Glutamate In Rat Hippocampal Slices Research Articles

Tonic activation of NMDA receptors by ambient glutamate of non‐synaptic origin in the rat hippocampus

In several neuronal types of the CNS, glutamate and GABA receptors mediate a persistent current which reflects the presence of a low concentration of transmitters in the extracellular space. Here, we further characterize the tonic current mediated by ambient glutamate in rat hippocampal slices. A tonic current of small amplitude (53.99 +/- 6.48 pA at +40 mV) with the voltage dependency and the pharmacology of NMDA receptors (NMDARs) was detected in virtually all pyramidal cells of the CA1 and subiculum areas. Manipulations aiming at increasing D-serine or glycine extracellular concentrations failed to modify this current indicating that the glycine binding sites of the NMDARs mediating the tonic current were saturated. In contrast, non-transportable inhibitors of glutamate transporters increased the amplitude of this tonic current, indicating that the extracellular concentration of glutamate primarily regulates its magnitude. Neither AMPA/kainate receptors nor metabotropic glutamate receptors contributed significantly to this tonic excitation of pyramidal neurons. In the presence of glutamate transporter inhibitors, however, a significant proportion of the tonic conductance was mediated by AMPA receptors. The tonic current was unaffected when inhibiting vesicular release of transmitters from neurons but was increased upon inhibition of the enzyme converting glutamate in glutamine in glial cells. These observations indicate that ambient glutamate is mainly of glial origin. Finally, experiments with the use-dependent antagonist MK801 indicated that NMDARs mediating the tonic conductance are probably extra-synaptic NMDARs.

Neurotoxicity Induced by Glutamate in Glucose-Deprived Rat Hippocampal Slices is Prevented by GMP

Guanosine-5'-monophosphate (GMP) was evaluated as a neuroprotective agent against the damage induced by glutamate in rat hippocampal slices submitted to glucose deprivation. In slices maintained under physiological conditions, glutamate (0.01 to 10 mM), Kainate, alpha-amino-3-hydroxi-5-methylisoxazole-propionic acid (AMPA), N-methyl-D-aspartate (NMDA), 1S,3R-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD), or L-2-amino-4-phosphonobutanoic acid (L-AP4) (100 microM) did not alter cell membrane permeability, as evaluated by lactate dehydrogenase (LDH) release assay. In slices submitted to glucose deprivation, GMP (from 0.5 mM) prevented LDH leakage and the loss of cell viability induced by 10 mM glutamate. LDH leakage induced by Kainate, AMPA, NMDA or 1S,3R-ACPD was fully prevented by 1 mM GMP. However, glutamate uptake was not altered in slices submitted to glucose deprivation and glutamate analogues. Glucose deprivation induced a significant decrease in ATP levels which was unchanged by addition of glutamate or GMP. Our results show that glucose deprivation decreases the energetic charge of cells, making hippocampal slices more susceptible to excitotoxicity and point to GMP as a neuroprotective agent acting as a glutamatergic antagonist.

Electrically evoked release of glutamate in rat hippocampal slices: effects of various drugs and fimbria-fornix lesions

A model of electrically evoked release of glutamate from rat hippocampus was developed and used to detect possible changes induced by lesions of hippocampal afferences. Neuronal glutamate in hippocampal slices was labelled by preincubation with [3H]glutamine. The slices were then superfused with physiological medium in the presence of the glutamate uptake inhibitor L-transpyrrolidine-2,4-dicarboxylic acid (100 microM or 3 microM) and stimulated twice electrically (S1, S2: 240 pulses, 3 Hz, 2 ms, 26-30 mA); various drugs were added before S2. In order to determine the basal and evoked outflow of [3H]glutamate only, the mixture of 3H-labelled compounds (glutamine, glutamate and GABA) was separated by ion exchange chromatography in superfusate fractions and slices. The electrically evoked overflow of [3H]glutamate was largely Ca2+-dependent and tetrodotoxin-sensitive and hence represented action potential-induced exocytotic release of [3H]glutamate. Evoked [3H]glutamate release was significantly increased by the adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 0.1 microM), suggesting the presence of endogenous inhibitory adenosine, and reduced by the A1 receptor agonist N6-cyclopentyladenosine (1 microM, antagonized by DPCPX, 0.1 microM). There was no evidence for a cholinergic, serotonergic, or adrenergic modulation of the evoked release of [3H]glutamate: the corresponding selective agonists (or antagonists) were ineffective. After aspirative lesions of the septohippocampal pathways the hippocampal noradrenaline content was markedly increased, whereas cholinergic and serotonergic markers were reduced. The evoked release of [3H]glutamate in hippocampal slices of lesioned rats was significantly increased by a mechanism which still has to be determined, but which is not related to alterations in A1 receptor function. It is concluded that the present model was able to detect lesion-induced differences in electrically evoked release of [3H]glutamate, but the relationship of these differences to changes of noradrenergic, cholinergic or serotonergic hippocampal innervations remains to be established.

Ontogenetic and Pharmacological Studies on Metabotropic Glutamate Receptors Coupled to Phospholipase D Activation

The present study was aimed at characterizing the metabotropic receptor subtype which is involved in the activation of phospholipase D (PLD) by glutamate in rat hippocampal slices. We first observed that the ontogenetic profile of glutamate-induced hydrolysis of phosphoinositides and of phosphatidylcholine was strikingly similar. Both pathways were significantly activated by glutamate in tissue taken from 3-, 8- and 15-day old rats, but not in adult rats. PLD activation was strongest in slices taken from 8-day old rats. At this age, quisqualate had a higher potency for PLD activation ( ec 50: 0.6 μM) than 1S,3R-ACPD ( ec 50: 16 μM) and DHPG, a specific activator of group I mGluR, was a full agonist at PLD activation ( ec 50: 3.5 μM) indicating an involvement of a group I mGluR (mGluR1 and 5). MCPG and AIDA, two pulative antagonists at mGluR1 receptors, caused a small but (in the case of MCPG) significant inhibition. DCG-IV, an activator of group II mGluR, was a weak partial agonist at PLD activation ( ec 50: 22 nM) while l-AP 4, an activator at group III mGluR, was totally inactive. Likewise, forskolin, a stimulant of cyclic AMP formation, was inactive either alone, or in combination with glutamatergic agonists. Pretreatment of the slices with pertussis toxin did not affect PLD activation. In summary, the glutamate-mediated activation of hippocampal PLD, which occurs transiently during postnatal development, is mediated by a group I mGluR, possibly involving mGluR5. © 1997 Elsevier Science Ltd. All rights reserved.

Adenosine triphosphate depletion reverses sodium-dependent, neuronal uptake of glutamate in rat hippocampal slices

Extracellular accumulations of excitatory amino acids (EAAs) may mediate ischemic neuronal damage. Metabolic insults can decrease Na+ and K+ plasma membrane gradients, thereby reducing the driving force for uptake of EAAs into cells by Na(+)-dependent EAA cotransporters. EAA accumulations could result from decreased uptake and increased release due to reversal of these cotransporters. ATP depletion, uptake, and release of EAAs were measured by HPLC in slices treated with metabolic inhibitors. Inhibition and reversal of cotransporters were determined by uptake or release of D,L-threo-beta-hydroxyaspartate (OH-Asp), an EAA analog with high affinity for cotransporters. Moderate ATP depletion (7 > ATP nmol/mg protein > 3) reduced uptake by cotransporters without increasing release of EAAs. When ATP was severely depleted (ATP < 2 nmol/mg protein), increased release of EAAs and preloaded OH-Asp occurred, consistent with reversal of cotransporters. Release of glutamine and asparagine was not increased, confirming that release was not primarily due to nonselective increased membrane permeability. ATP depletion and ouabain acted synergistically to produce EAA release, strongly suggesting release was largely mediated by inhibition of Na/K-ATPases. Severe ATP depletion decreased glutamate-like immunoreactivity primarily in axonal terminal-like structures, suggesting release occurred primarily from terminals. Moderate ATP depletion may increase extracellular EAAs by decreasing uptake. Severe ATP depletion may further increase EAAs by reversing uptake, thereby releasing cytosolic neuronal pools of EAAs.

Electrogenic uptake contributes a major component of the depolarizing action of L-glutamate in rat hippocampal slices.

1. A grease-gap technique has been used to measure d.c. potentials, in response to the application of excitatory amino acids and electrical stimulation of the Schaffer collateral-commissural pathway, in the CA1 region of rat hippocampal slices. The actions of L-glutamate (L-Glu) have been quantified and compared to those of structurally related compounds. 2. Perfusion of L-Glu (90s applications) depolarized the tissue with a threshold of approximately 50 microM and a maximum response in excess of 10 mM. L-Aspartate (L-Asp) produced a similar dose-response relationship. By comparison N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) were more potent excitants, producing dose-dependent depolarizations over the range 2-50 microM. 3. Application of the agonists depressed the amplitude of electrically-evoked synaptic responses; an effect that presumably reflects depolarization of neuronal tissue. However, for a given agonist-induced d.c. potential. L-Glu or L-Asp caused smaller depressions of synaptic responses than did either NMDA or AMPA. 4. The combined application of 50 microM D-2-amino-5-phosphonopentanoate (AP5) and 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) substantially depressed synaptic responses and antagonized responses to NMDA and AMPA producing mean (+/- s.e.) dose-ratios of 12.2 +/- 1.2 and 7.0 +/- 0.8, respectively. However, these compounds produced minimal antagonism of responses to L-Glu and L-Asp (dose-ratios of 1.5 +/- 0.1 and 1.5 +/- 0.2, respectively). 5. Responses to the stereoisomers of homocysteate (HCA) were compared over the range 50 microM to 10 mM. D-HCA was approximately 3.6 times more potent than L-HCA and was antagonized to a greater extent by the combined application of 50 microM AP5 and 10 microM CNQX; the dose ratios were 8.7 + 0.8 and 5.1 + 0.9 for the D- and L- isomers, respectively. 6. The application of high doses of an excitant (e.g., 50mM L-Glu or 5mM D-HCA) caused an irreversible loss of sensitivity to NMDA and AMPA and abolished synaptic transmission. Responses to the other excitants were depressed by this excitotoxic lesion in the following order: D-HCA > L-HCA > L-Glu = LAsp. In slices treated in this manner, L-Glu, L-Asp and L-HCA produced very similar dose-response curves. 7. Some slices were unresponsive to NMDA, AMPA and electrical stimulation from the onset of the experiment but had sensitivity to L-Glu, L-Asp and L-HCA similar to that of slices that had received an excitotoxic lesion. 8. Slices that were experimentally lesioned, such that they became unresponsive to synaptic stimulation, AMPA and NMDA, were depolarized by a variety of compounds when tested at 5 mm. The D- and L- enantiomers of Asp and Glu produced similar responses and were slightly more active than quisqualate and threo-hydroxy-L-aspartate (THLA). Glycine, L-serine, D-serine, L-alanine and 4-aminobutanoate (GABA) elicited responses 15-30% of the size, while L-lysine, alpha-amino-isobutanoate and L-proline produced depolarizations of less than 10% of the size of those induced by 5mM L-Glu. NMDA and dihydrokainate were either inactive or induced small negative shifts in the d.c. potential. 9. We conclude that when hippocampal slices are perfused with L-Glu (or L-Asp) the main source of the depolarization is due to an interaction with an electrogenic carrier system.

Extracellular accumulation of glutamate in the hippocampus induced by ischemia is not calcium dependent — In vitro and in vivo evidence

Calcium dependency of ischemia-induced increase in extracellular glutamate in the hippocampus was studied in vitro and in vivo. Perfusion of a low pO 2 medium without glucose (in vitro ischemia) induced an increase in extracellular glutamate in rat hippocampal slices. This increase did not depend on Ca 2+. which is in contrast with the observation that about 40% of membrane depolarization (50 mM KCl)-evoked release was Ca 2+-dependent. In vivo cerebral ischemia of 5 min duration in gerbils also caused Ca 2+-independent increase in extracellular glutamate in the hippocampus. The data suggest that the increase in extracellular glutamate induced by ischemia is not due to the enhanced release of neurotransmitter glutamate.

The release of [3H]GABA formed from [3H]glutamate in rat hippocampal slices: comparison with endogenous and exogenous labeled GABA.

to compare the storage and release of endogenous GABA, of [3H]GABA formed endogenously from glutamate, and of exogenous [14C]GABA, hippocampal slices were incubated with 5 microCi/ml [3,4-3H]1-glutamate and 0.5 microCi/ml [U-14C]GABA and then were superfused in the presence or absence of Ca+ with either 50 mM K+ or 50 microM veratridine. Endogenous GABA was determined by high performance liquid chromatography which separated labeled GABA from its precursors and metabolites. Exogenous [14C]GABA content of the slices declined spontaneously while endogenous GABA and endogenously formed [3H]GABA stayed constant over a 48 min period. In the presence of Ca+ 50 mM K+ and in the presence or absence of Ca2+ veratridine released exogenous [14C]GABA more rapidly than endogenous or endogenously formed [3H]GABA, the release of the latter two occurring always in parallel. The initial specific activity of released exogenous [14C]GABA was three times, while that of endogenously formed [3H]GABA was only 50% higher than that in the slices. There was an excess of endogenous GABA content following superfusion with 50 mM K+ and Ca2+, which did not occur in the absence of Ca2+ or after veratridine. The observation that endogenous GABA and [3H]GABA formed endogenously from glutamate are stored and released in parallel but differently from exogenous labelled GABA, suggests that exogenous [3H] glutamate can enter a glutamate pool that normally serves as precursor of GABA.

Inhibition by Diazepam and γ-Aminobutyric Acid of Depolarization-Induced Release of [¹⁴C]Cysteine Sulfinate and [³H]Glutamate in Rat Hippocampal Slices.

Effects of diazepam and γ-aminobutyric acid-related compounds on the release of [¹⁴C]cysteine sulfinate and [³H]glutamate from preloaded hippocampal slices of rat brain were examined by a superfusion method. Diazepam markedly inhibited the release of cysteine sulfinate and glutamate evoked either by high K⁺ or veratridine without affecting that of other neurotransmitter candidates, e.g., γ-aminobutyric acid, acetylcholine, noradrenaline, and dopamine; IC₅₀ values for the release of cysteine sulfinate and glutamate were about 20 and 7 μM, respectively. γ-Aminobutyric acid (1 to 10 μM) and muscimol (100 μM) significantly reduced high K⁺-stimulated release of glutamate. Bicuculline, which had no effect on the release at a concentration of 50 μM by itself, antagonized the inhibitory effects of diazepam and γ-aminobutyric acid on glutamate release. Similar results were obtained with the release of cysteine sulfinate except that a high concentration (100 μM) of γ-aminobutyric acid was required for the inhibition. These results indicate the modulation by γ-aminobutyric acid innervation of the release of excitatory amino acids in rat hippocampal formation, and also suggest that some of the pharmacological effects of diazepam may be a consequence of inhibition of excitatory amino acid transmission.