Transmitter Glutamate Release from Isolated Nerve Terminals: Evidence for Biphasic Release and Triggering by Localized Ca2+

The kinetics of Ca2(+)-dependent release of glutamate from guinea-pig cerebrocortical synaptosomes evoked by KCl or 4-aminopyridine are investigated using a continuous fluorimetric assay. Release by both agents is biphasic, with a rapid phase complete within 2 s followed by a more extensive slow phase with a half-maximal release in 52 s for KCl-evoked release and greater than 120 s for 4-aminopyridine-evoked release. The two phases of glutamate release may reflect a dual localization of releasable vesicles at the active zone and in the bulk cytoplasm. Decreasing depolarization depresses the extent rather than increasing the time for half-maximal Ca2(+)-dependent release. Both the fast and the slow phases of glutamate release require external Ca2+ and cytoplasmic ATP. KCl depolarization produces a transient "spike" of cytoplasmic free Ca2+ [( Ca2+]c), which recovers to a plateau; the major component of glutamate release occurs during this plateau. Predepolarization in the absence of added external Ca2+, to inhibit transient Ca2+ channels, does not affect the subsequent glutamate release evoked by Ca2+ readdition. Thus, release involves primarily noninactivating Ca2+ channels. For a given increase in [Ca2+]c, KCl and 4-aminopyridine cause equal release of glutamate, while ionomycin releases much less glutamate. This lowered efficiency is not due to ATP depletion. It is concluded that glutamate exocytosis is evoked by localized Ca2+ entering through noninactivating voltage-dependent Ca2+ channels and that nonlocalized Ca2+ entry with ionomycin is inefficient.     

Dendrotoxin, 4-Aminopyridine, and β-Bungarotoxin Act at Common Loci but by Two Distinct Mechanisms to Induce Ca2+-Dependent Release of Glutamate from Guinea-Pig Cerebrocortical Synaptosomes

The release of endogenous glutamate from guinea-pig cerebrocortical synaptosomes evoked by dendrotoxin, beta-bungarotoxin, and 4-aminopyridine is compared. Dendrotoxin and 4-aminopyridine cause Ca2+-dependent release, representing a partial depletion of the KCl-releasable transmitter pool. The decrease in the plasma membrane potential caused by 4-aminopyridine or dendrotoxin and the evoked release of glutamate from a transmitter pool accord with the inhibitory action of these agents on certain K+ conductances. In contrast, the massive release of glutamate evoked by beta-bungarotoxin is produced in the presence of Ca2+ but not of Sr2+, a result consistent with a generalised permeabilisation of synaptosomal plasma membranes. Although dendrotoxin inhibits the binding of beta-bungarotoxin and the resultant synaptosomal lysis, demonstration of a direct effect of beta-bungarotoxin binding per se on K+ permeability is impractical owing to its phospholipase A2 activity.     

Effect of Climbing Fiber Deprivation on Release of Endogenous Aspartate, Glutamate, and Homocysteate in Slices of Rat Cerebellar Hemispheres and Vermis

Aspartate (Asp) and/or glutamate (Glu) have been proposed as putative excitatory transmitters released from synaptic terminals of the olivo-cerebellar climbing fiber afferents to the Purkinje cells. Investigations of the climbing fiber transmitter(s) separately for hemispheres and vermis were performed to examine whether the current controversy over the role of Asp as a neurotransmitter in the climbing fibers may be due to topographic differences. K(+)-induced Ca2(+)-dependent release of endogenous substances was investigated in slices of cerebellar hemisphere and vermis of control rats and those deprived of climbing fibers by 3-acetylpyridine (3-AP) treatment. A release of Asp and Glu, as well as a small but significant release of homocysteic acid (HCA) was confirmed in control rats. Climbing fiber deprivation by 3-AP treatment reduced the stimulated release of Asp by 48% in slices of cerebellar hemispheres, but not in vermis. Climbing fiber deprivation completely abolished the release of HCA in both hemispheres and vermis. The release of HCA, Asp, and Glu from slices of control and climbing fiber-deprived rats evoked by 50 mM K+ was greater than 90% Ca2(+)-dependent. These results support the hypothesis that Asp is a transmitter candidate of the climbing fibers projecting to the cerebellar hemispheres, but not to the vermis, and provide the first evidence that HCA can be linked to a specific pathway.     

Repetitive Action Potentials in Isolated Nerve Terminals in the Presence of 4-Aminopyridine: Effects on Cytosolic Free Ca2+ and Glutamate Release

The mechanisms by which an elevated KCl level and the K+-channel inhibitor 4-aminopyridine induce release of transmitter glutamate from guinea-pig cerebral cortical synaptosomes are contrasted. KCl at 30 mM caused an initial spike in the cytosolic free Ca2+ concentration ([Ca2+]c), followed by a partial recovery to a plateau 112 +/- 13 nM above the polarized control. The Ca2+-dependent release of endogenous glutamate, determined by continuous fluorimetry, was largely complete by 3 min, by which time 1.70 +/- 0.35 nmol/mg was released. [Ca2+]c elevation and glutamate release were both insensitive to tetrodotoxin. KCl-induced elevation in [Ca2+]c could be observed in both low-Na+ medium and in the presence of low concentrations of veratridine. 4-Aminopyridine at 1 mM increased [Ca2+]c by 143 +/- 18 nM to a plateau similar to that following 30 mM KCl. The initial rate of increase in [Ca2+]c following 4-aminopyridine administration was slower than that following 30 mM KCl, and a transient spike was less apparent. Consistent with this, the 4-aminopyridine-induced net uptake of 45Ca2+ is much lower than that following an elevated KCl level. 4-Aminopyridine induced the Ca2+-dependent release of glutamate, although with somewhat slower kinetics than that for KCl. The measured release was 0.81 nmol of glutamate/mg in the first 3 min of 4-aminopyridine action. In contrast to KCl, glutamate release and the increase in [Ca2+]c with 4-aminopyridine were almost entirely blocked by tetrodotoxin, a result indicating repetitive firing of Na+ channels. Basal [Ca2+]c and glutamate release from polarized synaptosomes were also significantly lowered by tetrodotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of Glutamate Release by Arachidonic Acid in Rat Cerebrocortical Synaptosomes

The action of arachidonic acid on glutamate release in rat cerebrocortical synaptosomes was investigated. The Ca(2+)-dependent release of glutamate evoked by 4-aminopyridine (4-AP) was inhibited by arachidonic acid (0.5-10 microM), but the KCl-evoked release was not modified. The Ca(2+)-independent release of glutamate was insensitive to low concentrations of arachidonic acid, but higher concentrations of this free fatty acid (30 microM) induced a slow efflux of cytoplasmic glutamate. The decrease in the Ca(2+)-dependent release of glutamate by arachidonic acid was consistent with a reduction in both the depolarization and the subsequent rise in the cytoplasmic free Ca2+ concentration induced by 4-AP in the nerve terminal. The inhibitory action by arachidonic acid observed in glutamate release was reversed in the presence of the K(+)-channel blocker tetraethylammonium.     

Interorgan ammonia metabolism in liver failure

In the post-absorptive state, ammonia is produced in equal amounts in the small and large bowel. Small intestinal synthesis of ammonia is related to amino acid breakdown, whereas large bowel ammonia production is caused by bacterial breakdown of amino acids and urea. The contribution of the gut to the hyperammonemic state observed during liver failure is mainly due to portacaval shunting and not the result of changes in the metabolism of ammonia in the gut. Patients with liver disease have reduced urea synthesis capacity and reduced peri-venous glutamine synthesis capacity, resulting in reduced capacity to detoxify ammonia in the liver.The kidneys produce ammonia but adapt to liver failure in experimental portacaval shunting by reducing ammonia release into the systemic circulation. The kidneys have the ability to switch from net ammonia production to net ammonia excretion, which is beneficial for the hyperammonemic patient. Data in experimental animals suggest that the kidneys could have a major role in post-feeding and post-haemorrhagic hyperammonemia.During hyperammonemia, muscle takes up ammonia and plays a major role in (temporarily) detoxifying ammonia to glutamine. Net uptake of ammonia by the brain occurs in patients and experimental animals with acute and chronic liver failure. Concomitant release of glutamine has been demonstrated in experimental animals, together with large increases of the cerebral cortex ammonia and glutamine concentrations. In this review we will discuss interorgan trafficking of ammonia during acute and chronic liver failure. Interorgan glutamine metabolism is also briefly discussed, since glutamine synthesis from glutamate and ammonia is an important alternative pathway of ammonia detoxification. The main ammonia producing organs are the intestines and the kidneys, whereas the major ammonia consuming organs are the liver and the muscle.     

Regulation of Glutamate and Aspartate Release from Slices of the Hippocampal CA1 Area: Effects of Adenosine and Baclofen

Glutamate and/or aspartate is the probable transmitter released from synaptic terminals of the CA3-derived Schaffer collateral, commissural, and ipsilateral associational fibers in area CA1 of the rat hippocampal formation. Slices of the CA1 area were employed to test the effects of adenosine- and gamma-aminobutyrate (GABA)-related compounds on the release of glutamate and aspartate from this projection. Under the conditions of these experiments, the release of glutamate and aspartate evoked by 50 mM K+ was more than 90% Ca2+-dependent and originated predominantly from the CA3-derived pathways. Adenosine reduced the K+-evoked release of glutamate and aspartate by a maximum of about 60%, but did not affect the release of GABA. This action was reversed by 1 microM 8-phenyltheophylline. The order of potency for adenosine analogues was as follows: L-N6-phenylisopropyladenosine greater than N6-cyclohexyladenosine greater than D-N6-phenylisopropyladenosine approximately equal to 2-chloroadenosine greater than adenosine much greater than 5'-N-ethylcarboxamidoadenosine. 8-Phenyltheophylline (10 microM) by itself enhanced glutamate/aspartate release, whereas dipyridamole alone depressed release. These results support the view that adenosine inhibits transmission at Schaffer collateral-commissural-ipsilateral associational synapses mainly by reducing transmitter release and that these effects involve the activation of an A1 receptor. Neither adenosine, L-N6-phenylisopropyladenosine, nor 8-phenyltheophylline affected the release of glutamate or aspartate evoked by 10 microM veratridine. The differing effects of adenosine compounds on release evoked by K+ and veratridine suggest that A1 receptor activation either inhibits Ca2+ influx through the voltage-sensitive channels or interferes with a step subsequent to Ca2+ entry that is coupled to the voltage-sensitive Ca2+ channels in an obligatory fashion. Neither baclofen nor any other agent active at GABAB or GABAA receptors affected glutamate or aspartate release evoked by elevated K+ or veratridine. Therefore, either baclofen does not inhibit transmission at these synapses by depressing transmitter release or else it does so in a way that cannot be detected when a chemical depolarizing agent is employed.     

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.     

An Ion Channel Locus for the Protein Kinase C Potentiation of Transmitter Glutamate Release from Guinea Pig Cerebrocortical Synaptosomes

The mechanism by which protein kinase C (PKC) activates transmitter release from guinea pig cerebrocortical synaptosomes was investigated by employing parallel fluorescent assays of glutamate release, cytoplasmic free Ca2+, and plasma membrane potential. 4 beta-Phorbol dibutyrate (4 beta-PDBu) enhances the Ca(2+)-dependent, 4-aminopyridine (4AP)-evoked release of glutamate from synaptosomes, the 4AP-evoked elevation of cytoplasmic free Ca2+, and the 4AP-evoked depolarization of the plasma membrane. 4 beta-PDBu itself causes a slow depolarization, which may underlie the small effect of 4 beta-PDBu on spontaneous, KCl-evoked, and Ca(2+)-independent/4AP-evoked glutamate release. Because 4AP (but not KCl) generates spontaneous, tetrodotoxin-sensitive action potentials in synaptosomes, a major locus of presynaptic PKC action is to enhance these action potentials, perhaps by inhibiting delayed rectifier K+ channels.     

Regulation of transmitter release from retinal bipolar cells.

Mb1 bipolar cells (ON-type cells) of the goldfish retina have exceptionally large (approximately 10 microns in diameter) presynaptic terminals, and thus, are suitable for investigating presynaptic mechanisms for transmitter release. Using enzymatically dissociated Mb1 bipolar cells under whole-cell voltage clamp, we measured the Ca2+ current (ICa), the intracellular free Ca2+ concentration ([Ca2+]i), and membrane capacitance changes associated with exocytosis and endocytosis. Release of transmitter (glutamate) was monitored electrophysiologically by a glutamate receptor-rich neuron as a probe. L-type Ca2+ channels were localized at the presynaptic terminals. The presynaptic [Ca2+]i was strongly regulated by cytoplasmic Ca2+ buffers, the Na(+)-Ca2+ exchanger and the Ca2+ pump in the plasma membrane. Once ICa was activated, a steep Ca2+ gradient was created around Ca2+ channels; [Ca2+]i increased to approximately 100 microM at the fusion sites of synaptic vesicles whereas up to approximately 1 microM at the cytoplasm. The short delay (approximately 1 ms) of exocytosis and the lack of prominent asynchronous release after the termination of ICa suggested a low-affinity Ca2+ fusion sensor for exocytosis. Depending on the rate of Ca2+ influx, glutamate was released in a rapid phasic mode as well as a tonic mode. Multiple pools of synaptic vesicles as well as vesicle cycling seemed to support continuous glutamate release. Activation of protein kinase C increased the size of synaptic vesicle pool, resulting in the potentiation of glutamate release. Goldfish Mb1 bipolar cells may still be an important model system for understanding the molecular mechanisms of transmitter release.     

Kainic Acid Inhibits the Synaptosomal Plasma Membrane Glutamate Carrier and Allows Glutamate Leakage from the Cytoplasm but Does Not Affect Glutamate Exocytosis

Kainate inhibits the exchange of D-aspartate into guinea-pig cerebrocortical synaptosomes. Kainate inhibits the Ca2+-independent efflux of endogenous glutamate in the presence of a trapping system for the released amino acid but potentiates a Ca2+-independent net efflux of endogenous and labelled glutamate and aspartate in the absence of the trap. Dihydrokainate has a similar effect. No discrepancy is seen between the release of endogenous and exogenously accumulated amino acid. These results are consistent with the presence of a slow leak of glutamate or aspartate from the cytoplasm independent of the kainate-sensitive Na+-cotransport pathway. In the presence of the trap, glutamate effluxes by both pathways, whereas in the absence of the trap, the Na+-cotransport pathway opposes the leak. Neither in the presence or absence of the glutamate trap does kainate induce, inhibit, or otherwise affect the Ca2+-dependent release of endogenous glutamate. The results enable many of the apparent complexities in the presynaptic actions of kainate to be resolved.     

In Vitro Release of Endogenous Amino Acids from Granule Cell-, Stellate Cell-, and Climbing Fiber-Deficient Cerebella

Abstract: The K⁺-stimulated, Ca²⁺-dependent release of glutamate, aspartate, -γ-aminobutyric acid (GABA), alanine, taurine, and glycine was measured in slices of cerebella obtained from control, and granule cell-, granule cell plus stellate cell-, or climbing fiber-deficient cerebella of the rat. The 55 mm -K⁺-stimulated release of glutamate and GABA was 10-fold greater in the presence of Ca²⁺ than in its absence. The stimulated release of aspartate was 4-fold higher when Ca²⁺ was present in the bathing media, while the value for alanine was twice as high as the amount obtained in the absence of Ca²⁺. There was no stimulated release of either taurine or glycine from the cerebellar slices. Increasing the Mg²⁺ concentration to 16 HIM inhibited the K⁺-stimulated, Ca²⁺-dependent release of glutamate, GABA, aspartate, and alanine 85% or more. The K⁺-stimulated, Ca²⁺ dependent release of glutamate, aspartate, and alanine from x-irradiated cerebella deficient in granule cells was reduced to 50–57% of control value. Additional x-irradiation treatment, which further reduced the cerebellar granule cell population and also prevented the acquisition of stellate cells, decreased the release of glutamate by 77%, aspartate by 66%, alanine by 91%, and, in addition, decreased the release of GABA by 55%. The K⁺-stimulated, Ca²⁺-dependent release of glutamate, aspartate, GABA, and alanine was not changed in climbing fiber-deficient cerebella obtained from 3-acetylpyridine-treated rats. The data support a transmitter role for GABA and glutamate in the cerebellum, but do not support a similar function for either taurine or glycine. The data also suggest that alanine and aspartate may be co-released along with glutamate from granule cells.     

Glutamate as a Putative Transmitter in the Cerebellum: Stimulation by GABA of Glutamic Acid Release from Specific Pools

The aim of the present paper was to determine whether the release of glutamate from putative "glutamergic" terminals in the cerebellum is influenced by gamma-aminobutyric acid (GABA). In a group of preliminary experiments, we present biochemical evidence in favour of a neurotransmitter role of glutamate in the cerebellum: (1) endogenous glutamate was released from depolarized cerebellar synaptosomal preparations in a Ca2+-dependent away; (2) [14C]glutamate was synthesized from [14C]glutamine in cerebellar synaptosomes, and the newly synthesized [14C]glutamate was released released in a Ca2+-dependent way; (3) the elevation of cyclic GMP elicited by depolarization of cerebellar slices in the presence of Ca2+ was partly reversed by the glutamate antagonist glutamic acid diethyl ester, which probably prevented the interaction of endogenously released glutamate with postsynaptic receptors. GABA and muscimol at low concentrations (2--20 micrometers) potentiated the depolarization-induced release of D-[3H]aspartate (a glutamate analogue which labels the glutamate "reuptake pool") from cerebellar synaptosomes. The effect was concentration dependent and was largely prevented by two GABA antagonists, bicuculline and picrotoxin. The stimulation of D-[3H]aspartate release evoked by muscimol was linearly related to the logarithm of K+ concentration in the depolarizing medium. GABA did not affect the overall release of endogenous glutamate, but potentiated, in a picrotoxin-sensitive manner, the depolarization-evoked release of [14C]glutamate previously synthesized from [14C]glutamine. Since nerve endings are the major site of glutamate synthesis from glutamine, GABA and muscimol appear to exert their stimulatory effect at the level of "glutamergic" nerve terminals, probably after interacting with presynaptic GABA receptors. The possible functional significance of these findings is briefly discussed.     

Calcium-Dependent and-Independent Release of Glutamate from Synaptosomes Monitored by Continuous Fluorometry

An enzyme-linked fluorometric assay is described for the continuous monitoring of the unidirectional efflux of glutamate from guinea-pig synaptosomes. Glutamate efflux from freshly suspended, polarized synaptosomes occurs at 0.35-0.39 nmol min-1 mg of protein-1 and is not significantly affected by external Ca2+. KCl depolarization (30 mMKCl) in the absence of Ca2+ doubles this rate, whereas in the presence of Ca2+, the initial kinetics of the assay are consistent with the release in the first 5 s of 0.6 nmol mg of protein-1. The final extent of Ca2+-dependent release amounts to 1.9 nmol mg of protein-1, or 8.5% of the total intrasynaptosomal glutamate content. Preincubation of synaptosomes at 30 degrees C for 2 h before depolarization leads to a decrease in Ca2+-independent release and an increase in Ca2+-dependent release, consistent with an intrasynaptosomal relocation of the amino acid.     

N-Methyl-D-Aspartate Releases Excitatory Amino Acids in Rat Corpus Striatum In Vivo

There is a considerable amount of conflicting evidence from several studies as to the action of applied N-methyl-D-aspartate (NMDA) on the release of glutamate and aspartate in the brain. In the present study the effect of NMDA on extracellular levels of endogenous amino acids was investigated in conscious, unrestrained rats using intracerebral microdialysis. NMDA caused dose-related increases in extracellular levels of glutamate and aspartate; threonine and glutamine were unaffected. The NMDA-evoked release of glutamate and aspartate was significantly decreased by the specific NMDA receptor antagonist 3-[(+-)-2-carboxypiperazin-4-yl]-propyl-l-phosphonic acid. In addition, increasing the perfusate concentration (and therefore the extracellular concentration) of Ca2+ significantly enhanced the NMDA-evoked release of glutamate and aspartate, whereas removal of Ca2+ and addition of a high Mg2+ concentration to the perfusate caused a significant reduction in their NMDA-evoked release. Moreover, the NMDA-evoked release of glutamate and aspartate was reduced in decorticate animals. These results demonstrate that, in the striatum in vivo, NMDA causes selective release of endogenous glutamate and aspartate from neurone terminals and that this action occurs through an NMDA receptor-mediated mechanism. The ability of NMDA receptor activation to induce release of glutamate and aspartate, perhaps by a positive feedback mechanism, may be relevant to the pathologies underlying epilepsy and ischaemic and hypoglycaemic brain damage.     

Presynaptic Glutamate/Quisqualate Receptors: Effects on Synaptosomal Free Calcium Concentrations

Intracellular free [Ca2+]i was measured using fura-2 in synaptosomes prepared from cerebral cortices of adult male rats (12 weeks). L-(+)-Glutamate, D-(-)-glutamate, and quisqualate produced similar dose-dependent increases in [Ca2+]i, with EC50 values of 0.38 microM, 0.74 microM, and 0.1 microM, respectively, and maximum increases of approximately 40%. Ibotenate showed less affinity (EC50 4.4 microM) but had a greater maximum effect (57%). N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) did not increase [Ca2+]i. The increases in [Ca2+]i induced by quisqualate and ibotenate were not diminished in the absence of extrasynaptosomal Ca2+. L-2-Amino-4-phosphonobutyrate (L-AP4) (1 microM) completely blocked the changes in [Ca2+]i induced by L-(+)-glutamate, D-(-)-glutamate, quisqualate, or ibotenate. The effects of quisqualate and ibotenate on [Ca2+]i were also blocked by coincubation of synaptosomes with L-(+)-serine-O-phosphate (L-SP) (1 mM) (which, like L-AP4, blocks the effects of quisqualate and ibotenate on inositol phospholipid metabolism). 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) had no effect on agonist-mediated increases in [Ca2+]i when coincubated with either quisqualate or ibotenate. These data are consistent with the existence of presynaptic glutamate receptors (of the excitatory amino acid metabotropic type) which activate phospholipase C leading to the elevation of inositol 1,4,5-trisphosphate and release of Ca2+ from intracellular stores.     

Glutamate Stimulation of [3H]Dopamine Release from Dissociated Cell Cultures of Rat Ventral Mesencephalon

In dissociated cell cultures of fetal rat ventral mesencephalon preloaded with [3H]dopamine, glutamate (10(-5)-10(-3) M) stimulated the release of [3H]dopamine. Glutamate stimulation of [3H]dopamine release was Ca2+ dependent and was blocked by the glutamate antagonist, cis-2,3-piperidine dicarboxylic acid. Glutamate stimulation of [3H]dopamine release was not due to glutamate neurotoxicity because (1) glutamate did not cause release of a cytosolic marker, lactate dehydrogenase, and (2) preincubation of cultures with glutamate did not impair subsequent ability of the cells to take up or release [3H]dopamine. Thus, these dissociated cell cultures appear to provide a good model system to characterize glutamate stimulation of dopamine release. Release of [3H]dopamine from these cultures was stimulated by veratridine, an activator of voltage-sensitive Na+ channels, and this stimulation was blocked by tetrodotoxin. However, glutamate-stimulated [3H]dopamine release was not blocked by tetrodotoxin or Zn2+. Substitution of NaCl in the extracellular medium by sucrose, LiCl, or Na2SO4 had no effect on glutamate stimulation of [3H]dopamine release; however, release was inhibited when NaCl was replaced by choline chloride or N-methyl-D-glucamine HCl. Glutamate-stimulated [3H]-dopamine release was well maintained (60-82% of control) in the presence of Co2+, which blocks Ca2+ action potentials, and was unaffected by the local anesthetic, lidocaine. These results are discussed in terms of the receptor and ionic mechanisms involved in the stimulation of dopamine release by excitatory amino acids.     

Hyperammonaemia alters the mechanisms by which metabotropic glutamate receptors in nucleus accumbens modulate motor function

Activation of metabotropic glutamate receptors by injecting (S)3,5-dihydroxyphenylglycine (DHPG) in nucleus accumbens (NAcc) increases motor activity by different mechanisms in control rats and in rats with chronic liver failure due to portacaval shunt. In control rats DHPG increases extracellular dopamine in NAcc and induces locomotion by activating the 'normal' circuit: NAcc-->ventral pallidum-->medial-dorsal thalamus-->prefrontal cortex, which is not activated in portacaval shunt rats. In these rats, DHPG activates an 'alternative' circuit: NAcc-->substantia nigra pars reticulata-->ventro-medial thalamus-->prefrontal cortex, which is not activated in control rats. The reasons by which liver failure leads to activation of this 'alternative' circuit remain unclear. The aim of this work was to assess whether hyperammonaemia could be responsible for the alterations found in chronic liver failure. We injected DHPG in NAcc of control or hyperammonaemic rats and analysed, by in vivo brain microdialysis, the neurochemical responses of the 'normal' and 'alternative' circuits. In hyperammonaemic rats DHPG injection in NAcc activates both the 'normal' and 'alternative' circuits. In hyperammonaemia, activation of the 'alternative' circuit and increased motor response following metabotropic glutamate receptors activation in NAcc seem due to an increase in extracellular glutamate which activates AMPA receptors.     

Role of Excitatory Amino Acid Receptors in K+-and Glutamate-Evoked Release of Endogenous Adenosine from Rat Cortical Slices

K+ and glutamate released endogenous adenosine from superfused slices of rat parietal cortex. The absence of Ca2+ markedly diminished K+- but not glutamate-evoked adenosine release. Tetrodotoxin decreased K+- and glutamate-evoked adenosine release by 40 and 20%, respectively, indicating that release was mediated in part by propagated action potentials in the slices. Inhibition of ecto-5'-nucleotidase by alpha,beta-methylene ADP and GMP decreased basal release of adenosine by 40%, indicating that part of the adenosine was derived from the extracellular metabolism of released nucleotide. In contrast, inhibition of ecto-5'-nucleotidase did not affect release evoked by K+ or glutamate, suggesting that adenosine was released as such. Inhibition of glutamate uptake by dihydrokainate potentiated glutamate-evoked release of adenosine. Glutamate-evoked adenosine release was diminished 50 and 55% by the N-methyl-D-aspartate (NMDA) receptor antagonists, DL-2-amino-5-phosphonovaleric acid and (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine hydrogen maleate (MK-801), respectively. The remaining release in the presence of MK-801 was diminished a further 66% by the non-NMDA receptor antagonist, 6,7-dinitroquinoxaline-2,3-dione, suggesting that both NMDA and non-NMDA receptors were involved in glutamate-evoked adenosine release. Surprisingly, K+-evoked adenosine release was also diminished about 30% by NMDA antagonists, suggesting that K+-evoked adenosine release may be partly mediated indirectly through the release of an excitatory amino acid acting at NMDA receptors.     

Dopaminergic Modulation of Glutamate Release in Striatum as Measured by Microdialysis

Glutamate and aspartate are the primary neurotransmitters of projections from motor and premotor cortices to the striatum. Release of glutamate may be modulated by dopamine receptors located on corticostriatal terminals. The present study used microdialysis to investigate the dopaminergic modulation of in vivo striatal glutamate and aspartate release in the striatum of awake-behaving rats. Local perfusion with a depolarizing concentration of K+ through a dialysis probe into the rat striatum produced a significant increase in the release of glutamate, aspartate, and taurine. The D2 agonist LY171555 blocked the K(+)-induced release of glutamate and aspartate, but not taurine, in a concentration-dependent manner. The D1 agonist SKF 38393 did not alter K(+)-induced release of glutamate and taurine, but did significantly decrease aspartate release. Neither agonist had any effect on basal amino acid release. The D2 antagonist (-)-sulpiride reversed the inhibitory effects of LY 171555 on K(+)-induced glutamate release. These results provide in vivo evidence for a functional interaction between dopamine, the D2 receptor, and striatal glutamate release.