administration to intact animals, following adrenalectomy, and it was decreased by single hormonal administration to adrenalectomized rats. GABA synthesis in synaptosomes of hippocampus from putrescine was increased by single hydrocortisone and multiple hormonal administrations. GABA content was increased by multiple administration of both hormones and was decreased by adrenalectomy. Putrescine level was decreased by multiple hydrocortisone administration to intact and single administration to adrenalectomized rats; ornithine decarboxylase activity was decreased by multiple administration of both hormones.     

Neuronal dependence of extracellular dopamine,acetylcholine, glutamate,aspartate and gamma-aminobutyric acid (GABA) measured simultaneously from rat neostriatum using in vivo microdialysis: reciprocal interactions

Summary The neuronal origin of extracellular levels of dopamine (DA), acetylcholine (ACh), glutamate (Glu), aspartate (Asp) and gamma-aminobutyric acid (GABA) simultaneously collected from the neostriatum of halothane anaesthetized rats with in vivo microdialysis was studied. The following criteria were applied (1) sensitivity to K⁺-depolarization; (2) sensitivity to inhibition of synaptic inactivation mechanisms; (3) sensitivity to extracellular Ca²⁺; (4) neuroanatomical regionality; sensitivity to selective lesions and (5) sensitivity to chemical stimulation of the characterized pathways.It was found that: (1) Extracellular DA levels found in perfusates collected from the neostriatum fulfills all the above criteria and therefore the changes in extracellular DA levels measured with microdialysis reflect actual release from functionally active nerve terminals, and so reflect ongoing synaptic transmission. (2) Changes in neostriatal ACh levels reflect neuronal activity, provided that a ACh-esterase inhibitor is present in the perfusion medium. (3) Extracellular Glu, Asp and GABA could be measured in different perfusion media in the rat neostriatum and probably reflect metabolic as well as synaptic release. However, (4) the majority of the extracellular GABA levels found in perfusates collected from the neostriatum may reflect neuronal release, since GABA levels were increased, in a Ca²⁺-dependent manner, by K⁺-depolarization, and could be selectively decreased by an intrinsic neostriatal lesion. (5) It was not possible to clearly distinguish between the neuronal and the metabolic pools of Glu and Asp, since neostriatal Glu and Asp levels were only slightly increased by K⁺-depolarization, and no changes were seen after decortication. A blocker of Glu re-uptake, DHKA, had to be included in the perfusion medium in order to monitor the effect of K⁺-depolarization on Glu and Asp levels. Under this condition, it was found (6) that neostriatal Glu and Asp levels were significantly increased by K⁺-depolarization, although only increases in the Glu levels were sensitive to Ca²⁺ in the perfusion medium, suggesting that Glu but not Asp is released from vesicular pools. (7) Evidence is provided that selective stimulations of nigral DA cell bodies may lead to changes in release patterns from DA terminals in the ipsilateral neostriatum, which are in turn followed by discrete changes in extracellular levels of GABA and Glu in the same region. Finally, some methodological considerations are presented to clarify the contribution of neuronal release to extracellular levels of amino acid neurotransmitters in the rat neostriatum.     

Characterization of glutamate decarboxylase from a high gamma-aminobutyric acid (GABA)-producer, Lactobacillus paracasei

Gamma-aminobutyric acid (GABA) has several physiological functions in humans. We have reported that Lactobacillus paracasei NFRI 7415 produces high levels of GABA. To gain insight into the higher GABA-producing ability of this strain, we analyzed glutamate decarboxylase (GAD), which catalyzes the decarboxylation of L-glutamate to GABA. The molecular weight of the purified GAD was estimated to be 57 kDa by SDS-PAGE and 110 kDa by gel filtration, suggesting that GAD forms the dimer under native conditions. GAD activity was optimal at pH 5.0 at 50 degrees C. The Km value for the catalysis of glutamate was 5.0 mM, and the maximum rate of catalysis was 7.5 micromol min(-1) mg(-1). The N-terminal amino acid sequence of GAD was determined, and the gene encoding GAD from genomic DNA was cloned. The findings suggest that the ability of Lb. paracasei to produce high levels of GABA results from two characteristics of GAD, viz., a low Km value and activity at low pH.     

Functional regulation of gamma-aminobutyric acid transporters by direct tyrosine phosphorylation

Tyrosine phosphorylation regulates multiple cell signaling pathways and functionally modulates a number of ion channels and receptors. Neurotransmitter transporters, which act to clear transmitter from the synaptic cleft, are regulated by multiple second messenger pathways that exert their effects, at least in part, by causing a redistribution of the transporter protein to or from the cell surface. To test the hypothesis that tyrosine phosphorylation affects transporter function and to determine its mechanism of action, we examined the regulation of the rat brain gamma-aminobutyric acid (GABA) transporter GAT1 expressed endogenously in hippocampal neurons and expressed heterologously in Chinese hamster ovary cells. Inhibitors of tyrosine kinases decreased GABA uptake; inhibitors of tyrosine phosphatases increased GABA uptake. The decrease in uptake seen with tyrosine kinase inhibitors was correlated with a decrease in tyrosine phosphorylation of GAT1 and resulted in a redistribution of the transporter from the cell surface to intracellular locations. A mutant GAT1 construct that was refractory to tyrosine phosphorylation could not be regulated by tyrosine kinase inhibitors. Activators of protein kinase C, which are known to cause a redistribution of GAT1 from the cell surface, were additive to the effects of tyrosine kinase inhibitors suggesting that multiple signaling pathways control transporter redistribution. Application of brain-derived neurotrophic factor, which activates receptor tyrosine kinases, up-regulated GAT1 function suggesting one potential trigger for the cellular regulation of GAT1 signaling by tyrosine phosphorylation. These data support the hypothesis that transporter expression and function is controlled by the interplay of multiple cell signaling cascades.     

Substrate-induced regulation of gamma-aminobutyric acid transporter trafficking requires tyrosine phosphorylation

Neurotransmitter transporters regulate synaptic transmitter levels and are themselves functionally regulated by a number of different signal transduction cascades. A common theme in transporter regulation is redistribution of transporter protein between intracellular stores and the plasma membrane. The triggers and mechanisms underlying this regulation are important in the control of extracellular transmitter concentrations and hence synaptic signaling. Previously, we demonstrated that the gamma-aminobutyric acid transporter GAT1 is regulated by direct tyrosine phosphorylation, resulting in an up-regulation of transporter expression on the plasma membrane. In the present report, we show that two tyrosine residues on GAT1 contribute to the phosphorylation and transporter redistribution. Tyrosine phosphorylation is concomitant with a decrease in the rate of transporter internalization from the plasma membrane. A decrease in GAT internalization rates also occurs in the presence of GAT1 substrates, suggesting the hypothesis that tyrosine phosphorylation is required for the substrate-induced up-regulation of GAT1 surface expression. In support of this hypothesis, incubation of GAT1-expressing cells with transporter ligands alters the amount of GAT1 tyrosine phosphorylation, and substrate-induced surface expression is unchanged in a GAT1 mutant lacking tyrosine phosphorylation sites. These data suggest a model in which substrates permit the phosphorylation of GAT1 on tyrosine residues and that the phosphorylated state of the transporter is refractory for internalization.     

Characterization of gamma-aminobutyric acid and dopamine overflow following acute implantation of a microdialysis probe

The present study characterized the voltage and calcium dependence of gamma-aminobutyric acid and dopamine overflow after the acute implantation of a microdialysis probe. Probes were implanted in dorsolateral striatum and globus pallidus. Experiments were performed under light halothane anesthesia. Basal, extracellular levels of GABA were not affected by tetrodotoxin (TTX) and were increased to 140 percent of basal values by calcium free Ringer. Basal, extracellular levels of dopamine were reduced to 14 percent of basal values by the addition of TTX and to 30 percent of basal values by the removal of calcium from the Ringer solution. The results suggest that in this in vivo preparation basal extracellular dopamine is largely of vesicular origin while GABA is not.     

Morphofunctional effects of applications of glutamate and dopamine on the goldfish Mauthner neurons

In the goldfish, we studied the effects of intramedullar applications of glutamate (Glu), dopamine (DA), and of long-lasting rotational stimulation on the functional activity, dimensional characteristics, and ultrastructure of Mauthner neurons (MNs). Applications of Glu, especially when combined with rotational stimulation, were found to result in suppression of the function of MNs, in a decrease in their dimensions and lengths of desmosome-like contacts (DLCs, whose structure is determined by filamentous actin) in afferent mixed and chemical synapses, and in destruction of actin microfilaments in the cytoskeleton of MNs. Applications of DA, vice versa, induced an increase in the resistance to the effects of long-lasting stimulation and stabilized the dimensions of MNs; the length of DLCs increased in afferent synapses of both the above types, and the number of fibrillar actin bridges in the DLC cleft of mixed synapses also increased. Bundles of the actin filaments, which were preserved after stimulation, appeared in the cytoskeleton of MNs. Testing of the action of neurotransmitters on actin preparations in vitro showed that Glu entirely depolymerizes filamentous actin, while DA, vice versa, polymerizes monomeric actin. Thus, the Glu-and DA-induced reactions are similar in their types and are of a reciprocal nature both in the actin cytoskeleton of MNs in situ and in purified actin in vitro; these effects correlate with suppression of the functional state of MNs under the influence of Glu and with stabilization of this state under the influence of DA. These results agree with the concept on the roles of depolymerization and polymerization of actin in changes of the morphofunctional state of MNs and show that actin of the cytoskeleton of MNs is a cellular target for the actions of Glu and DA. The similarity between the effects of tested neurotransmitters on actin in MNs in situ and in cell-free preparations in vitro allows us to hypothesize that these transmitters can penetrate into the neuron. Neirofiziologiya/Neurophysiology, Vol. 38, No. 4, pp. 320–330, July–August, 2006.     

Effect of tetanus toxin on the content of glycine, gamma-aminobutyric acid, glutamate, glutamine and aspartate in the rat spinal cord

Tetanus toxin injected intramuscularly induced no significant changes in the levels of glycine, GABA, glutamate, glutamine or aspartate in extracts of spinal cord from rats killed at timed intervals during the development of local and generalized tetanus. The amino acid contents in the hemisegment (longitudinal one-half) of the spinal cord (L₂-L₆) on the injected side (left gastrocnemius muscle) did not differ significantly from the contents in the hemisegment of the spinal cord on the non-injected side. Nor were there any consistent changes in the contents of the amino acids in either hemisegment of the spinal cord as the tetanic symptoms became progressively more severe. Hence, the amino acid pool in the spinal cord was relatively stable despite the metabolic changes known to occur in tetanus. Our observations are consistent with the view of Johnston , De Groat and CURTIS (1969) who suggested that if glycine were indeed a spinal inhibitory neurotransmitter released by interneurons affected by tetanus toxin, the toxin should interfere with the release of the amino acid rather than deplete the transmitter stores.     

Ontogeny of glutamate and gamma-aminobutyric acid release in the hippocampus of the guinea pig.

The ontogeny of the L-glutamate (GLU) and gamma-aminobutyric acid (GABA) neuronal systems in the guinea pig hippocampus was investigated with respect to tissue amino acid content, and spontaneous and K(+)-stimulated release of GLU and GABA. Transverse hippocampal slices were prepared from the guinea pig fetus at day 45 (brain growth spurt), 55 and 63 of gestation (term, about 68 days), from the 5-days-old neonate, and from the young adult. GLU and GABA release was determined as efflux from hippocampal slices into Krebs'-bicarbonate medium using a dynamic, submerged, superfusion apparatus. Hippocampal GLU content decreased during development, whereas GABA content was constant for all the ages investigated. The magnitude of spontaneous GLU efflux decreased during development; there was no measurable spontaneous GABA efflux. The K+ concentration-GLU efflux response curve was bell-shaped for the fetus at the three selected gestational ages, and was curvilinear for the neonate and adult. The apparent EC75 of K(+)-stimulated GLU efflux was higher for the neonate and adult compared with the fetus. In contrast, the K+ concentration-GABA efflux response curve was curvilinear, and the apparent EC75 of K+ was similar for all the ages investigated. K(+)-stimulated efflux of GLU and GABA was Ca++ dependent, but this was not the case for spontaneous GLU efflux. These data indicate that, in the guinea pig hippocampus, the GLU neuronal system is developing throughout gestation, whereas the GABA neuronal system appears to mature before the brain growth spurt.     

Role of serotonin,gamma-aminobutyric acid,and glutamate in the behavioral thermoregulation of female tilapia during the prespawning phase

The role of hypothalamic neurotransmitter systems in behavioral thermoregulation was investigated in the prespawning female tilapia, Oreochromis mossambicus. Intrahypothalamic microinjection with serotonin (5-HT, 3 microliters of 1.0 x 10(-6) M) resulted in a significant increase in the selected temperature. This effect was mimicked by the agonist of 5-HT1A, 1B, and 2C receptors, N-3-trifluoromethylphenyl piperazine. Intrahypothalamic microinjection of tilapia with gamma-aminobutyric acid (GABA) resulted in a biphasic effect of the temperature selection, whereas microinjection with muscimol, an agonist of GABAA receptor, had no effect on temperature selection. Both agonist and antagonist of glutamate (Glu), N-methyl-D-aspartate (NMDA), and MK-801 (1.0 x 10(-6) M), a noncompetitive blocker of NMDA receptor, significantly decreased the preferred temperature. These results indicate that the hypothalamic 5-HT, GABA, and Glu systems play a role in the temperature selection of prespawning female tilapia.