dizocilpine-maleate and pyrrolidine-2-4-dicarboxylic-acid

Mechanisms of motor neuron loss in amyotrophic lateral sclerosis (ALS) are unknown, but it has been postulated that excitotoxicity due to excessive glutamatergic neurotransmission by decreased efficiency of glutamate transport may be involved in both familial (FALS) and sporadic ALS. Using microdialysis in vivo, we tested the effects of the glutamate transport inhibitor L-trans-pyrrolidine-2,4-dicarboxylate (PDC) and of 4-aminopyridine (4-AP), which stimulates glutamate release from nerve endings, in the hippocampus and motor cortex of wild type (WT) and transgenic SOD1/G93A mice, an established model of FALS. Perfusion of 4-AP induced convulsions, expression of the inducible stress-marker heat-shock protein 70 (HSP70) and hippocampal neuronal loss. These effects were similar in both WT and G93A mice, and, in both groups, they were prevented by the previous systemic administration of the NMDA receptor antagonist MK-801. In contrast, perfusion of PDC resulted in a large and long-lasting (2 h) increase of extracellular glutamate, but no convulsions, neuronal damage or HSP70 expression were observed in either the WT or the G93A mice. Our results demonstrate that SOD1 G93A mutation does not enhance the vulnerability to endogenous glutamate-mediated excitotoxicity in brain, neither by blocking glutamate transport nor by stimulating its release. Therefore, these data do not support the possibility that glutamate transport deficiency may be an important factor of brain neuronal degeneration in familial ALS.

Topics: 4-Aminopyridine; Amyotrophic Lateral Sclerosis; Animals; Biological Transport; Dicarboxylic Acids; Disease Models, Animal; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Extracellular Space; Glutamic Acid; HSP72 Heat-Shock Proteins; Humans; Mice; Mice, Transgenic; Microdialysis; Neurons; Neurotransmitter Uptake Inhibitors; Pyrrolidines; Superoxide Dismutase

Topics: Amyotrophic Lateral Sclerosis; Animals; Biological Transport; Cell Death; Dicarboxylic Acids; Disease Models, Animal; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glutamate Plasma Membrane Transport Proteins; Glutamates; Humans; Mice; Motor Neurons; Neurotransmitter Uptake Inhibitors; Pyrrolidines; Receptors, Glutamate

In contrast to vertebrates the involvement of glutamate and N-methyl-D-aspartate (NMDA) receptors in brain functions in insects is both poorly understood and somewhat controversial. Here, we have examined the behavioural effects of two noncompetitive NMDA receptor antagonists, memantine (low affinity) and MK-801 (high affinity), on learning and memory in honeybees (Apis mellifera) using the olfactory conditioning of the proboscis extension reflex (PER). We induced memory deficit by injecting harnessed individuals with a glutamate transporter inhibitor, L-trans-2,4-PDC (L-trans-2,4-pyrrolidine dicarboxylate), that impairs long-term (24 h), but not short-term (1 h), memory in honeybees. We show that L-trans-2,4-PDC-induced amnesia is 'rescued' by memantine injected either before training, or before testing, suggesting that memantine restores memory recall rather than memory formation or storage. When injected alone memantine has a mild facilitating effect on memory. The effects of MK-801 are similar to those of L-trans-2,4-PDC. Both pretraining and pretesting injections lead to an impairment of long-term (24 h) memory, but have no effect on short-term (1 h) memory of an olfactory task. The implications of our results for memory processes in the honeybee are discussed.

Topics: Animals; Bees; Cyclohexenes; Dicarboxylic Acids; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Learning; Limonene; Memantine; Memory; Mental Recall; Neural Pathways; Pyrrolidines; Receptors, N-Methyl-D-Aspartate; Smell; Terpenes

It is widely believed that long-term depression (LTD) and its counterpart, long-term potentiation (LTP), involve mechanisms that are crucial for learning and memory. However, LTD is difficult to induce in adult cortex for reasons that are not known. Here we show that LTD can be readily induced in adult cortex by the activation of NMDA receptors (NMDARs), after inhibition of glutamate uptake. Interestingly there is no need to activate synaptic NMDARs to induce this LTD, suggesting that LTD is triggered primarily by extrasynaptic NMDA receptors. We also find that de novo LTD requires the activation of NR2B-containing NMDAR, whereas LTP requires activation of NR2A-containing NMDARs. Surprisingly another form of LTD, depotentiation, requires activation of NR2A-containing NMDARs. Therefore, NMDARs with different synaptic locations and subunit compositions are involved in various forms of synaptic plasticity in adult cortex.

Topics: 2-Amino-5-phosphonovalerate; Animals; Aspartic Acid; Cerebral Cortex; Dicarboxylic Acids; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Glutamic Acid; Long-Term Potentiation; Long-Term Synaptic Depression; N-Methylaspartate; Neurons; Neurotransmitter Uptake Inhibitors; Phenols; Picrotoxin; Piperidines; Protein Subunits; Pyrrolidines; Quinoxalines; Rats; Receptors, Metabotropic Glutamate; Receptors, N-Methyl-D-Aspartate

Glutamate transporters are coupled with cystine/glutamate antiporters to supply cystine as a component of glutathione, an important antioxidant. We sought evidence that L-trans-pyrrolidine-2,4-dicarboxylate (PDC) enhances glutamate-induced neuronal damage not only via the N-methyl-D-aspartate (NMDA) receptor mediated pathway, but also through induction of oxidative stress. Cultured hippocampal cells were exposed to glutamate (100 microM) for 5 min, washed and incubated for 18 hr with PDC (200 microM). PDC, increasing the neuronal death to 147% of that induced by glutamate alone, depleted glutathione in the culture, and produced dichloro-dihydro-fluorescein-diacetate-positive reactive oxygen species in neurons. N-acetylcysteine (2 mM) not only reduced PDC-enhanced neuronal death but also recovered glutathione and abolished the reactive oxygen species in these neurons. Threo-beta-benzyloxyaspartate, another type of glutamate transporter inhibitor, also induced glutathione depletion in the glutamate-preloaded cells, suggesting the involvement of glutamate transporter blocking in glutathione depletion. The NMDA receptor antagonist MK-801, although partially effective in reducing PDC toxicity, slightly recovered glutathione level but did not reduce the reactive oxygen species even at a high concentration (100 microM). N-acetylcysteine, dimethylsulfoxide, alpha-phenyl-N-butyl nitrone and glutathione ethylester prevented neuronal death enhanced by PDC, but superoxide dismutase and catalase did not. Our study provides evidence that the block of glutamate uptake by PDC exerts toxicity on glutamate-pretreated neurons not only through the accumulation of extracellular glutamate and subsequent activation of the NMDA receptor but also through depletion of glutathione and generation of reactive oxygen species.

Topics: Acetylcysteine; Amino Acid Transport System X-AG; Animals; Antioxidants; Aspartic Acid; Cell Death; Cells, Cultured; Dicarboxylic Acids; Dizocilpine Maleate; Drug Interactions; Excitatory Amino Acid Antagonists; Fluorescent Dyes; Glutamic Acid; Glutathione; Hippocampus; Neurons; Neurotransmitter Uptake Inhibitors; Oxidative Stress; Pyrrolidines; Rats; Rats, Wistar; Reactive Oxygen Species

Prolonged inhibition of glutamate reuptake by L-trans-pyrrolidine-2,4-dicarboxylate (PDC), a specific glutamate transporter blocker, reduced the number of GABA positive neurons in a primary cerebellar culture by 54%. The disappearance of immunostaining for GABA was gradual and was partially prevented by the N-methyl-D-aspartate (NMDA) receptor blocker, MK-801, and the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor antagonist, NBQX. Combined blockade of NMDA and AMPA receptors restored the original proportion of GABAergic neurons observed in control cultures. Following the PDC exposure, expression of other GABAergic markers, such as glutamic acid decarboxylase (GAD) and vesicular GABA transporter (VGAT) was also dramatically decreased in an AMPA receptor-dependent manner. Loss of GABA or GAD immunostaining is commonly regarded as a sign of degeneration of GABAergic neurons. However, none of the GABAergic neurons were positive for propidium iodide uptake or showed abnormal nuclear morphology. Based on the above data we conclude that prolonged activation of ionotropic glutamate receptors by endogenously released glutamate was not toxic to cerebellar GABAergic neurons, but lead to the loss of their characteristic neurotransmitter phenotype.

Topics: Animals; Animals, Newborn; Carrier Proteins; Cell Death; Cells, Cultured; Cerebellum; Chromatography, High Pressure Liquid; Dicarboxylic Acids; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Interactions; Excitatory Amino Acid Antagonists; Fluorescent Dyes; GABA Plasma Membrane Transport Proteins; gamma-Aminobutyric Acid; Gene Expression; Glutamate Decarboxylase; Glutamic Acid; Indicators and Reagents; Membrane Proteins; Membrane Transport Proteins; Neurofilament Proteins; Neurons; Neurotransmitter Uptake Inhibitors; Organic Anion Transporters; Organic Chemicals; Propidium; Pyrrolidines; Quinoxalines; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Time Factors

Glucose is the main substrate that fulfills energy brain demands. However, in some circumstances, such as diabetes, starvation, during the suckling period and the ketogenic diet, brain uses the ketone bodies, acetoacetate and beta-hydroxybutyrate, as energy sources. Ketone body utilization in brain depends directly on its blood concentration, which is normally very low, but increases substantially during the conditions mentioned above. Glutamate neurotoxicity has been implicated in neurodegeneration associated with brain ischemia, hypoglycemia and cerebral trauma, conditions related to energy failure, and to elevation of glutamate extracellular levels in brain. In recent years substantial evidence favoring a close relation between glutamate neurotoxic potentiality and cellular energy levels, has been compiled. We have previously demonstrated that accumulation of extracellular glutamate after inhibition of its transporters, induces neuronal death in vivo during energy impairment induced by glycolysis inhibition. In the present study we have assessed the protective potentiality of the ketone body, acetoacetate, against glutamate-mediated neuronal damage in the hippocampus of rats chronically treated with the glycolysis inhibitor, iodoacetate, and in hippocampal cultured neurons exposed to a toxic concentration of iodoacetate. Results show that acetoacetate efficiently protects against glutamate neurotoxicity both in vivo and in vitro probably by a mechanism involving its role as an energy substrate.

Topics: Acetoacetates; Adenosine Triphosphate; Animals; Cell Survival; Cells, Cultured; Dicarboxylic Acids; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Administration Routes; Drug Administration Schedule; Drug Interactions; Embryo, Mammalian; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Female; Glutamic Acid; Glycolysis; Hippocampus; Iodoacetates; Male; Neurons; Neuroprotective Agents; Neurotransmitter Uptake Inhibitors; Pregnancy; Pyrrolidines; Pyruvic Acid; Quinoxalines; Rats; Rats, Wistar; Time Factors

Although neurons often fire in bursts, most of what is known about glutamate signaling and postsynaptic receptor activation is based on experiments using single stimuli. Here we examine the activation of ionotropic glutamate receptors by bursts at the parallel fiber to stellate cell synapse. We show that brief stimulus trains generate prolonged AMPA receptor (AMPAR)- and NMDA receptor (NMDAR)-mediated EPSCs recorded in whole-cell voltage clamp. These EPSCs contrast with the rapid AMPAR-mediated EPSC evoked by a single stimulus. The prolonged AMPAR-mediated EPSC is promoted by high-frequency and high-intensity trains and can persist for hundreds of milliseconds. This EPSC is also increased by l-trans-2,4-PDC, an inhibitor of glutamate transporters, suggesting that these transporters usually limit the synaptic response to trains. These prolonged EPSCs reflect both receptor properties and a long-lasting glutamate signal. In addition, several experiments demonstrate that glutamate spillover can contribute to receptor activation. First, imaging stimulus-evoked changes in presynaptic calcium establishes that distinct parallel fiber bands can be activated. Second, activation of parallel fibers that do not directly synapse onto a given stellate cell can evoke indirect AMPAR- and NMDAR-mediated EPSCs in that cell. Third, experiments using the use-dependent NMDAR blocker MK-801 show that these indirect EPSCs reflect glutamate spillover in response to trains. Together, these findings indicate that stimulus trains can generate a sustained and widespread glutamate signal that can in turn evoke large and prolonged EPSCs mediated by ionotropic glutamate receptors. These synaptic properties may have important functional consequences for stellate cell firing.

Topics: 2-Amino-5-phosphonovalerate; Animals; Cerebellum; Dicarboxylic Acids; Dizocilpine Maleate; Electric Stimulation; Evoked Potentials; Excitatory Amino Acid Antagonists; Glutamic Acid; In Vitro Techniques; Nerve Fibers; Neurons; Neurotransmitter Uptake Inhibitors; Pyrrolidines; Quinoxalines; Rats; Rats, Sprague-Dawley; Receptors, AMPA; Receptors, N-Methyl-D-Aspartate; Synapses; Synaptic Transmission; Time Factors

1. The main purpose of the present study was to investigate the effects of the neuroprotective agent riluzole on the electrically evoked release of [(3)H]-glutamate ([(3)H]-Glu) in mouse neocortical slices. The reported selectivity of riluzole for excitatory amino acids was tested in release experiments with further neurotransmitters. Also distinct species, mouse, rat and man were compared. 2. [(3)H]-Glu was formed endogenously during incubation of slices with [(3)H]-glutamine ([(3)H]-Gln). Released [(3)H]-Glu and tissue [(3)H]-Glu was separated by anion exchange chromatography. Electrically evoked [(3)H]-Glu release was strongly diminished by tetrodotoxin (TTX) and Ca(2+)-withdrawal. 3. Riluzole (100 microM) depressed the release of [(3)H]-Glu up to 77% (IC(50)=19.5 microM). Riluzole was also able to inhibit strongly the electrically evoked release of [(3)H]-acetylcholine ([(3)H]-ACh) (at 100 microM by 92%, IC(50)=3.3 microM, and [(3)H]-dopamine ([(3)H]-DA) (at 32 microM by 72%, IC(50)=6.8 microM). However, the release of [(3)H]-serotonin ([(3)H]-5-HT) was less diminished (at 100 microM by 53%, IC(50)=39.8 microM). Riluzole up to 100 microM did not affect [(3)H]-noradrenaline ([(3)H]-NA) release. 4. Between species, i.e. in mouse, rat and human neocortex, no significant differences between the effects of riluzole could be observed. 5. The NMDA-receptor blocker MK-801 (1 microM) and the AMPA/Kainate-receptor blocker NBQX (1 microM) did neither affect the electrically evoked [(3)H]-ACh release nor its inhibition by riluzole, indicating that effects of riluzole on transmitter release were neither due to modulation of ionotropic Glu receptors, nor due to indirect inhibition of Glu release through these receptors. 6. Taken together, riluzole inhibits the release of distinct neurotransmitters differently, but is not selective for the excitatory amino acid Glu.

Topics: Acetylcholine; Animals; Calcium; Dicarboxylic Acids; Dizocilpine Maleate; Dopamine; Dose-Response Relationship, Drug; Electric Stimulation; Excitatory Amino Acid Antagonists; Glutamic Acid; Humans; In Vitro Techniques; Mice; Neostriatum; Neuroprotective Agents; Neurotransmitter Agents; Norepinephrine; Pyrrolidines; Quinoxalines; Rats; Rats, Wistar; Riluzole; Serotonin; Tetrodotoxin; Tritium

Neuronal damage associated with cerebral ischemia and hypoglycemia might be the consequence of the extracellular accumulation of excitatory amino acids. In previous studies we showed that elevation of glutamate and aspartate extracellular levels by inhibition of its uptake in vivo is not sufficient to induce neuronal damage unless mitochondrial energy metabolism is compromised. In the present study we show that chronic systemic administration of the glycolysis inhibitor iodoacetate (25 mg/kg) induces no damage to the brain per se but enhances neuronal vulnerability to glutamate-mediated neurotoxicity in the hippocampus. Tissue injury is well protected either by antagonizing NMDA glutamate receptors with MK-801 or by administration of pyruvate, a substrate of the tricarboxylic acid cycle. In contrast to systemic treatment, local infusions through a dialysis probe of 5 mM iodoacetate into the hippocampus induced acute lesions not sensitive to MK-801. Iodoacetate intrahippocampal perfusion induced substantial increases in the extracellular levels of glutamate (3.5-fold), taurine (8.8-fold), and particularly aspartate (35-fold). Neuronal damage under this conditions occurs very rapidly as revealed by the histological analysis of animals transcardially perfused immediately after iodoacetate perfusion. Aspartate might contribute to neuronal damage since intrahippocampal administration of this amino acid (600 nmol/microl) induces extensive lesions. The present study might suggest that impairment of glucose oxidation through the glycolytic pathway in vivo facilitates glutamate neurotoxicity. Additionally, the results indicate that pyruvate might prevent as efficiently as glutamate receptor antagonists glutamate-mediated neuronal damage associated with ischemia/hypoglycemia.

Topics: Animals; Dicarboxylic Acids; Dizocilpine Maleate; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Excitatory Amino Acids; Glutamic Acid; Glycolysis; Hippocampus; Iodoacetates; Male; Neurons; Neuroprotective Agents; Neurotransmitter Uptake Inhibitors; Pyrrolidines; Rats; Rats, Wistar

The effect of diethylmaleate administration on ascorbic acid release following cerebral ischemia was investigated in anesthetized rat brain cortex. Cerebral ischemia, induced by ligating bilateral common carotid arteries and unilateral middle cerebral artery, significantly increased the extracellular ascorbic acid levels. Diethylmaleate (4 mmoles/kg, i.p.), which has been shown in earlier studies to decrease the ischemia-induced glutamate release, significantly reduced the ischemia-induced ascorbic acid release. The ischemia-induced ascorbic acid release was unaffected by perfusing NMDA receptor antagonist MK 801 (75 microM). Additionally, elevated extracellular glutamate levels, achieved by either externally applied glutamate solutions or by perfusing L-trans-pyrrolidine-2,4-dicarboxylate (PDC) (31.4 mM and 15.7 mM) to inhibit the glutamate uptake transporter, also significantly increased the extracellular ascorbic acid levels. These results suggested that ascorbic acid release in cerebral ischemia might be related to the elevated extracellular glutamate levels, which occurs following cerebral ischemia.

Topics: Amino Acid Transport System X-AG; Anesthesia; Animals; Ascorbic Acid; Brain Chemistry; Brain Ischemia; Carrier Proteins; Cerebral Cortex; Dicarboxylic Acids; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Extracellular Space; Glutamate Plasma Membrane Transport Proteins; Glutamic Acid; Glutathione; Infarction, Middle Cerebral Artery; Male; Maleates; Microdialysis; Neurotransmitter Uptake Inhibitors; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Symporters

In the present study, we have examined the effects of prolonged (up to 72 h) inhibition of high-affinity glutamate reuptake by L-trans-pyrrolidine-2,4-dicarboxylate (PDC; 100 microM) on glutamate receptor functions in primary cultures of rat cerebellar granule neurons. This was done by comparing the effects of various glutamate receptor agonists on neuronal 45Ca2+ uptake, free cytoplasmic Ca2+ concentration ([Ca2+]i), and cell viability. We also determined the parameters of[3H]MK-801 binding as well as the expression of the NMDAR1 subunit protein in control and PDC-exposed cultures. The blockade of glutamate reuptake by PDC led to a gradual increase of ambient glutamate to concentrations that are neurotoxic when applied acutely to control cells. In PDC-exposed cells, however, the acute glutamate-induced NMDA receptor-mediated calcium fluxes were strongly diminished and no toxicity was observed. The down-regulation of the functional effects of glutamate was dependent on the duration of PDC exposure and was accompanied by a reduced NMDAR1 subunit expression and decreased [3H]MK-801 binding, indicative of a pronounced structural rearrangement of NMDA receptors. The possibility that the decrease of NMDA glutamate receptor sensitivity can be explained on the basis of a reduced density or altered subunit composition of NMDA receptors is discussed.

Topics: Animals; Calcium; Cells, Cultured; Cerebellum; Cytoplasm; Dicarboxylic Acids; Dizocilpine Maleate; DNA Fragmentation; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Excitatory Amino Acid Antagonists; Glutamates; Histones; Neurons; Neurotoxins; Neurotransmitter Uptake Inhibitors; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Time Factors

Glutamate can be toxic to neurons although it is a neurotransmitter. Regulation of extracellular glutamate levels is essential for prevention of glutamate neurotoxicity. Astrocytes play a major role in clearance of glutamate released by neurons. A coculture system combining cerebellar cells and astrocytes was employed to investigate the astrocytic control of glutamate toxicity. Coculture of astrocytes with cerebellar neurons enhanced uptake of glutamate by astrocytes. Inhibition of glutamate uptake in a coculture system led to death of cerebellar cells. This toxicity could be inhibited by MK801. However, in the presence of the glutamate uptake inhibitor, there was no increase in glutamate in the cultures compared to when the neurons were not cocultured. This indicated that neurons become more susceptible to glutamate toxicity in the presence of astrocytes and thus become dependent on astrocytes for prevention of glutamate toxicity. Astrocytes treated with conditioned medium from cerebellar cells did not show an increase in glutamate uptake but medium from astrocytes exposed to neuron conditioned medium was toxic to cerebellar cells. This toxicity was due to glutamate present in the medium. This suggests that a soluble factor released by neurons signals to astrocytes that neurons are present and stimulates a signal back to neurons which causes an increased sensitivity to glutamate toxicity.

Topics: Animals; Astrocytes; Cell Communication; Cells, Cultured; Cerebellum; Culture Media, Conditioned; Dicarboxylic Acids; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glutamic Acid; Memantine; Mice; Neurons; Neurotoxins; Neurotransmitter Uptake Inhibitors; Pyrrolidines; Signal Transduction; Tritium

Prolonged blockade of glutamate reuptake by the specific inhibitor of glutamate transporters, L-transpyrrolidine-2,4-dicarboxylate (PDC), produces a dramatic decrease in NMDA-induced neurotoxicity in cerebellar granule cell cultures, and is accompanied by a down-regulation of NMDA receptors. We now report that cultured cerebellar granule cells treated with 100 microM PDC for 1, 2, 4, 8, 16 and 24h, respectively, show increased AP-1 DNA-binding activity as measured by electrophoretic mobility shift assay. This effect was blocked by the NMDA receptor antagonist, CGP 37849, indicative of a pivotal role of NMDA receptors in the PDC-evoked enhancement of AP-1 DNA-binding. Our results suggest that AP-1 may be involved in the transcriptional regulation of neuronal adaptation initiated by prolonged inhibition of glutamate reuptake.

Topics: 2-Amino-5-phosphonovalerate; Animals; Biological Transport; Cells, Cultured; Cerebellum; Dicarboxylic Acids; Dimerization; Dizocilpine Maleate; DNA-Binding Proteins; Excitatory Amino Acid Antagonists; Glutamic Acid; Neurons; Neurotoxins; Neurotransmitter Uptake Inhibitors; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; Pyrrolidines; Rats; Rats, Sprague-Dawley; Receptors, N-Methyl-D-Aspartate; Transcription Factor AP-1

This study examined the effects of chronic intrastriatal infusion of L-trans-pyrrolidine-2,4-dicarboxylate (PDC), a selective competitive inhibitor of high affinity glutamate transport systems, via osmotic minipumps in rats. Injection of PDC at the rate of 25 nmol/h for 14 days caused striatal lesion. Histological evaluation on frontal striatal sections showed that the lesion was circumscribed to a circular area showing a dramatic neuronal loss accompanied by gliosis and representing 30% of the whole striatal surface at the level of the injection site. A total loss of neurons expressing glutamate decarboxylase (GAD67), enkephalin or substance P mRNA was observed on a similar circular area, suggesting degeneration of the two populations of striatal efferent neurons. In the whole striatum outside the region devoided of hybridization signal, a selective 27% decrease in enkephalin mRNA expression occurred, suggesting a higher sensitivity of enkephalin neurons versus substance P neurons to glutamate uptake-mediated alterations. Injection of PDC at the rate of 25 nmol/h for 3 days produced striatal lesion of similar extent. In contrast, PDC at the rate of 5 nmol/h did not produce neuronal damage when administered over 14 days. This study provides new in vivo evidence that defective glutamate transport is one of the critical conditions that may give rise to toxicity of an endogenous transmitter system in the striatum, and may underlie neuronal death in neurodegenerative diseases.

Topics: Animals; Biological Transport; Corpus Striatum; Dicarboxylic Acids; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Female; Glutamic Acid; In Situ Hybridization; Infusion Pumps, Implantable; Molecular Weight; Nerve Degeneration; Neurotransmitter Uptake Inhibitors; Osmotic Pressure; Pyrrolidines; Rats; Rats, Wistar

It is well documented that neurons exposed to high concentrations of excitatory amino acids, such as glutamate and aspartate, degenerate and die. The clearance of these amino acids from the synaptic cleft depends mainly on their transport by high-affinity sodium-dependent carriers. Using microdialysis in vivo and HPLC analysis, we have studied the effect of the administration of inhibitors of the glutamate transporter (L-trans-pyrrolidine-2,4-dicarboxylate and dihydrokainate) on the extracellular concentration of endogenous amino acids in the rat striatum. In addition, we have analyzed whether the changes observed in the concentration of glutamate and aspartate were injurious to striatal cells. Neuronal damage was assessed by biochemical determination of choline acetyltransferase and glutamate decarboxylase activities, 7 days after the microdialysis procedure. In other experiments, pyrrolidine dicarboxylate and dihydrokainate, as well as two other inhibitors of the glutamate carrier, DL-threo-beta-hydroxyaspartate and L-aspartate-beta-hydroxamate, were microinjected into the striatum, and neuronal damage was assessed, both biochemically and histologically, 7 or 14 days after the injection. Dihydrokainate and pyrrolidine dicarboxylate produced a similar remarkable increase in the concentration of extracellular aspartate and glutamate. However, the former induced also notable elevations in the concentration of other amino acids. Clear neuronal damage was observed only after dihydrokainate administration, which was partially prevented by intraperitoneal injection of (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate or by intrastriatal coinjection of 2,3-dihydroxy-6-nitro-7-sulfamoylbenzo(f)quinoxaline. No cell damage was observed with the other three glutamate carrier inhibitors used. It is concluded that an increased extracellular glutamate level in vivo due to dysfunction of its transporter is not sufficient for inducing neuronal damage. The neurotoxic effects of dihydrokainate could be explained by direct activation of glutamate postsynaptic receptors, an effect not shared by the other inhibitors used.

Topics: Animals; Cell Death; Choline; Choline O-Acetyltransferase; Corpus Striatum; Dicarboxylic Acids; Dizocilpine Maleate; Extracellular Space; gamma-Aminobutyric Acid; Glutamate Decarboxylase; Glutamic Acid; Kainic Acid; Male; Microdialysis; Nerve Degeneration; Neurons; Neurotransmitter Uptake Inhibitors; Pyrrolidines; Quinoxalines; Rats; Rats, Wistar