THE SPONTANEOUS AND ELECTRICALLY EVOKED RELEASE, FROM SLICES OF GUINEA-PIG CEREBRAL CORTEX, OF ENDOGENOUS AMINO ACIDS LABELLED VIA METABOLISM OF d-[U-14C]GLUCOSE

Abstract— In an effort to identify neurotransmitters in slices of guinea-pig cerebral cortex, a study was made of the release of endogenous amino acids which had become labelled via metabolism of d -[U-¹⁴C]glucose. While incorporation of ¹⁴C into endogenous glutamate, aspartate, GABA, alanine and threonine-serine-glutamine (unseparated) was large enough to permit measurement of their release, that into other amino acids was not. In parallel experiments, the release of exogeneous labelled glutamate, aspartate, GABA and α-aminoisobutyrate was examined. Electrical field stimulation evoked a transient increase in the release of all the adequately labelled endogenous amino acids and all the exogenous amino acids. The stimulated ‘increase’ in the release of each of the endogenous ¹⁴C-labelled transmitter candidates (glutamate, aspartate and GABA) was larger than that of any other amino acid (except that of exogenous GABA). When the experiments were performed without the glucose (5 mm ) usually present in the medium bathing the slices, larger amounts of each labelled amino acid were released from the slices than in the presence of glucose. Moreover, the pattern of selective release of the endogenous labelled transmitter candidates was much more pronounced in the absence of glucose. It is likely that in the absence of glucose, release from the tissue was larger because cells in the slice were relatively depolarized and uptake of amino acids into cells was impaired. Because previous evidence suggests that over 90% of glucose consumption occurs in the ‘large metabolic compartment’ which is thought to be composed of neuronal elements, neurons were probably the main site from which the larger release of endogenous ¹⁴C-labelled transmitter candidates was evoked. The exogenous amino acids were probably released from several cellular elements in the slices. It was concluded that the pattern of a selective release of the endogenous labelled transmitter candidates may have been indicative of a transmitter releasing mechanism in nerve terminals.     

Modulation of Glutamate and Aspartate Release from Slices of Hippocampal Area CA1 by Inhibitors of Arachidonic Acid Metabolism

Abstract: Slices of hippocampal area CA1 were used to test inhibitors of arachidonic acid metabolism for their effects on glutamate/aspartate release from the CA3-derived Schaffer collateral, commissural, and ipsilateral associational terminals. Test compounds [3 µ M nordihydroguaiaretic acid (NDGA) and 1 µ M 3-[3-(4-chlorobenzyl)-3- tert -butylthio-5-isopropylindol-2-yl]-2,2-dimethyl-propanoic acid (MK-886)] that reduced the production and release of 5-lipoxygenase metabolites also selectively reduced the K⁺-evoked release of aspartate. In contrast, the cyclooxygenase inhibitor indomethacin (100 µ M ) selectively enhanced the release of glutamate. At a concentration (100 µ M ) that nonselectively depressed the release of arachidonic acid and its metabolites, NDGA markedly depressed the release of aspartate, glutamate, and GABA. An inhibitor of the 12-lipoxygenase and an inhibitor of nitric oxide synthase did not affect the K⁺-evoked release of any transmitter amino acid. These results suggest that a 5-lipoxygenase product selectively enhances aspartate release and a cyclooxygenase product selectively depresses glutamate release. They are also consistent with previous evidence that arachidonic acid and/or platelet-activating factor enhances the release and depresses the uptake of glutamate and aspartate. The K⁺-evoked release of excitatory amino acids is much more sensitive to modulation by lipid mediators than is GABA release. Activation of NMDA receptors may enhance the K⁺-evoked release of glutamate and aspartate from CA1 slices by stimulating the production and release of lipid modulators.     

In Vitro Release of Endogenous β- Alanine, GABA, and Glutamate, and Electrophysiological Effect of β-Alanine in Pigeon Optic Tectum

The efflux of 20 amino acids, induced by either high K+ concentration or veratrine, was determined in pigeon tectal slices. Ca2+-dependent, K+-induced release of beta-alanine, gamma-aminobutyric acid (GABA), and glutamate was observed. Veratrine caused release of the same amino acids plus glycine in a tetrodotoxin-sensitive manner. beta-Alanine had a strong inhibitory effect on the activity of tectal neurons which was blocked by strychnine but not by bicuculline. The results indicated a transmitter function for beta-alanine in the optic tectum, and were consistent with the previously proposed transmitter role of GABA and glutamate in this structure.     

β- Alanine,a Possible Neurotransmitter in the Visual System?

Abstract— The chemically evoked efflux of endogenous amino acids from perfused rabbit superior colliculus (SC) slices was studied. Amino acids in the perfusates were determined fluorimetrically with a precolumn derivatisation method and subsequent separation on an HPLC column. Potassium-induced depolarisation caused a calcium-dependent release of β-alanine, GABA, glutamate, and aspartate. Veratridine-evoked efflux was essentially similar in selectivity and could be blocked by tetrodotoxin (TTX). The results are indicative of a neurotransmitter role of β-alanine, GABA, glutamate, and aspartate in the SC of the rabbit.     

Effect of Motor and Premotor Cortex Ablation on Concentrations of Amino Acids, Monoamines, and Acetylcholine and on the Ultrastructure in Rat Striatum. A Confirmation of Glutamate as the Specific Cortico-Striatal Transmitter

At 1, 2, and 4 weeks after unilateral premotor and motor cortex ablation in rats, a significant and lasting decrease in glutamate levels in the ipsilateral versus contralateral striatum was observed. A significant corresponding fall in aspartate was seen only after 1 week. In contrast, there was a large increase in the striatal concentrations of lysine, threonine, alanine, and glutamine 1 week after the cortical ablation. This correlates with the extensive glial proliferation in the deafferented ipsilateral striatum. Four weeks after cortical ablation the GABA concentration was significantly increased. There was no decrease in other putative transmitters (dopamine, serotonin, acetylcholine, glycine and taurine), nor was a glutamate decrease observed in the hippocampus or in the hypothalamus, which do not receive direct premotor and motor cortical inputs. Both biochemical and morphological evidence for a minor contralateral cortico-striatal projection was obtained. Correlating with the fall in glutamate, ultrastructural observations indicated the degeneration of two types of striatal synapses, i.e., those of the axo-spinous type III and of the axo-dendritic type VII. Frontal cortex ablation clearly affects, in opposite directions, the metabolism of various striatal amino acids but not that of acetylcholine and the monoamine transmitters. The results strongly support the view that glutamate is the transmitter of the cortico-striatal fibers.     

N-Methyl-D-Aspartate-Mediated Injury Enhances Quisqualic Acid-Stimulated Phosphoinositide Turnover in Perinatal Rats

Previous work in our laboratory demonstrated that ischemic-hypoxic brain injury in postnatal day 7 rats causes a substantial increase in phosphoinositide (PPI) turnover stimulated by the glutamate analogue quisqualic acid (QUIS) in the hippocampus and striatum. To examine this phenomenon in more detail, we performed similar experiments after producing injury by unilateral intracerebral injections of the glutamate analogue N-methyl-D-aspartate (NMDA). The 7-day-old rodent brain is hypersensitive to NMDA neurotoxicity and NMDA injection causes histopathology that closely resembles that produced by ischemia-hypoxia. NMDA, 17 nmol in 0.5 microliter, was injected into the right posterior striatum of 7-day-old rat pups and they were killed 3 days later. Hippocampal or striatal tissue slices were prepared from ipsilateral and contralateral hemispheres from vehicle-injected control and from noninjected control rat pups. Slices were then incubated with myo-[3H]inositol plus glutamate agonists or antagonists in the presence of lithium ions and [3H]inositol monophosphate ([3H]IP1) accumulation was measured. The glutamate agonists, QUIS, L-glutamic acid, and (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, stimulated greater [3H]IP1 release in tissue ipsilateral to the NMDA injection compared with that in the contralateral side and in control pups. The glutamate antagonists, D,L-2-amino-7-phosphonoheptanoic acid, 3-[(+)-2-carboxypiperazin-4-yl]-propyl-1-phosphoric acid, kynurenic acid, and 6,7-dinitroquinoxaline-2,3-dione did not inhibit QUIS-stimulated [3H]IP1 release. The enhanced PPI turnover in the lesioned tissue was specific to glutamate receptors because carbachol (CARB) failed to elicit preferential enhanced stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of Glial Cells for the Basal and Ca2+-Dependent K+-Evoked Release of Transmitter Amino Acids Investigated by Microdialysis

The role of glial cells for the inactivation and synthesis of precursors for amino acid transmitters was studied in the brains of anesthetized rats in vivo using the microdialysis technique. The dialysis probes were inserted stereotactically into each neostriatum. One neostriatum was treated with the gliotoxin fluorocitrate, whereas the contralateral side served as a control. The basal efflux of amino acids, reflecting the extracellular level, was measured as well as the efflux during depolarization with 100 mM K+ in the dialysis stream. The potassium-evoked efflux of transmitter amino acids was calcium dependent and thus considered to reflect release from the transmitter pool. gamma-Aminobutyric acid (GABA) and glutamate release from the treated side was higher than the control value during the first 2-3 h, a result indicating an important role of glial cells in the inactivation of released transmitter. After 6-7 h with fluorocitrate, the release of glutamate was lower than the control value, a result indicating an important role of glial cells in the synthesis of precursors for the releasable pool of glutamate. The role of glutamine for the production of transmitter glutamate and GABA in vivo was further investigated by inhibiting glutamine synthetase with intrastriatally administered methionine sulfoximine. The release of gluatamate into the dialysis probe decreased to 54% of the control value, whereas the release of GABA decreased to 22% of the control value, a result indicating that glutamine may be more important for transmitter GABA than for transmitter glutamate.     

Effects of methylmercury on the spontaneous and potassium-evoked release of endogenous amino acids from mouse cerebellar slices

The effects of methylmercury on the spontaneous and potassium-evoked release of endogenous amino acids from mouse cerebellar slices have been examined. Methylmercury induced a concentration-dependent increase in the spontaneous release of glutamate, aspartate, gamma-aminobutyric acid, and taurine from mouse cerebellar slices. Glycine release was slightly increased, but not in a concentration-dependent manner. The spontaneous release of glutamine from mouse cerebellar slices was not altered by any concentration of methylmercury examined (10, 20, and 50 microM). The tissue content of glutamate, gamma-aminobutyric acid, glutamine, and taurine decreased after exposure to methylmercury. Exposure of cerebellar slices to 20 microM methylmercury resulted in a significant enhancement in glutamate release during stimulation with 35 mM K+. This increase could be accounted for by the methylmercury-induced increase in spontaneous glutamate release. The increase in spontaneous release of glutamate and gamma-aminobutyric acid was independent of the availability of extracellular calcium. These results suggest that methylmercury increases the release of neurotransmitter amino acids, particularly gamma-aminobutyric acid and glutamate, by acting at intracellular sites to increase release from a neurotransmitter pool. The increase in the potassium-stimulated release of glutamate may reflect an increased sensitivity of the cerebellar granule cell to the effects of methylmercury. It is suggested that alterations in amino acid neurotransmitter function in the cerebellum may contribute to some of the neurological symptoms of methylmercury intoxication.     

Amino acids and the synaptic pharmacology of granule cells in the dentate gyrus of the rat

Granule cells in the dentate gyrus in the hippocampi of anaesthetized rats were excited by stimulation of the contralateral hippocampus (the commissural input) and the ipsilateral entorhinal cortex (the perforant path). The cells were also activated by the electrophoretic administration of various amino acids. A selective antagonism of glutamate and perforant path excitations was obtained with glutamic acid diethylester, and of aspartate and other amino acid induced and commissural excitations with D- or DL-alpha-aminoadipate. An excitatory effect of alpha-aminoadipate which was sometimes observed was prevented by the gamma-aminobutyric acid antagonist bicuculline, and may be a disinhibitory phenomenon. The results lend support to the proposition that the transmitter of the perforant path is glutamate while that of the commissural fibres is aspartate.     

Role of various amino acids on the modulation of dopamine release from nigro-striatal dopaminergic neurones

The effects of various transmitter amino-acids on the striatal release of 3H-dopamine (3H-DA) were investigated both in vivo in cat and in vitro in rat striatal slices. When applied to the substantia nigra of the anaesthetized cat by means of a push-pull cannula, GABA induced an increase followed by a transient decrease of 3H-DA release in the ipsilateral caudate nucleus; glycine reduced 3H-DA release under similar experimental conditions. When added to the superfusion medium of rat striatal slices, GABA, glutamate and glycine increased the release of the newly synthetized 3H-DA, suggesting that these amino-acids are also directly or indirectly involved in the presynaptic modulation of striatal DA release.     

Effects of Ischaemia on Neurotransmitter Release from the Isolated Retina

Abstract: The effects of "ischaemia" (glucose-free Krebs-bicarbonate medium gassed with N₂/CO₂ on the release of glutamate and other major neurotransmitters in the retina were examined using the isolated rat and rabbit retina. Amino acid transmitters, acetylcholine, and dopamine were measured by HPLC. The release of glutamate, aspartate, GABA, and glycine from ischaemic retinas was more than doubled after 30 min, and after 90 min of ischaemia the release of amino acids was ∼ 15–20-fold that of control values. Ischaemia also produced large increases in the release of dopamine from both the rat and especially the rabbit retina. In contrast, the release of acetylcholine from the rat retina was significantly decreased by ischaemia, although the release of choline was increased. Because the ischaemia-induced release of glutamate, aspartate, and GABA from the rat retina was completely Ca independent, and exposure of the retina to high K (50 m M ) did not stimulate amino acid release, it is concluded that the mechanisms underlying the ischaemia-induced release do not involve an initial release of K or an influx of calcium.     

Enhancement of Endogenous Release of Glutamate and γ-Aminobutyric Acid from Hippocampus CA1 Slices After In Vivo Long-Term Potentiation

The effect of long-term potentiation (LTP) on endogenous amino acid release from rat hippocampus slices was studied. LTP was induced in vivo by application of a tetanus (200 Hz, 200 ms) to the Schaffer collateral fibers in unanesthetized rats. Endogenous release of glutamate and gamma-aminobutyric acid (GABA) was investigated 60 min after tetanization in CA1 subslices of potentiated and control rats. No significant effects of LTP were observed in basal and K(+)-induced Ca(2+)-independent release components of these amino acids. In contrast, K(+)-induced Ca(2+)-dependent release of both glutamate and GABA increased approximately 100% in slices from potentiated rats. No differences were observed in total content of glutamate and GABA between the subslices from control and LTP animals. These results suggest a persistent increase in the recruitment of the presynaptic vesicular pool of glutamate and GABA during LTP.     

The effect of fluorocitrate on transmitter amino acid release from rat striatal slices

In order to study the role of glutamine from glial cells for the synthesis of transmitter amino acids, the effect of the gliotoxic substance fluorocitrate on amino acid release from slices was investigated. In vivo treatment with 1 nmol fluorocitrate reduced the Ca²⁺ dependent K⁺ evoked release of endogenous glutamate and GABA from the slices, whereas the glutamine efflux decreased and alanine efflux increased. The K⁺ evoked release of [³H]d-aspartate increased during fluorocitrate treatment. The latter is consistent with an inhibited uptake ofd-aspartate into glial cells. Incubation of striatal slices with fluorocitrate (0.1 mM) decreased the glutamine efflux and increased the alanine efflux. Similar to the in vivo condition, fluorocitrate increased the K⁺ evoked [³H]d-asparate release, but the K⁺ evoked release of endogenous glutamate and GABA increased rather than decreased. The ratio between the K⁺ evoked release of exogenousd-aspartate to endogenous glutamate increased in both cases. The results suggest an important role of glial cells in the synthesis and inactivation of transmitter amino acids.Special Issue dedicated to Prof. Holger Hydén.     

Excitatory amino acid receptors and transmitter release in the retina

The effects of excitatory amino acids and some analogues on the release of GABA and ACh from amacrine cells were studied. The release of endogenous GABA from the isolated rat retina was measured by HPLC. When animals were pretreated with γ-vinyl-GABA (GVG), glutamate evoked a large efflux of GABA but kainate, quisqualate and (NMDA) were relatively ineffective. The glutamate evoked release of GABA was calcium dependent and was blocked by the antagonist, piperidine-dicarboxylic acid (PDA) indicating that activation of excitatory amino acid receptors was involved in the response. The release of [³H]ACh from the rabbit retina was strikingly increased by homocysteate and this effect was blocked by NMDA. Since NMDA also blocked the light evoked release of [³H]ACh but not the effects of exogenous glutamate or aspartate, it is possible that homocysteate may be a bipolar cell transmitter released onto cholinergic amacrine cells.     

Glutamic acid as a synpatic transmitter candidate in the dorsal sensory neuron: reconsiderations.

Two primary reasons which are emerging to suggest that glutamate is not a dorsal root transmitter are: 1) that free glutamate levels in the dorsal root vs. the ventral root are not sufficiently different to warrant a transmitter function in the dorsal root, and 2) the spinal cord glutamate levels do not significantly change (per g tissue) after dorsal root input section. Recent analyses suggest, however, that there is a highly significant depression of glutamate in the dorsal root when related to total free amino acid concentration changes after root injury. This is not seen in the peripheral nerve. Thus the dorsal vs. ventral root free glutamate concentration difference is highly significant metabolically. The failure to see a decrease in spinal cord gray glutamate levels after dorsal root section would appear to be explained by the fact that the spinal cord satellite cells and neurons have a higher free glutamate concentration than the entering dorsal roots along with a considerable perikaryal free amino acid pool for protein synthesis. This will mask any changes due to dorsal root section.Comparisons of excess free glutamate and substance P (the two leading dorsal root transmitter candidates) in the dorsal root compared to the ventral root have shown that there is a much larger excess of free glutamate in the dorsal root. This is true, even when considering the excitatory potency differences of these two substances. Thus, a very large free glutamate excess in the dorsal root is present with a relatively small concentration difference compared to the ventral root (where a transmitter role is not entertained). This fact could be of considerable metabolic significance in the regulation of transmitter levels of glutamate. The data available, therefore, are supportive of a possible glutamate transmitter role in a population of dorsal root fibers.     

Correlation between rotational behaviors and neurochemical changes associated with damage of rat striatum: Analysis using unilateral microinjection of kainic acid

The correlation between rotational behaviors and neurochemical changes associated with the striatal damage induced by an unilateral microinjection of kainic acid were investigated. Shortly after the unilateral striatal injection of kainic acid, rats exhibited contralateral rotational behaviors, and these changes were antagonized by the simultaneous striatal injection of haloperidol. On the other hand, systemic injection of methamphetamine to animals having the lesion on nigro-striatal dopaminergic neurons exhibited ipsilateral turnings. In addition, it was found that the release of [¹⁴C]dopamine from striatal slices was increased by the in vitro addition of kainic acid. Following 2 days after the striatal injection of kainic acid and thereafter, the rats exhibited ipsilateral rotational behaviors and microinjection of muscimol into the ipsilateral substantia nigra of these animals altered turning movements to a contralateral type. Simultaneous nigral injection of bicuculline antagonized to the muscimol-induced contralateral turnings. These results suggest that the increase of dopamine release from dopaminergic neurons in the striatum may be involved in the occurrence of contralateral turning behaviors observed shortly after the striatal kainic acid treatment. The present results also suggest that changes in the functional states of striatonigral GABA-ergic neurons may play an important role in the occurrence of ipsilateral rotational movements at a late stage following the striatal injection of this agent.     

Utilization of alpha-ketoglutarate as a precursor for transmitter glutamate in cultured cerebellar granule cells

Alpha-ketoglutarate together with an amino group donor (alanine) was shown to be able to serve as a precursor for the glutamate pool which is released by potassium-induced depolarization (i.e., transmitter glutamate) in cerebellar granule cells. However, these compounds could not be utilized as precursors for intracellular glutamate or for release of transmitter aspartate. The formation of transmitter glutamate was inhibited by the transamination inhibitor aminooxyacetic acid but not by phenylsuccinate, an inhibitor of the dicarboxylate carrier in the mitochondrial membrane. Both of these inhibitors have previously been found to inhibit synthesis of transmitter glutamate from glutamine. The results support the hypothesis that alpha-ketoglutarate and alanine undergo transamination in the cytosol to form pyruvate and glutamate, and that this glutamate pool is available for transmitter release of glutamate but does not constitute the major intracellular pool of glutamate.     

Decreased expression of vesicular GABA transporter, but not vesicular glutamate, acetylcholine and monoamine transporters in rat brain following focal ischemia

In nerve terminals, vesicular transporters pack neurotransmitters into synaptic vesicles, which is an essential prerequisite for transmitter release. To date, three distinct families of vesicular transporters have been identified which are specific for (a) excitatory amino acids (glutamate and aspartate), (b) inhibitory amino acids (GABA and glycine) and (c) acetylcholine and monoamines. The present study evaluated the effect of transient focal cerebral ischemia on the expression of these vesicular transporters in adult rat brain. Ischemia was induced by a 1 h transient middle cerebral artery occlusion (MCAO) in spontaneously hypertensive rats. At various reperfusion periods (3-72 h), mRNA levels of the vesicular transporters were estimated in the contralateral and the ipsilateral cerebral cortex by real-time PCR analysis. Following transient focal ischemia, mRNA expression of the vesicular GABA transporter (VGAT) decreased significantly by 3 h of reperfusion and remained at a significantly lower level than sham until at least 72 h of reperfusion. Western blotting showed a significant decrease in the VGAT immunoreactive protein levels in the ipsilateral cortex of rats subjected to focal ischemia and 24 h reperfusion. Immunohistochemistry demonstrated many VGAT immunopositive puncta in the contralateral cortex, which were significantly decreased in the ipsilateral cortex at 24 h reperfusion. Focal ischemia had no effect on the mRNA levels of the vesicular transporters specific for glutamate/aspartate, acetylcholine and monoamines at either 6 h or 24 h of reperfusion.     

BIOCHEMICAL CORRELATES OF TRANSMISSION MEDIATED BY GLUTAMATE AND ASPARTATE

Abstract— Glutamate and aspartate probably serve as transmitters of hippocampal perforant path and commissural afferents, respectively. We therefore used slices of hippocampal regions to evaluate certain biochemical properties as markers for sites of transmission mediated by these amino acids. In these studies content and accumulation of glutamate and aspartate were compared with their Ca²⁺-dependent effluxes.
Hippocampal regions varied little in their contents of glutamate and aspartate, but slices of regio superior and dentate gyrus accumulated and released more of each than slices of regio inferior. A commissurotomy or bilateral entorhinal lesion altered Ca²⁺-dependent efflux and accumulation in the same direction, but did not affect the glutamate or aspartate content of any hippocampal region. Elimination of hippocampal mossy fibers reduced the Ca²⁺-dependent efflux of glutamate and probably aspartate from slices of dentate gyrus, but not of regio inferior, where most mossy fiber synapses are located. The mossy fibers appeared relatively deficient in aspartate in both strains tested, but only in Purdue-Wistar rats were they enriched in glutamate. Removal of the perforant path input to the fascia dentata did not significantly change the activity of any of the enzymes most actively involved in glutamate synthesis.
These results suggest that accumulation or high affinity transport of glutamate or aspartate can be employed to localize afferents which use these amino acids as transmitters, although it is not so reliable or selective a marker as Ca²⁺-dependent efflux. Enrichment in either glutamate or aspartate content or in the activity of enzymes which synthesize them is not a reliable marker. Neither amino acid is likely to be used as a transmitter by the hippocampal mossy fibers.     

Effects of High-Potassium-Induced Depolarization on Amino Acid Chemistry of the Dorsal Cochlear Nucleus in Rat Brain Slices

High K⁺ was used to depolarize glia and neurons in order to study the effects on amino acid release from and concentrations within the dorsal cochlear nucleus (DCN) of brain slices. The release of glutamate, -aminobutyrate (GABA) and glycine increased significantly during exposure to 50 mM K⁺, while glutamine and serine release decreased significantly during and/or after exposure, respectively. After 10 min of exposure to 50 mM K⁺, glutamine concentrations increased in all three layers of DCN slices, to more than 5 times the values in unexposed slices. In the presence of a glutamate uptake blocker, L-trans-pyrrolidine-2,4-dicarboxylic acid (PDC), glutamine concentrations in all layers did not increase as much during 50 mM K⁺. Similar but smaller changes occurred for serine. Mean ATP concentrations were lower in 50 mM K⁺-exposed slices compared to control. The results suggest that depolarization, such as during increased neural activity, can greatly affect amino acid metabolism in the cochlear nucleus.